Drx PDFs

Lecture 37: Bacterial Metabolism and Growth

Major Features of Prokaryotes and Eukaryotes Bacteria grow in a variety of forms Bacterial Biofilms and Quorum Sensing Division 1. Cell wall extends 2.Chromosome replicates 3. Septum forms and DNA binds at the mesosom Solution to Stubborn Bacterial Infections Might Be Sweet Energy is generated through catabolism. Cellular constituents are synthesized through anabolism. Minimal growth requirements include: carbon, nitrogen, iron, etc. Growth requirements and products of metabolism are used in classification Ex. Carbon source Ex. Obligate vs. facultative intracellular growth Ex. Ability to grow in presence/absence of oxygen Ex. Ability to ferment specific sugars Catalase Test + Staphylococcus - Streptococcus Growth on MacConkeyAgar Lac -/Lac + Glycolysis(anaerobic): 2ATP TCA(aerobic): 38ATP Cytoplasmic Membrane Site of: Active transport of molecules Electron transport chain Synthesis of cell wall precursors Signal transduction Most actions at cell cell, ribosome, RNA synthesis, Folate metabolism, and DNA synthesis Peptidoglycan synthesis: soluble substrates activated, attached on undecaprenol phosphate, peptidoglucam units cross link on outside Transpeptidation reaction: MurNAC - Lys binds to second-t-last C=O D Ala (MurNAC flipped) Sites of actions of inhibitors of bacterial cell wall synthesis: Fosfomycin NAG to NAM, Cycloserin NAM to NAM, Bacitracin inhibits recycling of bactoprenol, Vancomyci, Vancomycin, Penicilin acts on B-lactam Lipopolysaccharide (LPS) is synthesized on the cytoplasmic side of the inner membrane (IM) and flipped to the periplasmic side by the ATP-binding cassette (ABC) transporter MsbA. LPS is then transported to the cell surface by the LPS transport (Lpt) pathway. This pathway consists of seven essential proteins, LptA, LptB, LptC, LptD, LptE, LptF and LptG. LPS is extracted from the IM in an ATP-dependent manner by the ABC transporter LptB2FG and transferred to LptC, which forms a complex with LptB2FG. LptC consists of a single membrane- spanning domain and a large periplasmic domain, which forms a periplasmic bridge with the soluble protein LptA and the amino-terminal region of LptD. LPS transverses the aqueous periplasmic space through this protein bridge and reaches the cell surface with the aid of the carboxy-terminal domain of LptD, which forms a β-barrel structure that is plugged by the outer membrane (OM) lipoprotein LptE. LPS is composed of lipid A, the inner and outer core oligosaccharides, and the O antigen, which is highly variable and absent in Escherichia coli K-12. The letters A-G in the figure correspond to the respective Lpt protein in the transport pathway. EtN, ethanolamine; Gal, D-galactose; Glc, D-glucose; Hep, L-glycero-D-manno-heptose; Kdo, 3-deoxy-D-manno-octulosonic acid; P, phosphate; Pi, inorganic phosphate.

Select Economic Aspects of Drug Use

Medicine spending below healthcare spending growth for 5 of the last 10 years $374 BN industry Government health care spending crowds out other taxpayer-funded priorities 58% $457 billion was spent on prescription drugs in 2015, comprising 16.7% of total healthcare spending. ASPE also broke down the drivers of increased drug spending, finding: - 10% was due to population growth; - 30% to increases in the number of prescriptions per person; - 30% to economy-wide inflation; - 30% to changes in composition of drugs prescribed towards higher-priced products or price increases for drugs that drove the average price increase above general inflation Increasing Utilization of Medications all ages but mine Specialty" Pharmaceuticals Spending 1/3 spending oncology autoimmune HIV MS VH other Drug Pricing Dominates the News - Still, and More to Come PHARMALOT SEPTEMBER 19, 2016 Sarepta to charge $300K for Duchenne drug. 'We tried to be reasonable,' CEO says Forbes Pharma and HealthCare August 21, 2016 Why Did Mylan Hike EpiPen Prices 400%? Because They Could The Wall Street Journal January 10, 2016 Drugmakers Raise Prices Despite Criticisms: Pharmaceutical companies cite high cost of research as reason for price increases Pharmaceutical Industry ■ ■ ■ ■ ■ Good Guys? Economic juggernaut Cure for Chronic Hepatitis C infection Disease modifying therapies for Cystic Fibrosis, many immune diseases Advances in treatment of cancer Bad Guys? ■ Price increases beyond inflationary factors ■ Accusations of Price gouging, cornering the market ■ CEO compensation ■ Drugs in United States costing significantly more than in other countries LantusTM Price Increases Oct 2012 - Nov 2014 188% Price Increase Nov 2014 - Jul 2016 O% Price Increase Insurance Who Pays - How? ■ ■ Pool of money to provide benefits to members of the pool (beneficiaries) Cost-sharing strategies: - Premiums • Employer ● Individual - Deductibles • Out-of-Pocket (OOP) payments before benefit kicks in - Co-Insurance • Percentage OOP payment for service - Co-Pays • Fixed dollar amount OOP payment for service Pharmacy Benefit Cost-sharing ■ Co-Pays - Tier 1 - Tier 2 - Tier 3 - Tier 4 Generics $10 Preferred Brands $25 Non-preferred Brands $50 Specialty Pharmacy 20% • AKA co-insurance Pricing / Cost / Value / Affordability ■ Price vs. Cost - Price - Sum of money asked or given - Cost - Value of resources consumed ■ List Price and Discounts ■ Value - Is it worth it? - Cost Effectiveness; Willingness to Pay ■ Affordability - Can we afford it? - Feasible; Sustainable MassHealth Overview (FY17) ■ Members - ~1.98 Million Total Including: - Medicaid Contracted Managed Care Organizations - Primary Clinician Care Plan ("in-house" managed care plan) - "Fee-for-Service" Members (Mostly Other Insurance) • Medicare Dual Eligible members • Members with commercial insurance • "True" FFS members ■ Dollars - - - State Budget - $39.15Bn MassHealth Budget - $15.4Bn • Plus Connector, Health Safety Net, other Pharmacy Spend - MCOs (~$1B) + PCCP/FFS ($654Mm) • Plus Dual Eligibles - Medicare Part D Clawback ($372Mm) BUSM Disease and Therapy August 1, 2017 MassHealth Rx Spending $1,200 MassHealth Pharmacy Spend FY 2000-2017 Retail Pharmacy, Fee-for-Service (Non-MCO) lower Brand - Generic Utilization Rate 2002 - 2016 % Claims December 2002 MassHealth is primary payer Avg claim vs generic claim omeprazole = most amount Harvoni - 22k Budget Impact Model: Axicabtagene ciloleucel $300,000 8 to 20 $2.4 to $6 million/year* ■ Approval of federal 1115 waiver ■ Development of Accountable Care Organization (ACO) Models - Model A: Integrated entity includes both the ACO provider and health plan (MCO) - Model B: ACO providers contract directly with MassHealth, which remains the insurer - Model C: ACO providers contract directly with MCOs Deploy ACO Pilot project - PCC Plan only (Dec 2016) - Boston Accountable Care Organization - Community Care Cooperative - UMass Memorial Healthcare, Inc - Partners Healthcare Accountable Care Organization - Children's Hospital Integrated Care Organization - Steward Medicaid Care Networ MassHealth Priorities Restructuring / Payment Reform ACO models: goals and principles ■ Materially improve member experience- ACOs expected to innovate and engage members differently ■ Strengthen the relationship between members and Primary Care Providers (PCPs) by attributing members to an ACO through their selection of a PCP ■ Encourage ACOs to develop high value, clinically integrated provider partnerships ■ Partner with MCOs, with expectations for MCOs to help administer the ACO program and work with providers in strengthening provider-based care management ■ Increase BH/ LTSS integration and linkages to social services in ACO models Value Frameworks for Medication ■ Analytical tool to measure the value of medications and determine potential impacts on healthcare system ■ Five Models: - - - - - American College of Cardiology / American Heart Association American Society of Clinical Oncology Memorial Sloan Kettering Cancer Center National Comprehensive Cancer Network Institute for Clinical and Economic Review Value and Affordability (ICER Definitions) ■ Care Value - Comparative clinical ■ Affordability - Short-term budget impact - Project uptake rate and competitive products - Benchmark budget impact ≤ GDP growth rate +1% ICER = Institute for Comparative Effectiveness Research effectiveness - Incremental cost- utility ($100- 150K/QALY) - Benefits / disadvantages - Other contextual issue Pharmacoeconomics ■ Identifying, measuring, and comparing the costs, risks, and benefits of programs, services, or therapies and determining which alternative produces the best health outcome for the resources invested. - Cost-Minimization Analysis - Cost-Benefit Analysis - Cost-Effectiveness Analysis - Cost-Utility Analysis The Generic Cliff Zyprexa - olanzapine Wholesale Acquisition Cost 95% REDUCTION Cost-Benefit vs. Cost-Effectiveness COST-BENEFIT COST-EFFECTIVENESS ■ ■ ■ ■ Uses dollar values for outcome measurements Maximizes benefit of investment Assumes limited resources Compares programs with different objectives ■ Use non-monetary outcome measurements ■ Minimizes cost of program ■ Assumes adequate resources ■ Compares programs with the same objective Sample Comparison Using Different Benefit- Cost Approaches Cost-Effectiveness Analysis Which drug is more cost effective for lowering blood pressure? Average Blood Pressure Cost/Month Reduction Drug X Drug Y $35 12mm Hg $50 15mm Hg Cost Effectiveness Ratio: Drug X: Drug Y: $35 / 12 = $2.91 / mmHg $50 / 15 = $3.33 / mmHg Cost-Utility Analysis Which chemotherapy regimen is preferred? Increased Lifespan of Life Average Quality Adjusted Life Years Gained 1.8 1.6 Quality Chemotherapy Regimen 1 * Chemotherapy Regimen 2 * 2 years 0.9 0.4 4 year * Assumes regimens are same cost The Medication Use Process Adjust MONITOR All PRESCRIBE Physician DISPENSE Pharmacist 40 ADMINISTER Nurse / Patient 1 IN 10 GETS DFRUG ERROR ■ Drug-related morbidity and mortality estimated to cost $76.7 billion annually in the ambulatory setting in the USA - 8.76 million hospitalizations (28%) @ $47.4 billion - Long-Term Care Admissions - Physician and Emergency Department Visits - Additional Prescriptions - 199,000 Deaths Governor declares an emergency on opiate abuse DEATHS INCREASE AND OPIOPD SOLD INCREASES AND ADMISSIONS Management strategies • Dose limits (120 MME as of March 8, 2016) • Duplicate short-acting and long-acting • Quantity limits • High dose short-acting monotherapy restriction Therapeutic Class Management (TCM) Workgroup • Reviews outlier member cases • Outreach to prescriber regarding treatment plans • Referrals to fraud, waste and abuse Recent legislation • Initial prescription day supply limits ■ ■ ■ Up to 50% of patients do not adhere to medication regimen Adds $100B in avoidable hospitalizations 89,000 premature deaths Increases total cost of care in U.S. by $290B Drug Expense Management ■ Follow the money... ■ Distribution of Dollar Revenue from a Retail Prescription - Manufacturer $0.76 - Pharmacy $0.22 - Wholesaler $0.02 Basic Strategies to Manage Pharmacy Costs ■ Control the Payment Rate - Manage the Reimbursement Rate to Providers ■ Control the Volume - Manage the Number of Units • Prescriptions or Tablets ■ Control the Utilization - Manage Selection of Drug Formulary (Preferred Drug List) - GenericEquivalence - TherapeuticEquivalence - PatientIncentives ■ Drug Use / Treatment Guidelines ■ Disease State or Care Management ■ Restrictions (e.g., Prior Authorization) ■ Drug Use Evaluation / Review - Prospective 49 BUSM Disease and Therapy August 1, 2017 - Retrospective

Introduction to Neoplasia Adaptive response, hyperplasia

Metaplasia - a reversible change from one adult cell type to another adult cell type: Adaptive Dysplasia- - Loss of normal orientation of one cell to another in a particular tissue - Preneoplastic - Associated with cellular "anaplasia" - Metaplasia, Gastroesophageal junction Barrett Esophagus Differentiation - the extent to which neoplastic cells resemble the parent cell of origin • "Well" differentiated - close resemblance to parent cell • "Poorly" differentiated - more primitive • "Moderately" - between well and poorly differentiated • For glandular tissues - ability to form glands • For squamous epithelium - ability to form keratin Epithelia Squamous - skin, oral mucosa cervix Glandular - ducts, GI tract, endocrine glands Transitional - bladder, ureters • Mesenchymal tissues Connective tissues - fibrous, muscle, bone, cartilage, blood vessels • Specialized tissues Gonads - germ cells Brain • Benign neoplasms • Cells resemble normal cells • Well circumscribed (encapsulated) • Localized - do not spread to other sites • Amenable to surgical excision • Mesenchymal - connective tissues - often designated by the suffix "oma" • Benign tumor of fibrous tissue - fibroma • Benign tumor of smooth muscle - leiomyoma • Benign tumor of adipose tissue - lipoma • Benign tumor of cartilage - chondroma • Benign tumor of endothelium - endothelioma Squamous - Papilloma • Glandular - Adenoma Benign • Dysplastic - Low grade - High grade • Carcinoma in situ • Invasive carcinoma • Metastatic carcinoma Could still be problematic - Due to location - Due to functional status - Due to complications - Due to "syndromic complexes" • Multiple endocrine neoplasia Adrenal gland tumor Familial Adenomatous Polyposis (FAP) MatureTeratoma - Arises from germ cells, and can give rise to different types of tissue. • Mixed tumor- may have neoplastic components of epithelial and mesenchymal (stromal) elements Carcinoma in situ of breast: Intact basement membranes, Invasive Adenocarcinoma, Invasive Squamous cell carcinoma, Malignant tumors, invasive, Pleomorphism + Mercedes benz Malignant tumors • Lack of differentiation of cells • Cellular pleomorphism • Nuclear pleomorphism • Increase in Nuclear cytoplasm ratio • Prominent nucleoli • Aneuploidy (abnormal DNA content) • Hyperchromasia of nuclei • Abnormal mitosis • Tumor giant cells • Anaplasia Lymphovascular Invasion: -how a tumor spreads in the body Tumor cells in a lymphatic space Tumor cells in a lymph node CANCEROUS: Lymphoma • Melanoma • Mesothelioma • Hepatoma • Epithelial cells - carcinomas (i) Glandular - Adenocarcinomas (ii) Squamous - Squamous cell carcinomas (iii) Urothelium - Urothelial carcinomas • Mesenchymal cells - sarcomas Fibrosarcoma Leiomyosarcoma Chondrosarcoma Osteosarcoma • Organ specific - Hepatocellular carcinoma (hepatoma) - Renal cell carcinoma - Papillary carcinoma of the thyroid • Germ cell tumors - Seminoma - Embryonalcellcarcinoma - Choriocarcinoma • Brain - Astrocytomas - Meningioma Grade: • Well differentiated - grade 1 • Moderately differentiated - grade 2 • Poorly differentiated - grade 3 Stage of tumor: • TNM criteria - Tumor - Nodes (Lymph nodes) - Metastasis Staging is more prognositc

Adaptation, Injury & Cell Death

-Cells & Tissues Respond to Stress: cells need to adapt to stress via hyperplasia, hypertrophy, atrophy, or metaplasia. i.e. hyperplasia of liver via TNF on Kuppfer cell = IL-6 = EGF/TGF-a/HGF/MET = proliferation. -Prostate gland hyperplasia via testosterone -Hyperplasia (#/vol): • Increased number of cells • Increases volume of the organ or tissue. • Only in cell population capable of division. • Physiologic or Pathologic. -Hypertrophy: increased load response, mechanical stretch, agonist a-angiotensin, IGF-1, synthesis of proteins, GF, genes, more performance. -• Increase in the size of cells • Increased synthesis of cellular components. • Can occur in fully mature non-dividing cells. • Usually caused by increased functional demand. • Physiologic or Pathologic Denervation Atrophy, Autophagy, Myocardial residual bodies, disuse, denervation, ischemia, inadequate nutrition, loss of hormonal stimulation, senile atrophy. Atrophy: • Loss of cell substance and or cell number. • Protein degradation (ubiquitination) exceeds synthesis • Autophagic vacuoles increase • Residual bodies increase • Physiologic or Pathologic. Metaplasia: Barrett's Esophagus, reveriusble change, adult cell flip, induces cytokines and gf, tissue and circulating stem cell. 1) aerobic respiration for the production of ATP - the mitochondria 2) Osmotic and ionic balance - membranes 3) Protein synthesis - ribosomes 4) Structural maintenance and intracellular transport - cytoskeleton 5) Functional genetic apparatus - nucleus Injury via: 1. Oxygen Deprivation 2. Physical Agents 3. Chemical Agents and Drugs 4. Infectious Agents 5. Immunologic Reactions 6. Genetic Derangements 7. Nutritional Imbalances Reversible Injury: RER swelling, hydronic change, steatosis, clumping of chromatin, membrane blebs Irreversible: lysosome rupture, swollen mitochondria, cell membrane rupture -Threshold of effect is reversible then gets worse: Cell death, ultrasutrual changes late, light microscope changes later, gross chances latest -Acute Myocardial Infarct Coagulation Necrosis, Gangrenous Necrosis, Lung Abscess Liquefactive Necrosis, Liquefactive Necrosis of Brain Coagulative (pink) vs Liquefactive Necrosis (grainy) Enzymatic Fat Necrosis (grainy near fat) Tuberculosis of Lung Caseous Necrosis (giant cells) Fibrinoid Necrosis (central ring) Types: 1. Coagulative Necrosis - Gangrenous Necrosis 2. Liquefactive Necrosis 3. Enzymatic Fat Necrosis 4. Caseation Necrosis 5. Fibrinoid Necrosis Methods of Cell DeathL Low ATP, Mitochrondial leakage, Ca+ Entry leads to leaking mito, high ROS damage, Membrane damage (PM. or lysosome), protein misfoliding or DNA damage.

Lecture 39: Pathology Overview

-First sign of anoxic cell injury: cell swelling -Reversible injury example is cytoplasmic vacuoles, chromatin clumping, cytoplasmic inclusions, and cellular edema -Irreversible injury includes pkynosis, rupture of cell membrane, karyolysis, and karyorrhexis -Splenic infarct: coagulative necrosis. -Ischemic infarct in brain: liquefactive necrosis -MI increases serum enzyme creatine kinase due to increases plasma membrane permeability from cell membrane rupture. -Squamous epithelium lining endocervial is an example of metaplasia -Papillary projects and fibromuscular storm lesion is an example of a epithelial and stroll hyperplasia response to stress in prostate resection sample. -Carb metabolism disorder causing increased hepatic glycogen and hypoglycemia is a result of absence of glucose-6-phosphatase. -Jewish splenomegaly lysosomal storage disease would be Gauchers Disease Type I. -Stored material in cells of infant dying from impaired Skelton devoted and heart failure is dermatan sulfate -Primary vascular mechanism for edema in acute inflammation is increased vascular permeability -Inflammatory response of a bronchospasm and edema due to bee sting is from a mast cell -Cell death from conversion of cell to acidophilic body + loss of nucleus but share is same will by just apoptosis -Yellow sclerae means abnormally high bilirubin in liver -Neutrophils are unable to recirculate once they leave the blood stream -Apriation of 1.050 means it is an effusion resulting from a serous exudate. -Acute apendicisti and acute inflame shows high neutrophils in blood. -Acute inflammation and fever is due to cytokines made by monocytes/macrophages -Infection by mycobacterium tuberculosis and peritoneum nodules will consist of macrophages and giant cells. -Bening tumor is low mitotic, nucleoli, encapsulation, different (normal N/C ration) -Injury with high ALT: Hepatocellular -Hepatic toxicity from acetaminophen overdose: NAPQI

The Pharmacology of Anti- inflammatory and Immunosuppressant Agents

-H1 Receptor Antagonists "Antihistamines" Histamine mediates •allergic and inflammatory reactions •gastric acid secretion •neurotransmission in parts of the brain Found in almost all tissues (lung, skin, gastric mucosa, CNS) Stored in secretory granules in mast cells and basophils (inactive and bound to heparin and an anionic protein) Histamine Release from Mast Cells and Basophils • Bradykinin • Complement components C3a and C5a • Antigen interaction with IgE • Basic drugs - Curare - Opioids - Vancomycin Histamine Release from Enterochromaffin-like Cells in Gastric Wall • Neurotransmitters - ACh • Hormones - Gastrin Discovery of Histamine Receptor Subtypes Development of "antihistamines" in 1940s with no effect on gastric acid secretion • Development in 1970s of histamine antagonists of gastric acid secretion with no effect on histamine-induced smooth muscle contraction Diphenhydramine and Loratadine are H1 receptor antagonists IP3DAGCA2+ Ranititnde is a H2 receptor antagonist cAMP H1 Recepter Activation should jusually increase permability, dilate via endothelial NO release, SM constrictio of larger arteries and veins and bronchioles (neural reflex increases sensitivity of asthmatics). Activating the H1 receptor causes sensory nerve ending stimulation of itch, flare, and pain, CNS arousal of histamionergic fibers from tuberomamillary nucleus diffusely to cortex. Intradermal Injection of Histamine: Redspotdueto vasodilation • Wheal: swelling of red spot due to increased vascular permeability • Flare due to axonally mediated reflex vasodilation Histamine-induced Reactions inclue itching, rhinitis, conjuctvites, angioedema, hives, and hypotension (direct drug effect i.e. morphine). Effects of H2 receptor activation: Stimulation of hydrogen ion secretion by gastric parietal cell, vascular smooth muscle relaxation, increate heart rate and force of contraction. H1 ANTAGONIST: 1st Gen: Diphenhydramine Benadryl 2nd Gen: Loratadine Claritin Aryl -X-Y-C-C-N 1st Gen: High selectivity for H1 vs H2, weak selectivity for H1 vs others (local anesthetic, antimuscarinin, a-adenrenegic, 5-HT), access to CNS sedative effect. Effect of H1 Antihistamines: Appetite, sedation, hypotension, dizziness, dry mouth, urine retention, sinus tachycardia. Use of H1 antagonist: ithcing, rhinitis, conjunctivity, hives, mastocytosis, myelogenous leukemia. DO NOT USE H1 ANTAG for astham or tpye 1 hypersensitivity. 1st Gen H1 can be used for nasuea, motion sickness, sedative. ATROPINE-LIKE toxicity of 1st gen h1: fixed dilated puples, dry mouith, tachycardia, urine retention, hallucinations, and fever bc blocks Ach. Advantages of 2nd gen H1: higher selectivity for H1 over others so less antimuscarinic effects, lower csedation Carboxylate residue limits access to CNS. Toxicity of 2nd gen: Ventricular arrhythmia, tosades de pointes, inhibition of K+ channel in heart. NSAIDS & Aspirin-like Drugs • Large class of agents used to treat inflammation, pain and fever. • Mechanism of action of agents is primarily mediated via inhibition of cyclooxygenase (COX), the enzyme involved in the production of prostaglandins (Pgs), thromboxanes (TxA) and prostacycline • Agents are called: nonopioid, nonnarcotic analgesics; antipyretic analgesics; nonsteroidal anti-inflammatory drugs (NSAIDs) - Inhibits COX/P's and Ts but not LOX Ls - PGE2: Fever, Pain, Edema -PGE2 + PGF2a: Increased GI motility, GI cytoprotection, decreased gastric secretion, uterine contract -Prostacyclin: inhibit platelet agrreagation, vasodilation TXA2: stimulate platelet aggregation, vasoconstriction Cyclooxygenase Pathway: COX-1 •Constitutively expressed •Gastric cytoprotection •Vascular homeostasis •Platelet aggregation •Kidney function COX-2: •Induced by cytokines, growth factors & tumor promoters •Associated with inflammation & cancer •Also constitutively expressed in brain, kidney, bone and gastrointestinal tissues Nonselective, Traditional NSAIDS (tNSAIDS): • Ibuprofen, Naproxen (& many others) • older agents that inhibit both COX-1 and COX-2 isozymes. • COX-2 inhibition is thought to mediate the antipyretic, analgesic and anti-inflammatory actions. • COX-1 inhibition largely (not entirely) mediates many adverse effects. COX-2-selective agent: • Celecoxib • designed and marketed to selectively inhibit COX-2 isoenzyme to provide antipyretic, analgesic and anti-inflammatory actions with less adverse effects (GI effects) Nonopioid, Nonnarcotic Analgesic agent: Acetaminophen • Antipyretic and analgesic activity • No anti-inflammatory activity Acetaminophen • Relatively weak cyclooxygenase inhibitory action compared with salicylates and NSAIDs • In analgesic doses its potency, elimination half- life, and duration of action are similar to salicylic acid (major metabolite of aspirin). • Lacks peripheral side effects observed with other cyclooxygenase inhibitors (no platelet effect, little GI toxicity) Analgesic and antipyretic actions due to reversible cyclooxygenase inhibition in the CNS as compared to peripheral sites • Actions at CNS receptors • Lack of anti-inflammatory effect possibly results from its poor ability to inhibit COX in the presence of peroxides found in sites of inflammation Therapeutic Uses: • Mild-to-moderate pain, fever • NOT an anti-inflammatory and less effective than NSAIDs in some conditions Analgesia •Antipyresis - e.g. chronic inflammatory conditions like rheumatoid arthritis • Preferable antipyretic-analgesic for patients at increased risk of NSAID toxicity Acetaminophen Biotransformation Metabolite causes centrilobular hepatic necrosis Risk increased by: CYP2E1, CYP3A4 inducers more quinone formed Alcohol CYP2E1 induction and hepatotoxicity Glutathione depletion less quinone detoxified •Drug of choice for children with viral infections & patients intolerant to aspirin and other NSAIDS "Traditional" NSAIDs • Aspirin* • Nonacetyatedsalicylates* • Ibuprofen*,naproxen*,fenoprofen, flurbiprofen • Diflunisal •Diclofenac,etodolac,sulindac,ketorolac • Meloxicam,piroxicam • Nabumetone • Indomethacin Salicylates: Aspirin irreversibly inactivates cyclooxygenase NSAIDS: Propionic Acid Derivatives: Ibuprofen (Advil, Motrin) (t1/2 ~ 2 h). Naproxen (Aleve, Naprosyn) (t1/2 ~ 14 h) Antipyresis Analgesia: Anti-inflammatory • Treatment of chronic musculoskeletal disorders • Do not arrest the progress of the disease or induce remission (often used in combination with DMARDS) • Reduce elevated body temperature • Relieve mild to moderate pain (headache, dysmenorrhea & dental procedures and pain originating from joints, skeletal muscle) Antipyretic Effect of NSAID • Decrease in elevated body temperature - Sweating - Increase in cutaneous blood flow • Response to reduced PgE synthesis in hypothalamus • Avoid salicylates in children with fever from viral illness due to association with Reyes'syndrome - Acetaminophen as preferred antipyretic-analgesic Analgesia •Inhibit peripheral PGE2 synthesis •Possible inhibition of constitutively expressed COX-2 in the dorsal horn of the spinal cord •Relieve mild to moderate pain •Limited maximal effect Opiod hass higher potency and max efficacy than NSAID + acetaminophen Anti-inflammatory reduce the inflammatory response to mechanical, chemical or immunological stimuli by inhibiting production of prostaglandins primarily by COX-2. Another proposed anti-inflammatory mechanism of salicylates includes the inhibition of activation of the transcription factor NF-kB - Decreased synthesis of proinflammatory mediators such as TNF-a - Decreased synthesis of COX-2 Inflammation of the joints - Rheumatoid, psoriatic, IBD-related arthritis - Osteoarthritis - Ankylosing spondylitis Therapeutic Uses of Aspirin Cardiovascular Use Aspirin is used prophylactically to decrease the risk of thromboembolic disorders such as transient ischemic attacks, and myocardial infarction Increased Bleeding Time •Inhibition of thromboxane synthesis o prolongs bleeding time by inhibiting normal formation of a platelet plug and platelet-induced acceleration of coagulation. o Aspirin and tNSAIDS prolong bleeding time o Aspirin irreversibly inactivates COX-1 in platelets o A single analgesic dose of aspirin doubles bleeding time for the life of the platelet (4 - 7 days) o Acetaminophen does not affect platelet function -81 mg/day: antiplatelet 650-100mg: analgesia 3 g/day: anti-inflammatory Renal Toxicity • Sodium retention and edema - NSAIDs - risk greater in patients with renal and CV disease. • Papillary necrosis and interstitial nephritis - "Analgesic nephropathy" - Acetaminophen and NSAIDs GI Toxicity • Most common side effect - est. 15-30% of regular users - Dyspepsia, GI bleeding, erosions, and ulcers • Risk caused by COX-1 inhibition: - reduced synthesis of gastroprotective prostaglandins in stomach wall - ulcerogenic effect of gastric acid secretion 'Aspirin Intolerance' • Bronchospasm, rhinorrhea and/or hives - INDUCED BY NSAIDS - NOT BY ACETAMINOPHEN • More common in asthmatics • Mediated by leukotrienes • Cross-sensitivity with other NSAIDS but not acetaminophen Selective COX-2 Inhibitors •Higher TI than non selective NSAIDS •Equal analgesic & anti-inflammatory efficacy with decreased risk of GI effects. COX-2 Inhibitors •No effect on platelets ROFECOXIB CELECOXIB The active center of COX-2 has a larger side pocket which can accommodate molecules with bulkier side chains than COX-1 Cox Selectivity: iC50 is much lower for COX-2 meaning higher potency juvenile and rheumatoid arthritis osteoarthritis ankylosing sponylitis primary dymenorrhea • GI Toxicity: lower risk than naproxen, and ibuprofen • Renal Toxicity: sodium retention and edema • Cardiovascular Toxicity: possible increased risk of heart attack and stroke •In platelets: there is no COX-2; TXA2 production is unchecked •In endothelial cells: COX-2 inhibition decreases PGI2; decreases PGI2-induced vasodilatation and inhibition of platelet aggregation •The risk of CV adverse effects increases with chronicity of dosing. •label contains boxed warning highlighting the risk of adverse CV events •lowest dose for the shortest period of time should be used •contraindicated for patients with cardiovascular disease •CV warning for all NSAIDs (except aspirin) Aspirin: High incidence of GI Effects; useful anti- platelet action at low doses Ibuprofen & naproxen: OTC nonselective NSAIDS with moderate effectiveness. Naproxen: longer-acting (t1/2 ~ 14 h) Celecoxib: selective COX-2 inhibitor; equal analgesic & anti-inflammatory efficacy; lower risk of GI effects; possible increased risk of heart attack and stroke

Cholinergic Drugs Sympathetic - exit from thoracic and lumbar region Parasympathetic - exit from cranial and sacral areas

Acetylcholine - ACh (cholinergic receptors) ACh Nicotinic (N) Muscarinic (M) Nicotinic postganlionic M on heart N on SM N on Epinephrine Norepinephrine - NE (adrenergic receptors) NE a1, a2, b1, b2 PS:, converse energy, bradycardia, vasodilation, low bp, high RBF, increase urine, increase salivation, I, low respiration, increase motlirty Sym: - Expenditure of energy - Tachycardia - Vasoconstrictor predominance - Blood pressure increased - Renal blood flow decreased - Urine output decreased - Salivation reduced - Respiration increased - GI motility and secretions reduced LOW HR: PS ACH M HIGH HR: S NE B1 HIGH GI: PS ACH M LOW GI: NE A2 block PS so no Ach no M GI low PS: Short Ciliary nerves to Spincter Pilillae contraction Iris sphincter muscle: Parasympathetic (miosis) S: Opthalmic nerve long ciliary nerve dilator iridis (Iris dilator muscle: Sympathetic (mydriasis)) Point and Shoot BLOOD VESSELS HAVE ONLY SYMAPTHETIC INNVERVATION Primarily parasympathetic NS Predominant Tone except blood vessels and sweat glands (sympathetic cholingergic) Dom PS: Hear (B1>B2 SA B1>B2 contractility), Iris (a1 dilates), Ciliary muscle (b2 contracts) GI tract (a1,a2,b1,b2 increases motility, a2 incrrases secretion, a1 relaxes sphincters), Urinary bladder (a1 ejaculation spincter, b2 bladder) Salivary glands(M,a,a1), Liver (b2,a1 increase sugar release into blood sym, a1-b3 increase fat cell lipolysis, kidney a1b1 increase renin). Dom Sym: Arterioles Veins(a1,a2,b1,b2 in organs and skin contract, b2 in muscle relaxes) NE, Sweat Ach. Sym nerve: a2 lower NE release PS: a2 lower Ach release In humans, the release of acetylcholine is responsible for which of the following postganglionic sympathetic responses? a. Activation of sweat glands

Acute Inflammation:

-A patient with acute inflammationPneumonia. Leukocytes found in Buffy coat. Granulocytes like NP, BP, EP: short lived nonrecirculating. -Monocytes in periphery, macrophage in tissue. Microglia in CNS< Histiocytes in CT, Kupffer cell in liver. Host response to injury or insult Infections Trauma Loss of blood - infarct (heart attack) Vaccinations Or - Immune reactions with blood flow Lasts hours to days. Neutrophils - hallmark inflammatory cell - part of innate immune system If you see neutrophils think acute inflammation Mediators of acute iflammation: made by preformed granules or de novo. Plasma protein derived present in inactive form or made in liver. -Mediators are Stimulate other cells Release secondary effector molecules Amplify or oppose given response Quickly destroyed or removed Activities in time and space are limited STEP OF ACUTE INFLAM 1. Blood vessel dilates to increase flow (Hyperemia in eyes) 2. Microvascular changes allow proteins and cells to leave the blood vessel 3. Emigration, accumulation and activation of leukocytes Vasodilators 1. Vasoactive amines (histamine, serotonin) 2. Cycloxygenase froma arachidonic acid (PG12), PGE2, PGD2, Prostacyclin. 3. NO 4. Bradykini 5. PAF (at low, at high PAF. causes vasoconstriction) 1.Vasoactive amines His/Ser are earliest mediators, preformed in granules, mast cell/platelets, histamine stimulated by trauma, cold, heart, binds ab to mast cells, C3a/C5a anaphylatoxin, substance P neuropeptides, dilates arterioles and increases permeability. Mast cell degranulation using allergen, heat, neuropeptide stimulus. 2. Cycloxygenase froma arachidonic acid (PG12), PGE2, PGD2, Prostacyclin. AA is a 20c pollyunsat FA phosphilids in cell membrane makes: prostaglandins, leukotrienes, and lipoxins. Glucocorticoids inhibit phospholipase so reduces the production a bit. COX-1/COX2 inhibit CO reducing just Prostaglandings and thromboxane. 3. Platelet Activating Factor is derivced form phsopholipids from leukocytes, mast cells endothelial cells, platelets, causes vasodilation in low levels, platelet aggeregation, bronchocontriction, leukocyte oxidative burst, leukocyte adhesion,c hemotaxis, and degranulation. 4. Nitric Oxide - NO: Synethesized from L-arginine + oxygen Constitutive, Inducible, or Endothelial isoforms. Vasodilation rleaxing SM, inhibit cellular components like PA WBC recruitment, microbicidal. 5. Biologically active proteins: Insulin, complem ent, coagulation proteins, proteolytic cleavage. Prekallikrein using Factor XIIa and HMWK Kallikrein makes Bradykinin a nonapeptide to increase vascular permeability, increase contraction of non-bv SM, dilate blood vessels, pain! Cell Derived Histamine, serotonin Cyclooxygenase products Nitric Oxide Platelet Activating Factor Plasma protein derived Bradykinin Most have additional biological activities Transudate: Fluid due to early inflam or hydro forces low protein <1.015 Exudate: Escames w high proteina nd high cell: +1.015. Effusion The escape of fluid into a defined cavity. Can be transudate or exudate. Edema The accumulation of fluid into the interstitium of tissues or alveolus. Can be transudate or exudate. Pulmonary Edema Types of Exudates: • Serous: Contains few cells. Clear fluid. Suggests mild vascular injury. • Purulent: Many inflammatory cells. "Pus" • Hemorrhagic: Contains red blood cells. Implies capillary damage. • Fibrinous: Exudate with a layer of fibrin deposited onto serosal surface. PLASMA LEAKAGE MEDIATORS: -Retraction of endothelial cells Histamine *Leukotrienes* Nitric oxide Bradykinin *Substance P* -Vasodilatation i.e. exert dual effects Histamine *Prostaglandins* Nitric Oxide Bradykinin Fibrinous exudate Bread & Butter Pericarditis Injury to endothelial cells Cytokines Bacterial Toxins (may be indirect by inducing cytokines) Leukocytes Leakage of Plasma Proteins Adhesion molecule expression AND Extravasation of neutrophils Leakage benefits: Plasma proteins are important - Kill invading pathogen (IgG FcyRI Fc kill) - Remove necrotic tissue Accumulate inflammatory cells PAMP DAMP TNF IL-1: 1. Margination 2. Rolling - Selectins & integrins 3. Tightadhesion - Integrins 4. Migration across vessel wall Adhesion Molcules: Pselectin - SLX RollingPMT Eselectin -SLX RA PMT Glycam1 -L selectin R PM ICAM-1 CD11/18 AdArMig PML VCAM-1 VLA-4 Int Adh EML Neutrophil chemotactic factors: 1. Bacterial products 2, C5a 3. LTB4 4. IL-8 CXC chemokines 5. PAF Chemotactic Cytokines • Chemoattractants for leukocytes • CXC - An amino acid between two cysteines. - Major neutrophil chemoattractant and activator • CC - No intervening amino acid - Attract monocytes and lymphocytes Vascular effects o C3a and C5a (anaphylatoxins) o Release histamine from mast cells o Vasodilation o Increased permeability C5a o Chemotaxis, esp o neutrophils, o Activation of lipoxygenase pathway of arachidonic acid o C3b: opsoninizes bacteria for phagocytosis Biological Activities of Inflammatory Mediators: Vasodilation: Histamine/Prostaglandins Increased vascular permeability: Histaminr, Serotonin, C3a, C5a, Leukotrienes C4, D4, E4 Chemotaxis, recruitment, activation: TNF, IL-1, chemokines, C3a, C5a, LTB4 Fever: TNF, Il-1,IL-6,Prostaglandins Pain: Prostaglandins, Bradykinin Tissue damage: Lysosome enzsymes, ROS Systemic Effects of Acute Inflammation 1. Fever: TNF, IL-1, IL-6, Prostaglandins on hypothalamic thermoregulatory center. 2. Leukocytosis: CSF increase leukocute release from bone marrow. 3. Tachycardia rapid heart rate 4. Tachypnea Rapid respiratoru rate 5. Acute phase reactants from hepatocyte release via IL-6. SOB: leakage of plasma proteins Green sputum: neutrophil degradation of bacteria Fever and malaise: CO metabolises and cytokines Purpose of acute inflammation Eliminate pathogen (s) Remove necrotic tissue Steps to accomplish these goals 1. Recognition 2. Removal 1. Extracellular traps 2. Intracellular killing a) Phagocytosis b) Phagolysosome c) Proteases d) Reactive oxygen and nitrogen Neutrophil Extracellular Traps: Product of a dying neutrophil Composed of strands of chromatin and proteins Bacteria stick to the chromatin Proteins have anti-microbial activity Phagolycosome: destroy gram negative rod Oxygen metabolites: Oxygen derivatives • Superoxide: O2• • Hydrogen peroxide: H2O2 • Hydroxyl radical: OH• • Main source • Leukocytes iNOS + Arginine + OH* + NADPH + Phagocyte Oxidase + MPO = OCL OH OONO kill G- rod. Tissue injury: more inflammation, release ROS, release protease, coagulation. Outcomes of acute inflammation 1. Complete resolution 2. Fibrosis 3. Chronic inflammation

MCQs lecture

1. Norepinephrine acts on beta-1 receptors to INCREASE heart rate. 2. NE acts on alpha-1 receptors to INCREASE blood pressure. 3. NE causes an increase in BP and decrease in HR if via IV; if muscarinic receptors are blocked then NE increases both BP (a1) and HR(b1). 4. Propranolol is a B1 antagonist/blocker alone causes no channge in BP (a1) and decrease in HR. 5. NE is a a1/b1 agonist alone causes rise in BP and baroreflex drop in HR. Miuscarinic blockade prevents M2-mediated baroreflex so the direct effect of NE is to increase HR via B1 and increase Bp via B1. 6.Isoproterenol is a B1 agonist and causes an increase in HR via B1. M2 is from the heart, M2 is from exocrine glands. 7. Adrenergic agonists can be used a) Phenylephrine: Nasal decongestants (a1; prevents dilation and engorgement of nasal mucosa blood vessels) b) Epinephrine a1 vasoconstriction reduces drug absorption to increase local anesthetic action c) Epinephrine a1/b1 resuscitation after cardiac arrest d) a1 vasoconstriction antidote to hypotensive agent overdose e) a1 vasoconstriction with fluids maintain blood pressure in spinal damage or anesthesia f) DA a1 restorate blood pressure after cariogenic shock g) Clonidine: a2 treat hypertension h) Phenylepinephrine a1 dilation for mydriasis i) a2/B1 reduce fluid generation for glaucoma j) a2 increases fluid outflow for glaucoma k) Allergy Asthma b2- Asthma bronchial smooth muscle relaxation l) a1-b1-b2 anaphylactic shock (b2 relaxes airway smoke muscle and inhibits mast cell release of anaphyslasic mediators, a1 maintains MAP) o) b2 relaxes uterine SM to delay premature labor q) a2 treats opioid withdrawals w) ADD via amphetamine methylphenidate z) Anaphylactic shock: g allergic reaction caused by severe hypersensitivity to insect venoms (e.g., bee), certain foods (e.g., peanuts), and certain drugs (especially penicillin). Acute treatment via epinephrine for a/b a1: vasoconstriction to increase BP and prevent glottal edema b1: increases CO/HR to help rise BP b2: increases bronchodilation -Albuterol most likely to induce bronchial dilation -NE: no B2 selectivity -Phenylephrine - a1 agonist vasoconstrictor -Clonidine a2 agonist - inhibits sympathetic sedative -Phenylephrine is a a1 agonist vasoconstrictor applying to nasal mucosal to induce vasoconstriction a1 side affects: vasoconstriction, ischemia, hypertensive reactions, cerebral hemorrhage, rebound nasal congestion a2 agonist: withdrawal syndrome b1: tachycardia, myocardial schema, cardiac ventricular arrhythmias b2: increased asthma exacerbation (b2 helps bronchodilate), hyperglycemia (b2 liver glycogenolysis) -Adrenergic agonists cause CNS stimulation side affect -Indirect Sympathomimetics will increase the availability of endogenous adrenergic transmitters: Dopamine(octopamine), amphetamine, methylphenidate, and cocain. -Indirectly acting adrenergic agonists: tyramine, ephedrine, amphetamine increase NE release leading to more postsynaptic action. Tyamine in food, cheese, red wine, cute: tyramine degraded by MAO, if MAO is inhibited (Parkinson's): hypertensive crisis by cheese/red Wien due to release of NE on b1, tyramine pressor response BP increases by 30mmHg. Longterm: tyramine converts to octopamine via dopamine b-hydroxylase and stores to replace NE which has little action so long term MAO therapy can impair sympathetic nervous system. -Indirect adrenergic agonists that increase NE include dextroamphetamine, methylphenidate via ADHD (NE DA release + a/b1 stimulate), ephedrine (release NE + a/b in nutritional supplements FDA regulated), pseudoephedrine used for decongestants. -Tricyclic antidepressants, coins (prevent NE DA reuptake) are central active leading to fatal CV VNS events. -Direct neuronal blockers: Resperine depletes NE stores by inhibits VMAT (inhibiting vesicular monoamine transport) in synapse, so no NE release, NE killed by intraneuronal MAO (monoanimine oxidase). Adrenergic Antagonists:AcH M on heart and SM, slow HR, no b1/b2/1 response. A1 Antagonist: The blockade or reduction of epinephrine and norepinephrine binding on alpha adrenoreceptors reduce arteriolar resistance and increase venous capacitance causes a reflex increase in heart rate. Depending on plasma concentration they may cause orthostatic hypotension. Alpha-1 blockers may decrease LDL and triglycerides and increase HDL. A2 Antagonist: Mechanistically, α2-blockers significantly increase adrenergic, dopaminergic and serotonergic neurotransmitters, and induce insulin secretion and decreases blood sugar levels. TCA. Phentoalamine a1/a2 a1/a2 irreversible: phenoxybenzamine/ethylemimonium a1 only: prazosin a2 only: Yohimbine Therapy: treat hypertension, treat pheochromocytoma, Raynaud reduce digital vasospasm, treat heart failure by lowering vascular resistance, treat BPH, decrease bladder muscle tone to make peeing easier, antidote to a-1 agonist vasocontsitiron. Side Effects: Postural hypotension (inhibit a-mediated vasoconstriction): reflex tachycardia, myocardial ischemia due to more O2 demand, B1 renal RAS reflex salt/water retention, peripheral edema, GI stimulation pain, inhibition of ejaculation. Beta Adrenergic Antagonists B1/B2/CNS: Propranolol (HR + BP) B1 only: Metoprolol (HR+) B1/B2/a1: Carbedilol B1/B2: Pindolol B1 short: Esmolol B1/B2/a1: Labetalol B1/B2: Propranalol B1: Atenolol Beta antagonists are used for Treatment of cardiac arrhythmias, reduce mortality after MI, shock to keep CO/perfusion, heart failure (carvedilol increases CO), hypertension (reduce CO inhibit renin), treatment of angina (to control O2 demand), treartment of hyperthyroidism (propranolol), reduce anxiety, migraine prophylaxis, OPEN ANGLE galucome. Timolol has a protective effect after MI. Carvedilol has a protective affect in heart failure. Metoprolol is less effective in protecting in CHF mortality. Toxic side affects of B-antagonists include bronchoconstirction, bradycardia, withdrawal increases angina, fatigue, cold extremities, epinephrine vasoconstriction, CHF increase via inhibition of adrenergic compensation, predispose new onset diabetes., -The reflex change in heart rate associated with administration of a low dose of isoproterenol could be blocked by administration of which one of the following agents? Propranol: Isoproterenol is a B1/B2 agonist causing vasodilation/tachycardia; effects can be blocked by a nonselective b-antagonist propanol. Atropine is a muscarinin antagonist. Prazosin is a a-1 antagonist. Physostigmine is a reversible cholinesterase inhibitor (increase Act) Apoptosis in liver lobule apaototic body is called councilman body. Necrosis: Unprogrammed/chaotic Numerous cells/field of injury Always pathological Cell membranes disrupted Inflammatory response Apopstosis: Programmed/controlled Scattered single cells Physiological or Pathological Cell membranes intact No inflammatory response Physiologic causes of apoptosis: Organogenesis - developmental involution and metamorphosis during embryogenesis. • Hormone-dependent involution e.g. the menstrual cycle and regression of the lactating breast after weaning. • Cell deletion in proliferating cell populations e.g. the crypts of the colon, granulocytosis Pathologic causes of apoptosis: Pathologic • Viral diseases, particularly viral hepatitis. • Pathologic atrophy following duct obstruction such as occurs in salivary gland, pancreas and kidney. • Cell death in some tumors • Cell death in response to DNA injury drugs • Cell death in response to misfolded proteins in the endoplasmic reticulum. Methods of Apoptosis: GF withdrawal, DNA damage, ER stress, Death Receptor Extrinsic Fas, TNF receptor, Apoptotic body results for phagocytosis. BCL2 increases BAX BAK and cytocrhrome C so caspase then apaostisis cell death. -Alzheimers disease cerebral generalized atrophy. -Drug (MMF) induced Apoptosis Colonic glands -Necroptosis (programmed necrosis): begins by ligation of TNFR1 by TNF = RIP1/RIp3 -> decreased mictochondrial ATP and higher ROS, membrane cell leakage. -Steatohepatitis Mallory's Hyaline.

Integ

Clinical Correlation DRx Foundations Robert C. Lowe, M.D. August 1, 2017 • 27 y.o. male with a history of IVDU presents with three days of R arm pain and redness. • He reports chills, but has not taken his temperature at home. • He uses 5-10 bags of heroin per day, shooting up in his arms and legs. • He has no other past medical history Physical exam • T 101.0 P 95 BP 120/70 RR 14 O2 sat 99% • HEENT normal • Chest CTA • Card RRR S1+S2 with no M/R/G • Abd soft, NT/ND, no HSM • Ext: no c/c/e • Skin... Cellulitis • Skin infection involving dermis and subcutaneous tissue • Erythema, edema, pain, warmth • May be associated with abscess • Commonly due to Strep and Staph in normal hosts • Diabetics = much broader range of bacteria www.goldbamboo.com Levels of SSTI Green, et al. Chest 1996; 110 219-29 Levels of SSTI Green, et al. Chest 1996; 110 219-29 All that is inflamed is not infected CCJM 2012 Aug;79(8):547-552. Venous stasis dermatitis Necrotizing Fasciitis • Infection of deep fascia - poorly vascularized • Type 1 - anaerobes, Gram negatives • Type 2 - Group A strep (+/- Staph) • Severe pain, bullae, purple color, anesthesia of surface • Surgical debridement is essential Levels of SSTI Green, et al. Chest 1996; 110 219-29 Contrasting Pathologic Findings Cellulitis Necrotizing Fasciitis • Non-necrotizing • Acute inflammation- PMNs • Architecture largely intact • Dermis, upper subcutaneous tissue involved • Necrosis of fascia and fat • Patchy acute inflammation • Extensive destruction • Can see bacteria in tissue sfghed.ucsf.edu/Education/ClinicImages/infections.htm Labs 16.5 250 138 104 24 3.6 24 0.9 110 40.1 Poly 90% Lymph 8% Monos 1% Eos 1% Plasma Protein Vessel Lumen PMN Endothelial cell Damage Damaged endothelial cell Tissue Leakage of Plasma Proteins AND Extravasation of neutrophils 19 Steps in Acute Inflammation 1. Blood vessel dilates to increase flow 2. Microvascular changes allow proteins and cells to leave the blood vessel 3. Emigration, accumulation and activation of leukocytes Which of the following receptors mediates the rolling of neutrophils along the endothelium? P-selectin Migration Across The Vessel Wall Which of the following is found in Gram positive bacteria, but NOT in Gram negative bacteria ? . Teichoic acid Bacterial Cell Walls Mycobacteria produce a thick mycolic acids-rich outer covering which functions as an exceptionally efficient barrier. Penicillin Methicillin blactam ring Vancomycin Spectrum: gram positive aerobes, including MRSA No gram negative activity, molecule too large to cross the outer membrane in gram negative organisms • Use: - Gram positive infections resistant to beta lactams - Gram positives infections in persons unable to tolerate beta lactams - Second line therapy for C. difficile colitis (oral) Vancomycin • Pharmacokinetics - Poor oral absorption, only use PO for C. difficile colitis - Good distribution (CSF poor) - Minimal metabolism, renal excretion - Monitor trough levels • AdverseEffects - Infusion related effects (slow rate of infusion) - Hematologic - Hypersensitivity - Ototoxicity/Nephrotoxicity rare

Pediatric Pathology I- CongenitalAnomalies(DevelopmentalGenetics) II- Prematurity 1. Neonatal Respiratory Distress Syndrome 2. Necrotizing Enterocolitis 3. Intraventricular and Germinal Matrix Hemorrhage III- Sudden Infant Death Syndrome IV- Pathologic Findings in Cystic Fibrosis (Molecular Genetic Abnormalities and Genetic Screening and Prenatal Diagnosis) • Pediatric pathology is considered a unique subspecialty within pathology since it is defined by an age group and not by an organ system or process. • Groups all diseases that occur during embryogenesis until early adulthood. • Knowledge of normal versus abnormal processes is extremely important.

Congenital Anomalies • Morphologic defects present at birth • Most common cause of mortality in the first year of life. • Contribute significantly to morbidity and mortality throughout the early years of life. • Include malformations, disruptions, deformations, sequence and syndromes. Trisomy 13: Patau Syndrome • Cleft lip/palate • Polydactyly • Microcephaly • Microphthalmia • Rocker bottom feet • Holoprosencephaly A) Micrognathia (Trisomy 18) B) Simian crease (Trisomy 21) C) Prominent occiput (Trisomy 18) D) Cleft lip and palate (Trisomy 13) E) Overlapping fingers (Trisomy 18) Prematurity - Gestational age of less than 37 weeks and weight less than 2500 grams. • Second most common cause of neonatal mortality (primary cause are congenital anomalies). • American College of Obstetrics and Gynecology (ACOG) estimates that 12% of all births in the United States are preterm deliveries. - Preterm premature rupture of placental membranes (PPROM) - Intrauterine infection - Uterine, cervical, and placental structural abnormalities - Multiple gestation (twin pregnancy) • Neonatal respiratory distress syndrome (hyaline membrane disease) • Necrotizing enterocolitis • Sepsis • Intraventricular and germinal matrix hemorrhage • Long-term complications, including developmental delay. Neonatal Respiratory Distress Syndrome (RDS) • Commonly observed in preterm infants, male gender and those delivered by cesarean section (decreased release of fetal glucocorticoids). • An estimated 24,000 cases of RDS are reported annually in the United States. • PE: dyspnea and fine rales heard on both lung fields. • Maternal history of diabetes (elevated fetal insulin). Diffuse bilateral atelectasis (alveolar collapse) causing a ground glass appearance. Immaturity of the lungs is the most important substrate on which this condition develops. • Main defect is deficiency of pulmonary surfactant. • Leads to increased surface tension, collapsed lungs (collapsed alveoli), progressive atelectasis and reduced lung compliance. • Results in deposition of a protein/fibrin-rich exudate in the alveolar spaces (hyaline membranes). Treatment: - Maternal steroids before birth - Artificial surfactant for infant - Mechanical ventilation (high frequency) - Oxygen supplementation Complications of Oxygen Therapy • Retrolental Fibroplasia (Retinopathy of Prematurity) • Intraventricular hemorrhage • Bronchopulmonary Dysplasia (BPD) RIB What is the pathogenesis of BPD? • Prolonged assisted ventilation at high oxygen concentrations. • Oxygen toxicity and mechanical ventilation damage the developing lung, leading to poor alveolar development. What is the pathogenesis of Retrolental Fibroplasia (Retinopathy of Prematurity)? Disorganized growth of blood vessels Oxygen toxicity leads to endothelial injury, resulting in fibrosis and neovascularization (disorganized growth of blood vessels is noted). Scar retraction causes the retina to detach Necrotizing Enterocolitis (NEC) • Incidence of disease is inversely proportional to gestational age. • Approximately 2500 cases occur annually in the United States. • Clinical presentation: - Abdominal distention - Absent bowel sounds - Bloody stools - Possible perforation (free air in abdomen) A) Duodenal atresia ("double bubble": dilated stomach, proximal duodenum on x-ray. Associated with Down syndrome). B) Hirschsprung disease (congenital megacolon. Lack of ganglion cells/enteric nervous plexuses in distal segment of colon. Normal portion of colon proximal to aganglionic segment is dilated. Associated with Down syndrome). C) Meckel diverticulum (persistence of the vitelline duct. May contain ectopic acid-secreting gastric mucosa and/or pancreatic tissue. Most common anomaly of the GI tract. The rule of 2's: 2 times as likely in males, 2 inches long, 2 feet from the ileocecal valve, 2% population, 2 first years of life, 2 types of epithelia: gastric/pancreatic). D) Pyloric stenosis (is an uncommon condition in infants that blocks food from entering the small intestine due to thickening of the pylorus muscles. It can lead to forceful vomiting, dehydration and weight loss). E) Necrotizing enterocolitis Intraventricular and Germinal Matrix Hemorrhage What are the predisposing factors for intraventricular hemorrhage? Prematurity Hypoxia Breech delivery What clinical symptoms may result from this? Apnea Lethargy Poor muscle tone Seizures SIDS is a disease of unknown cause. The National Institute of Child Health and Human Development defines SIDS as "the sudden death of an infant under 1 year of age which remains unexplained after a thorough case investigation, including performance of a complete autopsy, examination of the death scene, and review of the clinical history". • Infections (e.g., viral myocarditis or bronchopneumonia) are the most common causes of sudden "unexpected" death, followed by an unsuspected congenital anomaly. • Considered a multifactorial condition that reflects a delayed development of arousal and cardiorespiratory control. Lungs Cystic Fibrosis • Autosomal recessive disease. • Most common lethal genetic disease that affects Caucasian populations. • Primary defect is abnormal function of the epithelial chloride channel CFTR on chromosome 7. • Deletion of ∆F508 (most common mutation among Caucasians). • Affects fluid secretion in exocrine glands (salivary glands) and epithelial lining of respiratory, gastrointestinal (pancreas, liver, small bowel) and reproductive tracts. • Other: nasal polyps, meconium ileus in newborns, infertility in males (bilateral absence of vas deferens), and fat soluble vitamin deficiencies (A, D, E, K). • Refer to medical genetics course lectures

Lecture 38: Bacterial Gene Expression and Transfer A dreaded superbug found for the first time in a U.S. woman This STD is becoming 'smarter' and harder to treat Perdue Aims to Make Chickens Happier and More Comfortable Perdue Sharply Cuts Antibiotic Use in Chickens and Jabs at Its Rivals Mew guidelines released by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA) and published in the journal Clinical Infectious Diseases.

-ART IN US 2014: first snapshot of burden of ab-res germs on human health (2 million; 23k die per year; most deaths in healthcare setting/nursing homes). -National Action Plan for Combating AB-Res Bacteria: slow emergence, prevent spread, strengthen surveilance, advance rapid diagnotic tests, accelerate research for ab, vaccines, treatments, improve international collaboration, reduce in CRE, MRSA, and CDiff (prevent spread of threat, eliminate ab use in a animals, increase surveillance) 2020: Reduce 50% incidence CDiff, reduce 60% CRE hospital-acquired, maintain gonorrhoeae below 2%, reduce 35% MRSA in hospital acquired, reduce 50% MRSA total, reduce 25% Salmonella, reduce 15% TB, reduct 25% pneomocol in youth + elderly. BMC appropriate AB us Sterwardship more practical; less focused on cost-savings. -Law School Guides Global Effort to Create New Antibiotics CARB-X - $350 million to combat antibiotic-resistant superbugs -Definition of virulence gene, pathogenicity island, Differential expression of virulence genes, Examples of signals that regulate bacterial gene expression -The Lac Operon: An inducible operon under positive and negative regulation -Quorum sensing for bacteria in biofilms (plaktonic; adhesion; acyl-HSL signal, mature biofilm) -4 major sources of DNA that can be transferred between bacterial cells: 1. Plasmids 2. A simple transposon contains insertion sequences (IS) that include a transposase (tnp) gene flanked by inverted repeats. A composite transposon contains additional gene(s) 3. Bacteriophages 4. Bacterial DNA from Lysing Cells 5. Conjugation F+ cell 6. Lytic Phage and Generalized Transduction: These phage can infect new cells, but this will not result in more phage. 7. Transformation (free dan in enviornment: Homologous vs. Non-homologous Recombination) 1. Tranformation: Take up naked DNA (SHiN: S pneumonia, H inf B, Neis) 2. F+ - F- : Incorp sex pilus 3. Hfr - F-: F+ via Her cell 4. Transposition: DNA jump from one to another Transduction: Packaging phage then excision and insertion (Group A Step ET, BT, CT, DT, ST)

DRx Foundations Clinical Correlation

-poorly controlled with inhaled corticosteroids and beta-agonists. Also with recurrent sinus infections and episodic joint aches -In acute bacterial pneumonia, what cell type is most commonly seen infiltrating the alveolar spaces: neutrophils -WBC HGB/HCT Plt -NA/K CL/HCO3 BUN/Cr Gluy -Eosinophil w large granules fights against parasites -Eosinophil makes major basic protein -Pancreatic acinar cells make elastase -TNF alpha is made mainly by macrophages + more -Myeloperoxidase (MPO) is made by neutrophil granulocytes -IL 2 is made mainly by CD4 T Cells -25% Eosinophilia (usually 1%): NNAACP: Neoplasm, allergic diseases, asthma, addisons disease, collagen vascular disease, parasites, (worms, wheezes, weird diseases). -Asthma, sinus, pulm infil, palpable purpura, eosinophilia, renal insufficiency. -Eosinophilic Granulomatosis with Polyangiitis: Asthma, sinusitis, palpable purport, peripheral neuropathy, eosinophilia, granulomatous vasculitis, P-ANCA positive (anti-neutcytoab) - Eosinophilic Granulomatosis with Polyangiitis allergic granulomatosis, angitits, inflammation of small/medium arteries, starts with 95% asthma/rhinitis, then pulmonary infiltrates/eosinophilia (+1500 EOS), and vasculitis of other organs. Treat with oral prednisone until low inflammation taper over 12-18 months, corticosteroid potential for life, cyclophosphamide anti-neoplastic agent with immunosuppressive properties,. -Mechanism: interact w/ cytosolic hGR alpha, translocate into nucleus, bind GRE to promote gene expression, interact with GR with transcriptional regulators (inhibit NF-kb gene expression) -Glucocorticoids are ant-inflammatory by lowering conversion of arachidonic acid to prostaglandins/leukotrienes, decrease extravasation of leukocytes in injury site, lower fibrosis with reduced production of collagen. -Glucocorticoid long term side affects: osteoporosis with fractures, muscle wasting, hypertension, hyperglycemia, candida infections, weight gain, infection, impaired wound healing, cataracts, behavioral disturbances, slowed growth in kids, -Mustard grads are blinding, biting Cl----N/S----Cl with do Bis(chloroethyl)amine alkylation of N7 of guanine in DNA - Alkylating agents are primarily DNA-directed: Mechlorethamine, Melphelan (multiple myeloma, ovarian & breast cancer), chlorambucil (chronic lymphocytic leukemia, lymphoma), Cyclophosphamide, and Ifosfamide (testicular cancer, sarcomas). Monoalkylated, interstrand crosslink, intrastrand crosslink. Cyclophosphamide: must be activated to 4-hydroxy-cyclophosphamide by liver cytochrome P450. Used in leukemia/lymphoma, breast, ovarian cancer. Used in rheumatologic disorders to suppress immune system. Side effects: nausea and vomiting, bone marrow suppression; sterility; hemorrhagic cystitis Pretreatment: 100% death; now: 70% 5-year survival.

Lecture 41: Mechanisms of Bacterial Pathogenesis

1. A 15-y.o. male presents to the ED with several days of fever, malaise, and generalized myalgias. On the day of admission, he acutely developed painful swelling of his right wrist. On exam, a lesion is noted on his left shin just below the knee and several other lesions are on his right arm near the elbow. 2. LD50 (lethal dose) for Strain A is lower therefore stain A is more portent. Max Lethality is 100% so does not matter. 3. Surface-Associated Virulence Factors: -Fimbriae/pilli: Adhesion, antiphagocytic -Capsule/slide: Adhesion, antiphagocytic, anticomplement, camouflage -Peptidoglycan (muramyl peptides); immunomodulation, induction of inflammatory mediators -LPS/LOS: Anticomplement, induction of inflammatory cytokines, endotoxic shock systemic inflammatory response syndrome -Teichoic acid: Adhesion, sequester divalent cations, induction of inflammatory mediatorys -Flagella/axial filaments: Chemotaxis, penetration of mucus -Outer membrane protein: Adhesion, sequester iron, intracellular survival, invasion -Surface proteins: Adhesion, binding Fc region of immunoglobulins -Iceberg concept of disease: Classic disease, less severe disease, asymptomatic infection (most) -Early response: does not cross multiplication threshold for disease so no disease -Delayed response: crosses multiplication threshold so some disease shows in 8 days) -Immunodeficiencies increase susceptibility to infection/disease: B cell deficiency leads to more bacterial infections, T cell deficiency leads to more viral/fungal infections, B and T cell deficiency leads to more bacterial, viral, fungal, and protaozoal infections. There are also phagocytic deficiencies and complement deficiencies. AIDS, diabetes, immunosuppressive drugs, genetic disorders, old age, poor nutrition, stress, pregnancy, splenectomy, tobacco use, drug & alcohol abuse, some medications can cause immunodeficiency. Acute infections come and end. Persisitent infections virus stats for a while but symptoms only show at end. Slow virus has an acute symptom phase then a end of life symptoms phase but the middle has low virus production. Stages of disease include 1. incubation period 2. prodrome 3. clinical illness 4. recovery. Stages of bacterial pathogenesis include: 1. transmission and entry 2. evasion of host defense 3. adherence 4. colonization 5. disease 6. host immune response 7. disease progression or resolution. Determinants of bacterial pathogenesis include: 1. method of transmission and entry; infection can shed via conjuctiva, respiration, capillary for arthropods, scratch injury, skin, alimentary tract->urinogential tract + anus. Normal microbiota of conductive, nose, mouth/oropharnx, small intestine, large intestine, vagina, urethra, skin, stomach, and outer ear is huge. 2. Evasion of host defenses include tear lysozyme, respiratory mucus/ciliated epithelium/alveolar macrophages, skin anatomic barrier and antimicrobial secretion, stomach acidiity/flora/bile, washing urine/acidity/lysozyme, vaginal lactic acid. Oral mucosa has saliva, keratin, granular layer, BM, leukocytes, antibodies, blood vessels. Crevicular fluid (IgG,IgM,IgA,proteins, complement, enzymes, electrolytes, b/t/Mac/poly) and salivary gland secretion (IgA, proteins, enzymes, electrolytes) into whole saliva (IgA, IgG, Proteins, Enzymes, Electrolytes, Polymorphs). 3. Adherence to cell surfaces (Pili/fimriae, MSCRAMMS, capsules, LTA) 4. Colonization. 5. Invasion, growth, biofilms (i.e. abscess, erysipelas): Phagocytosis IgG + C3b receptor -> NADPH+Oxidase+Cl,OH,H2O2,O2*. Escapes phagocytosis by inhibiting opsonization, inhibiting chemotaxis, killing phagocyte, inhibiting phagocytosis, inhibiting lysocsomal fusion, escaping lysosome to grow in plasma, resist antibacterial lysosomal action and multiply, block activation by interferon-y. Bacteria can damage tissue by invasion and inflammation (pyogenic (Necrotizing faciitis: Aeromonas hydrophila/Sepsis: Streptococcus pyogenes, Group A Strep) or granulomatous) or with toxin production (EXOTOXIN: secreted, encoded by plasmid or phage genes, very toxic in low concentration, good antigens!, toxoids, act variously some A-B subunit). A unit inactivates elongation factor = prevents protein synthesis = cell death. Hyperactivation: A1-A2 increase adenylate cyclase = increase cAMP = high Na, H2O, Cl, K, HCO3 = loss of cell nutrients = diarrhea. There is super antigen binding to external regions of the T-cell receptor and MHC II right in the middle like a slug. G+ cocci: GI enterotoxin super antigen. G+ rod: GI depolymerize actin, inhibit Act release, inactive respiratory protein. G- rod: GI stimulate adenylate cyclase, Resp inactive protein synthesis, stimulate ac or gc. ENDOTOXIN: not secreted, part of bacterium, encoded by genes on bacterial chromosome, not as toxin as exotoxins, poor antigens, no toxoids or vaccines, causes of septic shock. Endotxoin (Lipid A, LPS on outer membrane of cocci and rod): Activate macrophage (IL-1 fever, TNF fever/hypotension hemorrhagic tissue necrosis , NO hypotension shock), Activate complement Alternative Pathway (C3a: hypotension edema C5a neutrophil chemotaxis), activates tissue factor (coagulation cascade DIC). E:Edema, N: NO D: DIC, O: Outer membrane, T: TNF-a, O: O-antigen/hypotension, eXtremely heat stable, Il-1, Neutrophil chemotaxis , by-products of bacterial growth, immunopathogeneis.

Chronic and Granulomatous Inflammation Inflammation that persists for more than a few days. Typical causes Prolonged exposure to injury/irritants etc. Immune mediated diseases (autoimmune, allergies) Persistent infections (results in granulomatous inflammation)

Acute: All but fibroblasts Chronic: All but neurtophils Wound healing phases: Note that the first phases are in minutes while later phase are weeks - months. 9 Sources of monocytes\macrophages: Bone Marrow, Blood monocyte, skin macro, resident cell macro, yolk sac. Macrophage - Critical Cell: 1. Classic activation: IFNy ROS NO Lysozyme: Bacteria and Fungi die. IL-1,IL-12,IL-23 Inflammation. M2: TGF-B fibrosis. IL-10,TGF-B: anti-inflammatory (activation by Il-13 IL-4). Acute inflammation becoming chronic Layers in ulcer = Sequence 1. Top Layer - Fibrinopurulent exudate - fibrin strands + PMNs 2. Middle layer - Granulation tissue , Angiogenesis, Mixture of Inflammatory cells 3. Bottomlayer-Scar- Red collagen, trapped nerve fiber, Granulation tissue - gross: Healing wound Skin excised and allowed to heal Red appearance due to blood flow on base of wound Chronic Inflammation Rheumatoid arthritis: Swollen Joint: Purple spots are collections of macrophages + lympohycytes, synovium on outside. Complex lymphocyte macrophage interaction: Active TH1 Th17 using IL17 TNF Active macrophage using TNF Il1 MEDIATORS: TNF: local [c]> systemic, inhibition improves chronic outcomes, IL-1 local+, overlap fx tnf TNF inhibitors approved for chronic inflammation: 4 Ab TNF, TNFsolublereceptors Examples of diseases treated with cytokine inhibitors • Rheumatoid arthritis • Crohn's disease • Psoriatic arthritis • Ankylosing spondylitis • And others Infliximab Anti-TNF Ab Kufperr cells induce Interleukin-6 which increases acute phase protein synthesis • Synthesis - Macrophages (especially Kupffer cells), fibroblasts, endothelial cells, also by activated T-cells • Biological actions - Stimulates synthesis acute phase reactants in liver - Systemic effects: fever - Growth of antibody-producing B lymphocytes Acute Phase Proteins (indicates how much the reactant increases) Alpha 1 Anti-trypsin (x 2-3) Alpha 1 Anti-chymotrypsin (x 2-5) Haptoglobin (x 1-2) Ceruloplasmin (x 2-5) Fibrinogen C3 C-Reactive Protein (x 5-500) Hemopexin Serum Amyloid A Protein Erythrocyte Sedimentation Rate ESR, Sed Rate APR: # tests/year at BMC CRP - 10,000 ESR - 11,000 Haptoglobin - 1,400 Asthma - chronic inflammation Asthma - Peribronchial Inflammation Asthma Attack Acute exacerbation of a chronic disease Granulomatous Inflammation • Granuloma: At a minimum, an aggregate of macrophages usually arranged concentrically around a pathogen or irritant. • Typical description - Focus of epithelioid macrophages surrounded by a collar of lymphocytes • Only associated with certain specific pathologic conditions. Attempt by the host to contain and eliminate an insult too great for a single cell alone. Combination of cells Epithelioid macrophages Lymphocytes Multinucleated giant cells Fibroblasts Infectious Mycobacteria (tuberculosis, leprosy) Coccidiomycosis Blastomycosis Spirochetes Many others Foreign Body Idiopathic - Sarcoidosis, Crohn's Disease Granulomas - Tuberculosis in Lymph Node: Caseuous Necrosis: Epitheliod macro, lympocytes, multinucleated giant cells RA: TNF inhibitor blocked the chronic inflammation. The response to therapy was measured by CRP However, blocking the inflammatory response allowed re-activation of the tuberculosis by allowing the mycobacteria to escape the inflammation keeping the pathogen under control. -Granulomatous Inflammation Coccidiomycosis - -Kidney Spherule with endospores -Granulomatous Inflammation Lung - Blastomycoses - Skin - Granulomatous Leprosy -Foreign Body Suture Granuloma, Polarized Light Sarcoidosis Crohn's disease - inflammatory disease of bowel Wegener's granulomatosis - necrotizing vasculitis, anti-neutrophil abs Chronic granulomatous disease - inherited deficiency of NADPH oxidase

Lecture 36: Introduction to Bacteria: Classification, Structure & Virulence Factors, Part 2 Streptococcus pneumoniae vs Neisseria meningitidis

Cell Wall: Peptidoglycan Outer membrane of Gram-negative bacteria Lipid A endotoxin Polysacc Major surface antigen Surface fibers in some Gram-positive bacteria Teichoic acid LPS-like, CM: Lipoprotein bilayer without sterols**: Site of oxidative and transport enzymes. Ribosome: Protein synthesis; site of action of aminoglycosides, erythromycin, tetracyclines & chloramphenicol. Periplasm of Gram-negative bacteria: Contains many hydrolytic enzymes, including β-lactamases. G- = LPS, Pore, OM, PPS PPG NBP, CM, G+ = PPG, TA, LTA, CW, CM Lipopolysaccharide, LPS In OM of G- Endotoxin Composed of 3 units O-antigen repeats, core sugar, lipid A Most Common ID's seen at BMC E. Coli UTIs HIV Staph bacteremia Pneumonia Meningitis Cellulitis Diabetic foot infections Osteomyelitis Spontaneous peritonitis Infections in IC hosts, e.g. cancer patients on chemo Tuberculosis Teichoic Acid Polymers of ribitol or glycerol connected by phosphates. Found in outer layer of G+ cells. "LPS-like" in some G+ bacteria Can mediate attachment May contain antigenic determinants Cytoplasmic Membrane Phospholipid bilayer like eukaryotic cells, except it does not contain... Site of: Active transport of molecules Electron transport chain Synthesis of cell wall precursors Signal transduction Cytoplasm containing DNA (nucleoid), ribosomes (70S), granules, plasmids, transposons, etc. Capsule Polysaccharide* Protects against phagocytosis Pilus or Fimbrae Glycoprotein 2 types: (1) Fimbrae mediates attachment to cell surfaces; (2) sex pilus mediates attachment of 2 bacteria during conjugation & transfer of genetic material. Flagellum Protein Motility Secretion Systems (Type I- VI) Protein Transfer of virulence factors Spore Keratin-like coat; dipicolinic acid Provides resistance to dehydration, heat & chemicals; germination gives rise to single cell. Plasmid DNA Contains a variety of genes for antibiotic resistance and toxins. Nutrient Storage Granule Glycogen, lipids, polyphosphates Site of nutrients in cytoplasm. Glycocalyx Polysaccharide Mediates adherence to surfaces. Streptococcus pneumoniae Quellung reaction Capsules inhibit Phagocytosis Capsule-based vaccines promote opsonization E. coli is a common cause of UTIs Clostridium tetani with terminal spores diff types of secretion Spores: Keratin-like coat Dipicolinic acid is a Ca++ chelator Highly resistant to heating: are not killed by boiling (1000C), but are killed at 1210C. Medical supplies must be heated to 1210C for at least 15 minutes to be sterilized. Highly resistant to many chemicals, including most disinfectants. Must use "sporicidal" solutions to kill spores. Can survive for many years, especially in soil. Wounds contaminated with soil can get infected with spores and cause diseases such as tetanus and gas gangrene (caused by Clostridium species). Metabolically inactive. Antibiotics are ineffective against spores; plus they cannot penetrate coat. Formed when nutrients are limited, but germinate when nutrients become available. Usually see bacteria, not spores, in specimens from wounds (why?). Produced by members of only 2 medically important genera, Bacillus and Clostridium (Gram-positive rods). Infections resulting from spores can be attributed to either Bacillus or Clostridium species.

Laboratory Medicine • Laboratory medicine is the integration of laboratory testing into the practice of medicine. • Laboratory medicine primarily involves testing done on "fluids" such as blood, urine, body fluids; and in a few instances "tissues" (microbiology, flow cytometry). • Anatomic pathology involves histopathologic evaluation of "tissues". • Most of the results resulted by the lab is performed by medical technicians, with a physician (clinical pathologist) interpreting a minority of cases. • 70% of medical decisions (ranging from diagnosis, monitoring of treatment, decision to admit or discharge a patient) are based on laboratory testing! • Sections: • Chemistry • Hematology • Microbiology • Blood bank • Point of care • Sendout / reference lab • Information technology (IT)

Chemistry Lab • Most chemistry tests involve measurement of a chemical or protein. • Common chemistry tests include blood gas, electrolytes, liver function, cardiac function, renal function, pancreas function, hormones, autoimmune disease, tumor markers, immunoglobulin, toxicology, and therapeutic drug monitoring tests. Hematology Lab • Common hematology tests include blood counts, leukocyte differential (peripheral smear and body fluids), urinalysis (especially the microscopic component), routine coagulation, special coagulation, hemolysis, hemoglobinopathy, and flow cytometry tests. Microbiology Lab • Microbiology testing involves identification of microorganisms (bacteria, mycobacteria, virus, fungi, parasites) through culture, stains, chemical reactions, molecular techniques, and proteomics. As well as other tests such as antibiotic susceptibility, viral load and genotype testing. lood Bank • The blood bank's primary responsibility is to provide blood products for transfusion (pRBC, PLT, FFP) and performing tests that ensure safe transfusion (type, screen, crossmatch). • Some blood banks perform blood replacement procedures (plasmapharesis and red cell exchange). • Also some blood banks have a blood donor center. Point of Care • Point of care (POC) involves tests that are performed by clinicians at the patient bedside which is not performed in the lab by a medical technologist (MT). • POC tests are easy to perform and easy to interpret. • POC tests include fingerstick glucose, Guaiac, urine pregnancy test, etc. • Some tests need to be made POC in some situations where a rapid test result is needed. • The lab still has to oversee the operational and compliance aspect of POC testing. Send Out / Reference Lab • There are many tests that are not performed in a hospital lab. • Send out tests are often technically difficult to perform, rarely performed, or costly. • Smaller hospital labs will have to rely more on reference labs. Laboratory Information System • Laboratory information system (LIS) is a software that laboratories use to minimize human errors and maximize efficiency. • Direct orders from electronic medical record (EMR) into the lab. • Notify phlebotomist what type of blood tube and how many blood tubes are needed for a test. • Partially automate receipt and accessioning of a specimen. • Direct interfacing with a laboratory instrument for test order and result. • Potentially catch mistaken patient identity based on previous results (delta check). • Report results into the EMR. Steps in Laboratory Analysis • Pre-analytic phase: The step before the specimen is either received in the lab or being tested. • Analytic phase: The step when the specimen is being tested and a result is generated. • Post-analytic phase: The step when the generated result is seen by the clinician who then uses the result in making a decision. Pre-Analytic Variables for Laboratory Testing • Proper patient identification and specimen labelling. • Time of day specimen is collected (i.e. fasting state for glucose and lipid testing, peak and trough sampling for therapeutic drug monitoring). • Right tube for the given test. Color Additive What Happens Used For Red (glass) None After clot has formed, serum is collected Some chemistry tests, some toxicology Gold Clot activator, serum separator gel After clot has formed, gel separate serum from clot Most common chemistry tube Lavender or Pink EDTA EDTA chelates calcium, because calcium is needed in coagulation, it acts as an anticoagulant and allows testing to be done on whole blood Lavender tops are used for hematology and molecular tests. Pink tops are used for blood bank. Green Heparin Heparin inhibits thrombin (through antithrombin III) and acts as an anticoagulant, allowing testing to be done on whole blood Some chemistry tests, cytogenetics Blue Citrate Citrate chelates calcium and acts as an anticoagulant, it allows the extraction of plasma Coagulation tests Gray Glycolysis inhibitor Inhibits glycolysis Forensics and some glucose studies Pre-Analytic Variables for Laboratory Testing • Quality of the specimen • Minimizing hemolysis. • Adequate mixing of anticoagulated blood tests (lavender, blue, pink, green top tubes). • Sometimes more is better, especially for microbiology tests (i.e. submitting tissue or an aspirate rather than a swab). • Not collecting blood downstream of an infusion site. • Collecting blood for coagulation testing that is free of line anticoagulants (i.e. flushing lines kept open with heparin). • Minimizing specimen transport time or time between draw and receipt in lab. Analytic Variables for Laboratory Testing • Manual vs automated testing. • Quantitative vs qualitative test results. • Most tests are quantitative and are resulted by MT without review by pathologist. • Some qualitative and interpretational tests are reviewed by a pathologist (i.e. problematic peripheral smears or body fluid cytospins, bone marrow aspirate smears, flow cytometry, SPEP/UPEP with or without immunofixation, ANA, abnormal T&S, transfusion reactions, etc). • Interference • For tests dependent on optical transmittance, factors that change the color or clarity of serum/plasma such as hemolysis, icterus, and lipemia may interfere with the assay. • For immunoassays, rarely false positive results can be seen in patients who have heterophile antibodies (human anti-animal antibodies that react with antibodies used in laboratory testing that are generated in animals). • Difficulty of a case (some tests may be difficult to interpret either due to equivocal findings, rarity of a case, multiple concurrent disease processes, etc) Post-Analytic Variables for Laboratory Testing • The time it takes for the generated lab result to transfer from the LIS to the EMR. • The time it takes for the ordering clinician to see the result in the EMR. Some lab values are deemed to be life-threatening and are designated as a critical alert value (CAV). • Laboratory accreditation and regulatory agencies (i.e. CAP, Joint Commission) stipulate that CAVs must be relayed from the lab to the ordering clinician within 30 minutes of the lab result. • The idea behind CAV is to minimize the post-analytic phase and possibly prevent death, especially for outpatients. • In our hospital, CAVs include a K+ <2.8 or >5.8, hemoglobin <7.0, a positive blood culture, and many others. Turnaround Time • The time it takes from specimen draw or specimen receipt in the lab to the generation of the lab result is the turnaround time (TAT). • TAT can vary based on specimen transport time, how often a test is run (some tests are 24/7/365, others are batched), the actual running of a test, etc. • Some tests require a fast TAT such as blood gas, whereas others may not such as molecular testing for genetic counselling. Quality Control • Quality control (QC) is a standard step of running designated QC reagents to make sure that a test (whether automated, semi-automated, or manual) is generating correct or reasonably accurate results. • Even for an automated test, just because a test is run on a machine does not mean it is always right. Instruments are prone to "drifting" and have to be kept in check with QC. • QC optimizes the analytic phase of lab testing. • Manufacturers sell QC reagents which have a known range for an acceptable result. • Some tests have three ranges (low, normal, and high i.e. WBC) or two ranges (normal and high i.e. PTT) of QC reagents. • Qualitative tests have two QC reagents (positive and negative i.e. malaria antigen test). • Example of performing QC before a run: • The range for normal platelet (PLT) count QC reagent is between 250 - 350K. • If your QC reagent yields a result of 300K, your QC is acceptable and you can your run patients. • If you QC reagent yields a result of 200K, your QC has failed. • When QC has failed, you can not run any patient samples until troubleshooting has occurred and the repeat QC has passed. • Troubleshooting can involve examination of reagents, examining MT performance (consulting procedure manual), recalibrating the instrument, etc. • For most tests, QC is run at the beginning of every shift (3 times a day). • For low volume or batched tests, QC is run before each run. QC can also be evaluated as an overall trend (in addition to daily operations) to make sure there is no systemic bias or drift in results. • Levey-Jennings plot is a graphical representation of results from QC reagents over time. • Westgard rules are rules recommending re-evaluation of a test if QC results over time show too much departure from the mean, systemic bias, or too much variation. • Quality assurance (QA) is the systematic approach overseen by the pathologist/medical director and laboratory supervisor to improve laboratory function including pre- analytic, analytic, and post-analytic variables. • QA can be achieved globally as well as monitoring performance in proficiency surveys and engaging in QA projects. • Many QA projects aim for improving TAT but other things can be monitored such as proper labelling of specimens, minimizing corrected reports, minimizing contamination rates, expeditious reporting of CAV, etc. • When a test/instrument is being replaced or a new test/ instrument is brought to a hospital, many things have to be considered. • Automation: Ideally a more automated test allows for greater ease, improved efficiency, and minimization of human errors. • Accuracy: Measures how close a method is in providing a test value that is close to the "true" value. • Precision: Measures the reproducibility of the assay such that if a test is performed multiple times for a given sample, the results should be similar. • Validation: Whenever bringing on a new test/instrument, validation has to be performed comparing the new with either the previous test/instrument or a "gold standard". Reference range: A reference range has to be created for every test using your patient demographics. The reference range includes values within 2 standard deviations of the mean (95% of normal or non-diseased patients). Rarely published reference ranges can be used for a test. Reference ranges can differ based on age, gender, time of year, time of day, etc. • Linearity: The reportable range for a given test that a lab feels comfortable in reporting. The manufacturer will recommend a reportable range. However the lab should also have a sense of kind of range reported is accurate. Hence sometimes lab results may be reported as "greater than __" or "less than __". • CLIA 88 or the Clinical Laboratories Improvement Act is a law passed in 1988 which mandates medical director oversight of clinical labs, use of FDA-approved reagents and instruments, and periodic inspection of labs by an accrediting agency (CAP, Joint Commission, Food and Drug Administration). • In an ideal world, all results generated by the lab would reflect what is happening in the patient. • However that is usually not the case and test results have to be compiled with history, physical, and imaging to make the correct diagnosis. • Just like pathologists have to use test performance statistics in deciding what is the best methodology, reagent, or instrument to bring to their lab; clinicians also have to use these same statistics in deciding which tests to perform. • Statistics is used by pathologists in deciding on validation results for a new test/instrument and by clinicians in deciding what test to order. • The test in question is compared to a "gold standard". In real life, there are very few gold standard tests and the gold standard might actually entail a compilation that includes history, physical, imaging studies, lab tests, and even looking at these factors in a retrospective manner. • Prevalence = Disease ÷ (Disease + No Disease) • Sensitivity is how good an abnormal test result detects a disease process. • Sensitivity = TP ÷ (TP + FN) • Specificity is how good a normal test result detects a normal process. • Specificity = TN ÷ (TN + FP) • Positive Predictive Value (PPV) is the probability that a positive test result predicts the patient has disease. • PPV=TP÷(TP+FP) • Negative Predictive Value (NPV) is the probability that a negative test result predicts that the patient does not have disease. • NPV = TN ÷ (TN + FN) • In the ideal world a test would have close to 100% sensitivity, specificity, PPV, and NPV. • In the real world, this is not the case. • Usually improvements in sensitivity causes specificity to suffer and vice versa. • A receiver operator characteristic (ROC) curve plots the true positive rate (sensitivity) vs the false positive rate (1 - specificity) and is a graphical representation of finding the best cutoff (where sensitivity and specificity are maximized) in discriminating disease from no disease. Integration of Laboratory Results in Medicine • Screening tests should have good sensitivity, whereas specificity isn't as important. • Confirmatory tests should have good specificity, whereas sensitivity isn't as important. • Ideally the appropriate number of tests are ordered in order to make the right diagnosis. • The laboratory is a very important component in medicine. • Consult the clinical pathologist if you have questions about a test, test algorithm strategies, or even bouncing off ideas on how to manage or treat a patient. • Although frequently right, the laboratory isn't always right. If there is a lab result that is incongruous with the clinical picture, contact the pathologist.

Evaluation of Dietary Supplements • Long history of use • Poorly regulated • Herb and supplement use is common • A few show promise; many do not • Up-to-date evidence-based information is available

Conventional Pharmaceuticals Derived from Natural Sources • Atropine • Colchicine • Taxol • Pseudoephedrine • Salicylin • Vincristine • Lovastatin Dietary Supplement Health and Education Act • DSHEA passed by Congress in 1994 • Dietary supplements include herbs, vitamins, minerals, metabolites, or extracts • Companies not required to prove efficacy or safety before marketing • Burden of proving inefficacy or lack of safety fell to FDA Name of product (w/ the word "supplement") • Fulldisclosureofallingredients - Identity & strength • Ingredient name (source), quantity (per serving), plant part • Net quantity of contents • Name and place of business of manufacturer, packer, or distributor • Directions for use • Must have disclaimer: not evaluated by FDA, not intended to diagnose, cure or prevent disease -Variation of Ginseng Preparations -Prevalence of Metals in 70 Ayurvedic Drugs: lead, Mercury, Arsenic, any metal 11+ 50% to kids GOOD NEWS - New regulatory framework - Companies have to follow GMP guidelines BAD NEWS - Manufacturers individually define the GMP guidelines and quality specifications, not FDA - Lack of enforcement provision - Ability for some companies to be exempt - Difficult for consumer to distinguish good from bad companies Varibles Affecting Quality • Plant species • Part of plant • Where, when, and how cultivated • Environmental contamination • Storage • Extraction, formulation, & packaging • Standardization of contents Data on Quality • Research post 2010 on the quality of herbal supplements in the American market sparse • From 2008 - 2012, increased number of dietary supplement recalls due to use of unapproved pharmaceutical ingredients (Harel, et al. JAMA 2013) • 465 drugs - class I recall (possible serious adverse health consequences or death) • Weight loss supplements • Bodybuilding • Sexualenhancements -20% of US - Perception of nature as benevolent and healing - "If it's natural, it must be safe" - Treat conditions w/ no conventional "cure" -Top Conditions Treated with Supplements and Perceived Helpfulness esp URI GI -Advertisements promising spectacular results -Recommendations by family & friends • Cultural, religious, & economic reasons Echinacea, ginsend, ginkgo biloba, garlic, glucosamine, st jogns, peppermint, omega 3 Evidence ↔ Public Use • Many pre-2000 positive trials - Low sample size - Inadequate description of supplement - No assessment of blinding • Post-2000 well-designed large NIH-funded negative trials - Saw palmetto, Echinacea, St. John's wort, Gingko, Glucosamine • Dissemination of evidence resulting in change in public use Large Negative Studies Saw palmetto for benign prostatic hypertrophy1 Ginkgo for dementia2-4 Echinacea for prevention/treatment of URI5-7 St. John's wort for major depression8-11 Glucosamine sulfate for osteoarthritis12 Promising Supplements: Fish Oils / Omega-3 Fatty Acids • Contains DHA and EPA • 20-50% ↓ triglycerides (Sirtori, Atheroscl 1998) • 15% ↓ nonfatal MI and death in post-MI patients (GISSI-Prevenzione, Lancet 1999) • Effective adjunct for cardiac risk factor reduction and treatment of post-MI patients (AHA Scientific Statement, Circulation 2002) • Brands: Berkley & Jensen (BJ's), CVS, GNC • Dose: 1-3 grams/day Supplements for Hyperlipidemia • Psyllium (Plantago ovata) - Decreases enterohepatic circulation of cholesterol • Walnuts - ↓LDL 8-16% (Sabate J et al, NEJM 1993) • Plant sterols (beta-sitosterol) and stanols - Prevent incorporation of cholesterol into micelles - ↓ LDL 5-17% (Law M, BMJ 2000) - Impact on CAD controversial - Marketed in spreads (Benechol) and supplements oy (Glycine max) • 1995 meta-analysis shows soy protein 25 g/dreduces TC 9%, LDL 13%, triglycerides 11% (Anderson, NEJM 1995) • > 40 studies and 1 meta-analysis since 1995 consistent w/ these findings (Zhan S et al, Am J Nutr 2005) Ginger • 0.5, 1, or 1.5 g supplementation 2x daily significant reduction in acute nausea during day 1 of chemotherapy (Ryan et al, 2012) • Ginger one hour before, and three and eight hours after start of chemotherapy significantly reduced incidence of acute and delayed emesis (Pillai et al, Pediatr Blood Cancer 2011) • Reduction of nausea up to 24 hours after chemotherapy with ginger supplementation (1.5 g 4x daily) (Panahi et al, Integr Cancer Ther 2012) Probiotics • Microorganisms that have beneficial properties for the host • Potential therapeutic effect for diseases including: - Pouchitis - Antibiotic-related diarrhea - Infectious diarrhea - Irritable bowel syndrome - Clostridium difficile toxin-induced colitis • Multiple systematic reviews demonstrated modest reduction in duration of infectious diarrhea Melatonin • Most thoroughly studied for jet lag • Systematic review of 10 placebo-controlled trials consistently showed reduced jet lag scores (Herxheimer & Petrie, Cochrane Database Syst Rev 2002) • Most trials have used 5 mg doses Xenadrine RFA-1 Citrus Aurantium peel (bitter orange, standardized for 4% synephrine) 125 mg Ma Huang (standardized for 6% ephedrine) 335 mg Guarana extract (standardized for 22% caffeine) 910 mg White Willow Bark extract (standardized for 15% salicin) 105 mg Acetyl L-Carnitine 100 mg L-Tyrosine 80 mg Ginger root 50 mg Vitamin B5 (pantothenic acid) 40 mg Common Dietary Supplements for Weight Loss • Ephedra-related compounds showed 0.9 kg weight loss/mo > placebo but 2.2-3.6 fold increase palpitations, psychiatric, autonomic, and GI symptoms (Shekelle, JAMA 2003) • Chitosan - blocks clinically insignificant amount of fat absorption • Chromium - may play a role in insulin sensitivity but not helpful for weight loss • Hydroxycytricacid-evidenceequivocal St Johns Wart reduces efficacy of HIV meds Certain herbal medicines may impact: - Coagulation: ginkgo, ginseng, garlic - Cardiovascular stability: ephedra - Glucose: ginseng - Anesthesia: valerian, kava - ↑Metabolism of perioperative meds: St. John's wort • Recommend avoiding for > 2 weeks prior to surgery >60% of patients did not disclose their CAM use to their physician (Eisenberg, Ann Intern Med, 2001) • Identify potential interactions, ineffective or unsafe therapies • "What prescription medications, over the counter medicines, vitamins, herbs, or supplements do you use?" AND • "Why?" • Empathy • Compassion • Warmth • Attentiveness • Listening • Competence • Confidence • 200 patients with non-specific complaints randomized to: 1. Positive Consultation, treatment 2. Positive Consultation, no treatment 3. Negative Consultation, treatment 4. Negative Consultation, no treatment • At 2 wks 64% receiving a positive consultation improved vs. 39% receiving a negative consultation (p = 0.001) • No difference between those treated and those not treated (p = 0.50) The Healer's Art Elective Spring Semester Supports medical students in recognizing, valuing, enhancing, and preserving the human dimension of health care • Sustaining idealism and altruism • Medicine as a calling to serve others • Learning how to grieve and heal loss • Preventing stress & burnout • Cultivating compassion and listening skills

Pharmacology of Anti-inflammatory Steroids

Glucocorticoids A class of steroids with anti-inflammatory and immunosuppressant effects A type of disease-modifying anti-rheumatoid drugs (DMARDs) Principal Adrenal Steroids • Glucocorticoids (GCs): cortisol (hydocortisone), corticosterone • Mineralocorticoids (MCs): aldosterone Regulation of Endogenous Glucocorticoid Synthesis • CRH from hypothalamus stimulates corticotropin (ACTH) release from anterior pituitary • ACTH increases synthesis of GC from cholesterol in adrenal cortex • GC inhibits CRH release and inhibits synthesis of proopiomelanocortin (source of ACTH) Output of endogenous steroids depends on the stimulatory effect of ACTH ACTH acts on a GPCR in the adrenal cortex to activate a signaling pathway resulting in the increased synthesis and release of cortisol GC Selectivity of Cortisol (Hydrocortisone): Equal affinity for GC and MC receptors: Converted to cortisone - in kidney, salivary gland and colon - by 11B hydoxysteroid dehydrogenase so Cortisone has low affinity for MC receptors GC action: Interactionwithcytosolic hGR alpha • Translocationintonucleus • Binding to GRE • InteractionofGRwith transcriptional regulators • Change in gene expression -Inhibitors of arachdon stuff: Increased synthesis of phospholipase A2 inhibitor • Increased synthesis of NF-kB inhibitor (IkB) • Blocked transcriptional effects of NF-kB • Decreased synthesis of adhesion factors that affect leukocyte localization Molecular Mechanisms of Anti-inflammatory and Immunosuppressant Effects of Glucocorticoids Block in release of arachidonic acid and its metabolites - Increased transcription of gene for lipocortin (inhibitor of phospholipase A2) - Decreased transcription of COX-2 Antagonism of NF-KB - Increased transcription of gene for I-kB (inhibitor of NF-kB translocation into nucleus) - Nuclear binding by ligand-bound GC receptor Anti-inflammatory Effects of GCs Blockade of the early manifestations of inflammation pain and vasodilation enhanced vascular permeability and edema extravasation of leukocytes Blockade of later stages of inflammation vascular proliferation fibroblast activation fibrosis and collagen production Immunosuppressive Effects of GCs • Inhibition of macrophage activity • Inhibition of cytokine secretion by macrophages and T cells - Interleukins 1-6,8 - TNFa - Cell adhesion factors • Inhibition of T and B cell replication • Decreased IgG production Greater selectivity GC selective: dexamethasone, prednisone MC selective: fludrocortisone Longer half-life and duration of action Cortisol: duration 8-12 hr Dexamethasone: duration 36-72 hr Therapeutic Uses of Glucocorticoids • Replacement therapy for adrenal disorders • Anti-inflammatory effect RA (rheumatoid arthritis) & LE (lupus erythematosis) • Immunosuppressant effect Autoimmune disorders (RA, LE), organ transplants, or immune cell malignancies Adrenocortical insufficiency - Acute • High doses of hydrocortisone - Chronic (Addison's disease) • Physiologic doses of hydrocortisone and fludrocortisone GC Insufficiency • Anorexia • Nausea and vomiting • Fever • Joint or muscle pain • Postural hypotension GC can be used for: Asthma, COPD, alergic reactions, RA, LE, collagen vasc idsorders, dermatologic disorders psoriasis, GI UC Chrons, cerebral edema, bells palsy inflammation, immune cell malignancies, tissue grafts, premature lungs. • Single large dose relatively safe • Beneficial effects only palliative • Prolonged treatment is NOT safe - Toxicities - Suppression of HPA axis • Abrupt termination is life-threatening Months after stoping GC: ACTH peaks then falls: cortisol palteus: GC-induced atrophy of the adrenal cortex Adverse Effects of Long-term GC Use Infection • Impaired wound healing • Osteoporosis with fractures • Muscle wasting • Hypertension • Hyperglycemia • Altered fat deposition • Cataracts • Ulcers • Behavioral disturbances: psychosis • Slowed growth in children Cushing Syndrome Caused by: • Chronic administration of large doses of glucocorticoids • Tumor of the pituitary or adrenal gland Measures to Reduce Risk of GC Toxicity • Local administration - MDI aerosol delivery with spacer for lung - Nasal sprays for allergic rhinitis - Topical preparations for skin, eye, oral cavity - Injection into joint - Intrathecal for post-herpetic neuralgia - Slow release capsule with high first pass effect for IBD (budesonide) • Minimize use to essential indications, avoid prolonged use, and taper on withdrawal from chronic use Other Immunosuppressants Small, Low MW molecules Purine/pyrimidinesynthesisinhibitors: -(Antimetabolites): Methotrexate (MTX) MTX Mechanism of Action •Inhibition of thymidine-MP synthesis -High dose antineoplastic effect -Reduces the availability of folate for thymidylate and DNA synthesis -Causes arrest of DNA replication and cell division •Inhibition of de novo purine (inosine-MP) synthesis -Low dose antirheumatic effect -Possibly mediated by increased adenosine that acts as the anti- inflammatory agent CalcineurinInhibitor:Cyclosporin Calcineurin inhibitors: Cyclosporine, Tacrolimus • Mechanism: Block T cell signaling for growth and differentiation by inhibiting enzymatic activity of calcineurin required for NFAT induction of T-cell IL- 2 synthesis • Indicated for organ transplants (cyclosporine-first- in-class) • mTORInhibitor:Sirolimus Mechanism: Block protein kinase (mTOR) activity required for cell cycle progression from G1 to S phase in T lymphocytes • Indicated for organ transplants and contained in coronary stents Large, High MW Proteins: Biologics TNFa Antagonists (Infliximab Mab, Etanercept SOlRec) • High molecular weight proteins that bind and block the inflammatory actions of TNFa • IV or SC administration • Long half-lives • Indicated for arthritis and inflammatory bowel disease • Increases the risk of infection • Expensive • IL-6 Antagonist Abatacept (ORENCIA) - IgG1 like protein that binds to Antigen-Presenting Cell (APC) and prevents T cell activation - Approved for rheumatoid arthritis - Increases risk of infection Mechanism • Two signals are required for T cell activation. • Signal one: T-cell receptor binds to the major histocompatability complex antigen (MHC) on the antigen presenting cell (APC) • Signal two: a co-stimulatory ligand B7 (CD80/CD86) on the antigen presenting cell binds the co-stimulatory receptor on the T cell (CD28). • Abatacept inhibits the binding of B7 (CD80/CD86) to CD28 and T cell activation • Anti-Integrin Antibody (Natalizumab) Integrin a4 Antagonist Natalizumab • 95% humanized antibody targeting integrin a4 on lymphocytes • Blockslymphocyteinteraction with adhesion molecules (MAdCAM-1) on vascular endothelium in inflamed regions of intestine • Approved for multiple sclerosis and Crohn's Disease (Vedolizumab)

Disorders associated with defects in enzymes • Lysosomal Storage Diseases Intracellular digestive tract • Enzymes are manufactured in Endoplasmic reticulum • Acquire "address label" in the golgi apparatus • Transport to lysosomes • Intracellular(Autophagy) • Christian de Duve was awarded the Nobel Prize in Physiology or Medicine in 1974 for the discovery of the lysosome • the 2016 Nobel Prize in Physiology or Medicine to Yoshinori Ohsumi for his discoveries of mechanisms for autophagy • Extracellular (Heterophagy)

Features common to most of the Lysosomal Storage Disorders Autosomal recessive transmission • (except Hunters X linked recessive) (Hunter marks the spot with an X) • Patient population consisting of infants and young children • Storage of insoluble intermediates in the mononuclear phagocyte system, giving rise to hepatosplenomegaly • Frequent CNS involvement with associated neuronal damage • Cellular dysfunctions, caused not only by storage of undigested material but also by a cascade of secondary events triggered, for example, by macrophage activation and release of cytokines The distribution of stored material (organs) is determined by two factors • Site where most of the material to be degraded is found - Gangliosides in brain •Location where most degradation normally occurs - RES (macrophages) in lymph nodes, spleen and liver 1. Sphingolipids • Gangliosides • Cerebrosides • Sphingomyelin 2. Mucopolysaccharides • Heparan Sulfate • Dermatan Sulfate • Keratan Sulfate • Chondroitin Sulfate 3. Glycogen • Tay - Sachs Disease •Missingenzyme - Hexosaminidase •Accumulatedmaterial - Gangliosides •Microscopic - Cytoplasmicvacuoles • EM - lysosomes filled with "onion-skin" membranes • Tissues involved - CNS - Retina (cherry red spot) - Autonomic nerves - Heart, Liver, Spleen etc. -Hypotonia • Niemann- Pick Disease • Missing enzyme - Sphingomyelinase • Accumulated material - Sphingomyelin • Tissues involved - CNS and RES in the spleen, liver, lymph nodes and bone marrow • Type A variant - 75-80% of cases - severe infantile form with extensive neurologic involvement, marked visceral accumulation of sphingomyelin, progressive wasting and death by age three • Type B variant - Patients have organomegaly but no CNS involvement - they usually survive into adulthood •Gaucher Disease • Missing enzyme - Glucocerebrosidase - cleaves glucose from ceramide • Accumulated material - Glucocerebrosides • Tissues involved - spleen, bone, liver and sometimes neurons in the CNS • Three clinical subtypes Type 1 - adult - spleen and skeletal involvement, reduced enzyme, slightly decreased longevity Type 2 - infantile - cerebral, no enzyme, death at an early age Type 3 - intermediate between type1 and type 2 • Hepatomegaly - 2 to 3 times normal size • Splenomegaly - up to 15 times normal size • Thrombocytopenia - excessive bruising and bleeding • Anemia - fatigue • Low White Cells - susceptibility to infections • "Bone Crisis" - insufficient blood circulation • Bone thinning/fracture - "Erlenmeyer flask" - Enzyme replacement • Mucopolysaccharidoses (MPS I-VII) • MPS I - Hurlers Syndrome • Missing enzyme - alpha-L-iduronidase • Accumulated material - Dermatan sulfate, Heparan sulfate • Tissues involved - connective tissues - RES, endothelial cells, smooth muscle cells, fibroblasts (balloon cells) • Clinical - Hepatosplenomegaly, skeletal deformities, valvular lesions, subendothelial arterial deposits (myocardial infarction) •Glycogenoses - two metabolic pathways •Cytoplasmic pathway •Lysosomal pathway - glucosidase (acid maltase): G6Phosphatase Von Gierke, Muscle phos McArdle, Acid Maltase Pome disease Type II Von Gierke Disease • Missing enzyme - glucose -6- phosphatase • Accumulated material - glycogen (lipid in liver cells) • Tissues involved - hepatocytes, renal tubular cells • Clinical - convulsions (hypoglycemia) - hepatomegaly - growth retardation - infections - hyperlipidemia, hyperuricemia • Pompe Disease • Missing enzyme - alpha-glucosidase (acid maltase) • Accumulated material - glycogen • Tissues involved - generalized involvement - heart and brain cause most prominent symptoms • Clinical - cardiac enlargement with progressive heart failure, muscular weakness, neurologic symptoms, death in infancy.

DrugAction,DoseEffect,rugSeelctivity Receptor Classes • Physiological ReceptorsPhysiological Receptors - Ligand-gatedionchannels - G-ProteinCoupledReceptors(GPCRs) - GrowthFactor/CytokineReceptors - IntracellularHormoneReceptors • Enzymes BLOCK: Penicillin (-lactam antibiotic) - transpeptidase Zidovudine - reverse transcriptase Physostigmine - acetylcholinesterase Disulfiram - aldehyde dehydrogenase • Transporters - Cocaine: Inhibits uptake of catecholamines and serotonin SERT NET DAT - Fluoxetine: Inhibits serotonin uptake SERT • Voltage-gated Ion Channels - Local anesthetics: inhibit voltage gated Na channels :Lidocaine, benzocaine, cocaine Cachannelblockers:inhibitvoltagegatedCachannels Nifedipine, Diltiazem • Structural Proteins vinblastine: Interfere with microtubule polymerization • Nucleic Acid - e.g. Cyclophosphamide: DNA alkylating agent used for immunosuppression and cancer chemotherapy • Biological Mediators (interact directly with signaling molecules) - Etanercept (soluble "decoy" TNF-α receptor, autoimmune diseases (psoriasis, rheumatoid arthritis)) Molecular structures which are receptors for endogenous substances Have a physiological signaling role Regulate physiological functions Can transduce signal from extracellular to intracellular space or from intracellular space to nucleus Agonists: bind receptor and generate effect, electrical or biochemical change ("2nd messenger") Ligands include neurotransmitters, hormones, and growth factors

GABA a receptor GPCR: CRF Adenosine CAnnabinoid Adrenergic Opioid • Therapeutic Effect (beneficial effect resulting from a drug treatment) • Non-therapeutic Effects • Placebo Effect Dose-intensity (graded) - How much effect? • X-axisdrug concentration or dose • Y-axis% effect • Dose-frequency (quantal) - How many affected? • X-axisdrug concentration or dose • Y-axis% of subjects responding Dose Dose-Intensity Curves (graded) KD is the drug concentration at which 50% of receptors are occupied 1:1 [Effect] / [Effectmax] [Drug]/ EC50 +[Drug] Why semilogarithmic plots? • Because linear plots are more difficult to be handled: close to the zero the effect changes rapidly • Semilogarithmic curves have a large middle portion close to the EC50, which is linear and easier to work with (easier to make accurate determinations) Emax vs EC50 Potency = EC50 = [ ] of a drug able to give the 50% of its maximal effect Efficacy = Emax = maximum effect of a drug achievable at high dose Potency ≈ how well the drug binds the receptor Efficacy ≈ how well the drug produces the effect Selectivity can be due: to a difference in binding affinity (Kd) of that drug for different receptors to the number of spare receptors ( spare receptors EC50) differences in drug access to receptors on different tissues Full agonist: a drug that binds to a receptor and produces a maximal biologic response Partial agonist: a drug that has efficacy greater than zero but less than that of a full agonist Neutral antagonist: a drug that does not induce a biological response and reduces/blocks the response induced by the agonist Inverse agonist: a drug that induces a pharmacological response opposite to that of the agonist Chemical Antagonism (see Etanercept) Occurs when the antagonist interacts with the agonist to render the agonist inactive. Pharmacokinetic Antagonism Occurs when the antagonist accelerates the metabolism or the elimination of the agonist. Physiological Antagonism Occurs when the antagonist activates a different mechanisms that opposes the effects of the agonist Pharmacological Antagonism (occurs at a receptor level) 1. Reversible competitive 2. Noncompetitive 3. Irreversible competitive 1. Reversible competitive the antagonist binds the same receptor site bound by the agonist the two drugs compete for the same site both agonist and antagonist bindings are reversible right-shift of the dose-response curve (higher ED50) unchanged Efficacy competitive antagonism is surmountable, meaning that it is always possible to "overcome" inhibition by increasing the concentration of agonist, in order to achieve the same magnitude of effect as before 2. Noncompetitive the antagonist binds the receptor to a different site than the site bound by the agonist (insurmountable) the antagonist does not interfere with the binding of the agonist decreased Efficacy (lower Emax) unchanged Potency noncompetitive antagonism is insurmountable. One cannot necessarily restore the original response of the agonist by increasing the concentration of the agonist 3. Irreversible competitive resembles noncompetitive typically insurmountable decreased Efficacy (lower Emax) unchanged Potency usually results from covalent binding of antagonist to receptor effect of drug may long outlast its presence in the body and is determined by the time required for the body to reconstitute the drug's target Spare Receptors Agonist concentration=Kd 50% receptor occupancy A certain response Agonist concentration<Kd ~20% receptor occupancy The same response as in 1 Receptor excess Limiting step downstream to binding An agonist may produce a maximum response even though the agonist concentration is not high enough to fully occupy all of the available receptors Spare receptors increase the apparent potency of agonists, because the response reaches 50% of Emax at a concentration at which less than 50% of receptors are occupied by agonist (i.e. EC50 < Kd). Receptor is assumed to exist in two conformations: inactive (Ri) and active (Ra) Nature of the drug (agonist, antagonist, etc) arises from differences in affinity constant for the active state and the inactive states Drugs act stabilizing either Ri or Ra conformation Inverse agonist: a drug that induces a pharmacological response opposite to that of the agonist. If there is an elevated basal response in the absence of ligand (agonist- independent constitutive activity), then activity will be inhibited and A will then be an inverse agonist (Negative Efficacy) Summation (the joint effect of the two drugs is the algebraic sum of their individual effects) • Synergism (the joint effect of the two drugs is greater than the algebraic sum of their individual effects) • Potentiation (an inert drug potentiates the effect of another drug. Ex.: Benzodiazepines and GABA) - Increasing potency - Increasing efficacy Allosteric modulators: bind receptor at site other than that of endogenous ligand and potentiate or inhibit the effect of the ligand Diazepam (a benzodiazepine) potentiates ( potency) the effects of GABA on GABAA receptor function (current, chloride channel) Flumazenil acts as a competitive inhibitor of an allosteric modulator competing with Diazepam (not GABA!) • Tolerance - Cross-tolerance (tolerance can develop for other drugs with the same mechanism of action e.g. same receptor) - Tachyphylaxis (the effect of the drug declines very rapidly. Timescale of minutes) - Desensitization (mechanism for decreased effect (including tachyphylaxis)) • Sensitization (cocaine, locomotor sensitization) To clarify the proper dose of a given drug that produces the desired therapeutic effect while limiting harmful side effects • To determine therapeutic and hazardous drug dosages Dose-Frequency curves (quantal) • Quantal dose-response curves are all or nothing: the effect is scored in a binary (present/absent) fashion • IntegratedGaussianbell • A continuous effect may be converted to quantal by defining a threshold effect intensity criterion (effect ≥ threshold present) • How many (what percentage of) subjects respond at a particular dose? Shallower slope indicates greater variability • Different slopes imply different sources of variability, suggesting difference in route or mechanism of action Nature of variation - Pharmacokinetic (Clearance, Distribution) - Pharmacodynamic (Receptor number, type, location, endogenous mediators) • Sources of variation - Genetic heterogeneity (different receptors, different receptor distribution, different enzymes of biotransformation, etc.) - Disease (impact of renal or hepatic disease on the effect of drugs) - Physiological state - Drug interactions (older people) Side effects - Pharmacologic - Pharmacokinetic - Allergic (hypersensitivity) - Idiosyncratic • Toxiceffects - Overdose Represents an important aspect of the post marketing surveillance of drugs (pharmacovigilance) • Unintended or unexpected • Noxious • Licituse • Severe • Ascribable to drug - Known effect of drug or class - No other cause - Timing consistent - Blood levels consistent - Reproducible ED50 = Dose producing an effect in the 50% of the subjects LD50 = Dose producing death in the 50% of the subjects Therapeutic Index as a measure of safety: LD50/ED50 Compare T1>T2 = Drug 1 sis safer than Drug 2 Importance of population variability: Same Therapeutic Index, different risk Standardized safety margin: LD1-ED99/ED99 Sources of placebo "response" A therapeutic "response" to an inactive drug or treatment • Spontaneous improvement ("most things get better on their own") • Subsiding toxic effect of previous treatment • Bias (often unintentional) • Power of suggestion? (controversial topic) • Use of placebo and ethical concerns Placebo controlled studies: • The "gold standard" of pharmaceutical research is the double-blind placebo-controlled study, in which some patients receive placebos and some receive active drug, but neither the patient nor the researcher knows which is which until the end of the study. • The double-blind design also controls against experimenter bias in selection of subjects or analysis of data • A valid placebo control must be indistinguishable from active drug in all respects (e.g. size, taste, color). Albuteral, antidepressants, Rofecoxib,Open vs. hidden drug administration

Multiple Dosing Kinetics Cpss = Infusion rate / ClT

Kinetics of Continuous Infusion • Constant rate input, first order output, single compartment • Cp = Cpss [1-e-(kelt)] • At steady state, input rate = output rate infusion rate = Cpss ClT • Cpss = Infusion rate / ClT (LEARN!) • Time (t) when Cp is 50% of Cpss is one elimination half-life • Time (t) when Cp is 93% of Cpss is four elimination half-lives Time required to reach Cpss depends on elimination half-life not input rate A drug with an elimination half-life of 4 hours is administered by continuous intravenous infusion. How long will it take for the drug to reach ~93% of its final steady-state level? 4*4 Cpss = Infusion rate / ClT decreased clearance with unaltered infusion rate normal clearance decreased clearance with lower infusion rate iof clearance is lower you hshould lower the infusion rate Drug A has a half-life of 4 hours and a recommended infusion rate of 10 mg/min in normal 70-kg subjects In a 70-kg patient with renal failure, the volume of distribution of drug A is like that found in normal subjects, but its total clearance is only half as large. An appropriate infusion rate of drug A for this patient would be: . 5 mg/ml Loading Dose is the initial dose administered to achieve therapeutic Cp at start of regimen Loading Dose is important for: - Indication requiring rapid onset of effect - Drugs with long elimination half-lives and slow attainment of steady-state by continuous infusion • Loading dose does NOT affect the Cpss achieved by continuous infusion loading dose (IV bolus) is a tsaper from top to Cpss maintenance dose (IV infusion) is a taper from bottom to cpss First-order elimination, single compartment distribution • Constant rate input, D / t maintenance dose D / dosing interval t • Each dose D diluted into Vd adds C0 to Cp D/Vd = C0 • Cp rises from Cpmin to Cpmax with each dose Cpmin + C0 = Cpmax e Kinetics Accumulation Kinetics Cmin is 'trough' Cp. Cmaxss to Cminss is 'therapeutic window' Accumulation from First Dose to Steady-State • The shorter the dosing interval, relative to the elimination half-life, the greater the drug accumulation • If dosing interval is greater than 7 half-lives, less than 1% remains and accumulation is.... NEGLIGIBLE • If dosing interval is about 1 half-life, then the accumulation is......TWO-FOLD If the therapeutic window of a drug is 125 - 500 mg/l and the t1/2 is 12 hr, what is the appropriate dosing interval for fluctuation specifically from this peak to this trough?00 If the therapeutic window of a drug is 125 - 500 mg/l and the t1/2 is 12 hr, what is the appropriate dosing interval for fluctuation specifically from this peak to this trough?00 24hr Fluctuation around a Cp"average" from Cmaxss to Cminss • Cp"average" = ( D / t ) / ClT • About the same Cp"average" can be achieved with infusion, with small dose at frequent intervals, or with high dose at long intervals. • What are the advantages and disadvantages of each regimen? Oral Formulations • solutions, tablets, controlled release capsules • absorption half-life and bioavailability dependent on disintegration and dissolution rates • dissolution rate dependent on factors such as drug crystal form, excipients (inactive ingredients), tablet compression force Rate Limiting Steps in Drug Absorption Disintegration & release Solid drug particles Rate processes for solid, oral, immediate-release products: •disintegration of drug product and release of drug •dissolution of drug in aqueous environment •absorption across biological membranes into the systemic circulation Dissolution Absorption Drug in drug product Drug in solution Drug in body Tablet Formulations Oral Formulations:Factors that affect dissolution rates Excipients \ Starches • Excipients are inactive substances in drug products (diluent) • Starches are added to drug products to enhance disintegration, the breakup of the tablet. • Starches bind water and swell, enhancing disintegration of the tablet and dissolution of the active drug. Oral Formulations:Factors that affect dissolution rates Drug crystal forms Chloramphenicol has several crystal forms that have different aqueous solubilities and absorptions. The b form is more aqueous soluble and better absorbed The oral suspension that contains the highest concentration of the b polymorph is absorbed the greatest. serum levels vs. time Oral Formulations:Factors that affect dissolution rates Particle/Crystal Size • the smaller the particle size, the greater its surface area, the faster its dissolution rate • the smaller the crystal size of chloramphenicol, the faster the absorption rate Oral Formulations:Factors that affect dissolution rates Compression Force • the greater the compression force used to create the tablet, the longer it takes for the tablet to dissolve Oral Formulations\ Buccal Administration Buccal Fentanyl Tablet • Buccal administration is intended for absorption in the oral cavity. • Buccal surface has large blood flow, provides rapid absorption for lipid-soluble drugs. • Venous drainage from the mouth bypasses the portal circulation and protects drug from first-pass metabolism. Oral Controlled Release Devices • Purpose: slow absorption and a more sustained duration of action • Technologies: - 'Gastrointestinal Therapeutic Systems' with release based on osmotic pressure of core - Capsules containing beads manufactured with differing dissolution rates Oral Controlled Release Devices Osmotic Pump Gastrointestinal Therapeutic System (GITS) Oral Controlled Release Devices Osmotic Pump Gastrointestinal Therapeutic System (GITS) PROCARDIA® (nifedipine) Osmotic Pump: GITS two 10 mg capsules 60 mg dose in the GITS Oral Controlled Release Devices • Drug product comprises a core (2 drug compartments and one compartment with osmotically active polymer) a surrounding semipermeable membrane, and an overcoat of immediate-release drug. • The drug overcoat dissolves in the GI tract providing the initial drug dose. • Water diffuses into core, expands the polymer, pushing dissolved drug out of the pore. • Over time, drug in compartment 2 dissolves and is pushed out of the pore, maintaining plasma drug levels for once-daily dosing Methylphenidate Special Delivery Devices to enhance safety, ease, and duration of administration • Ophthalmic • Uterine and vaginal • Subcutaneous • IV • Transdermal • Pulmonary Special Delivery Devices Vaginal Ring • NuvaRing® is a flexible plastic ring containing an estrogen and a progesterone inserted into the vagina • Hormones diffuse out of the ring, enter the bloodstream, and prevent ovulation for 3 weeks • On ring removal menstruation occurs Special Delivery Devices Subdermal Implants for long-term birth control Implanon® a progestin-containing rod implant for subdermal use indicated for women for the prevention of pregnancy. The rod consists of a copolymer core containing the synthetic progestin etonogestrel surrounded by a copolymer shell. Progestin is released into the circulation providing a long-acting (up to 3 years), reversible, contraception. Drug Infusion Pumps for Long-term Outpatient Use Insulin Pump and Catheter • Exteriorized programmable pumps for s.c. insulin administration in diabetics Drug Infusion Pumps for Long-term Outpatient Use Programmable Implanted Pump • Surgically implanted pump for spinal analgesic or antispasmodic agents Port for IV Chemotherapy Nanotechnology and Drug Delivery Wang, AS, Langer, R, Farokhzad, OC Ann. Rev. Med. 63:185-98. 2012 Liposomes: ex. the antifungal agent amphotericin B Albumin-bound nanoparticle: ex. antitumor agent paclitaxel Coronary Vessel Stent: Drug- coated to inhibit restenosis • Stents maintain patency of the coronary vessels to occlusions caused by atherosclerosis. • Active agent is embedded in a polymeric matrix that releases drug directly to the target site in the vessel. • Direct drug delivery into the vessel reduces systemic toxicity of cytotoxic drugs that act to inhibit the proliferation of smooth muscle that cause occlusion. • Inhibitors of cell replication include sirolimus (rapamycin, an mTOR inhibitor) and paclitaxel (taxol, a microtubule inhibitor). Transdermal Drug Delivery • Provides prolonged delivery for drugs with short half- lives to achieve effective levels • Avoids problem of low oral bioavailability due to first- pass effect • Allows for drug device removal to avoid drug tolerance • Useful for lipophillic agents with low mw Nitroglycerin-containing Transdermal Patches Inhalation Delivery Devices for Drug Particulates • Improves selectivity of bronchodilators and anti-inflammatory agents for asthma and chronic obstructive lung disease (COPD) • Permits routine outpatient use • Metered dose inhalers, nebulizers, and diskus differ in design, cost, and ease of use Pulmonary Devices for Targeted Delivery to Airways Metered Dose Inhaler • Metered dose inhalers and nebulizers release small aerosolized particles of drug • Small fraction gains access to deeper regions of the airways for efficacy in asthma and COPD • Much more selective effect than oral route; reduces risk of systemic adverse effects Pulmonary Devices for Targeted Delivery to Airways Ultrasonic Nebulizer for Drug Administration by Inhalation • Localized delivery improves drug selectivity for asthma and COPD Pulmonary Devices for Targeted Delivery to Airways Diskus • Delivery device for anti- inflammatory and bronchodilator agents for asthma • Advair Diskus® contains fluticasone, an anti- inflammatory steroid and salmeterol, a bronchodilator for asthma

TISSUE REPAIR, CELLULAR GROWTH, FIBROSIS AND WOUND HEALING

Labile Cells: Surface Epithelia Stable Cells: Liver, SM, Vessels Permanent Cells: Neurons and Skeletal muscles Proliferation Zones Ki67 IHC (G2 S G1) of Tonsil -Stem cells are undifferentiated cells known for self-renewal issues included fx integration and immunogenicity. Embryonic stem cells are the 32 cell blastocyst which is pluripotent. Adult stem cells have niches, markers. -Mature Cells Can be Reprogrammed to become Pluripotent: four retrovirally delivered genes, Oct4, Klf4, Sox2, and cMyc. (genes for stemness) -Disease issues by fibrosis: MI (myocardial fibrosis): cardiac failure, CVH (cirrhosis): portal hypertension liver failure, Diffuse alveolar damage pulm intersitital fibrosis (res failure). -Cirrhosis of Liver -Pulmonary Fibrosis -Chronic Ulcer of Foot -Cutaneous Wound Healing first intension blood brings neutrophils or second intention neutrophils bring blood. -Factors affecting wound healing: local: blood supply; denervation; infection; foreign body; necrotic tissue; mechanical stress; surgical technique; type and location of tissue. Systemic: diabetes; malnutrition; steroids; uremia; hypoxia; vitamin C deficiency; malignant disease -Chronic Peptic Ulcer H&E stain, Trichrome stain I, II, III, IV layers. I: Inflammation Necrosis and Acute Exudate II: Cell Prolif Granulation Tissue III: Synth ECM Granulation Tissue and ECM IV: Fibrous Scar Layer I Acute inflammation and Necrosis: Fibrin, Neutrophils (MPO), Macrophages, Platelets, Cytokines (interleukins, Interferons, TNF), Growth Factors (PDGF, FGF). Regeneration of Surface Epithelium - Ki67 2. Cell Proliferation and Migration - Layer II Granulation Tissue Capillaries, fibroblasts, macrophages, Growth factors: EGF-TGFa, PDGF, FGF, VEGF, TGF-B, Angiopoietins 3. ECM Functions: • Tissue Texture -turgor, elasticity, rigidity • Reservoir - growth factors • Substratum/matrix - cell adherence and migration 1. Basal Membrane 2. Interstitial Matrix -Fibrous structural proteins Collagens Elastins -Cell Adhesion Proteins (CAMs) Cadherins Integrins Fibronectin Laminin -Gel of proteoglycans and hyaluronan -Fibronectin: Large glycoprotein, produced by fibroblasts, endothelial cells, monocytes - specific binding domains -Laminin Most abundant in BM, cross-shaped, spans BM and links specific receptors to collagen IV and heparan sulfate Ingerins: B,A Vinculin, Talin, Paxillin, Tensin, Actin, ECM Gel: Proteoglycans Core proteins linked to disaccharide polymers Heparan sulfate, chondroitin sulfate, Regulate ECM structure and permeability, Hyaluronan Large molecule of repeating disaccharides Associates with various proteins & receptors Binds water to give tissues turgor pressure Abundant in matrix of actively migrating cells Events in Layer IV: Remodeling of ECM and formation of collagen plates - Metalloproteinases, gelatinases,stromolysins,Wound contraction - Myofibroblasts • Regression of blood vessels - Apoptosis

MICROBIOME

Lecture 40: The Microbiome August 1, 2017 1 Normal Flora Microorganisms that are frequently found on, or in, the body of healthy persons; an "organ"? Outnumber human cells 10-fold? Maybe not... -Mostly bacteria, but fungi, viruses (phages and human), and parasites can be found; culturable vs. non-culturable "peacekeeping organisms", "pathobionts" Human Microbiome Project Rationale: determine "healthy" microbiome; is it different from microbiome in some diseases? Can restoring "healthy" microbiome treat diseases due to dysbiosis? Tracked 242 healthy men and women over 2 years; sequenced genetic material from 15-18 anatomic sites (e.g. skin, nose, mouth, gut). Results: No 2 microbiomes are alike Microbial diversity is great even in one person But there are a few "signature" bacteria found at specific body sites in most people, with similar functions, e.g. carbohydrate processing. 2 Normal Flora -Fetus develops in a "sterile" environment, but quickly becomes colonized during and after birth. Vaginal microbiome changes during pregnancy Flora differs in vaginal vs. C-section delivery; breast-feeding vs. formula 3 Taxonomic distribution of mo's in mother and baby 4 Nature Reviews Microbiology Vol 9, Jan. 2011 5 Nature Medicine Vol 22 Number 7, July 2016 How is the normal flora established? 6 From: Metro Boston 1 From: Bagg et al. 2006 8 Plaque Formation 2 From: Bagg et al. 2006 9 Formation of Dental Plaque From: Prescott et al. 2005 10 Commensal Microorganisms Opportunistic Pathogens Strict Pathogens Colonizing Microorganisms 11 Normal Flora -Exposure to microorganism may result in: a. transient colonization b. permanent colonization c. disease production -Normal flora varies with age, environment, immune status, diet, hormone levels, medications -The large intestine has the most bacteria; other popular sites: skin, URT, mouth, vagina, anterior parts of the urethra -Most normal flora bacteria are anaerobes. -Normal flora provide essential benefits to humans 12 Normal Flora 13 EMBO reports vol 7 no 7 2006 The Gut Microbiota Affects Drug Metabolism Science Vol 336 6/8/12 14 15 NEJM 375:24 12/15/16 Nat Med 22;10:1079 2016 16 Antibiotic depletion of microbiota is sufficient to predispose mice to allergic disease Nat Medicine vol 18, No 4, 2012 17 18 NEJM 375:24 12/15/16 Normal flora serves as a source of opportunistic infections Prescott, et al. Microbiology 6th ed. 2005 19 From: Prescott et al. 2005 How does the normal flora co-exist with the immune system? 20 The Immune System Controls the Microbiota... Science Vol 336 6/8/12 21 Ex. The immune system monitors and regulates intestinal bacteria 22 EMBO reports vol 7 no 7 2006 NEJM July 7, 2016 23 And commensals have mechanisms for suppressing the immune response against them... 24 EMBO reports vol 7 no 7 2006 And the Microbiota Influences the Immune System... The hygiene theory Science Vol 336 6/8/12 25 Location Conjunctiva Skin Nose Mouth Dental plaque Gingival crevices Throat Stomach Colon Vagina Urethra Medically Important Members of the Normal Flora Important Organisms1 Staphylococcus aureus & coag-neg Staph, Corynebacterium species Staphylococcus epidermidis S. aureus3 Viridans streptococci Streptococcus mutans Various anerobes, e.g. Bacteroides, Fusobacterium, streptococci, Actinomyces Viridans streptococci Bacteroides fragilis, E. coli Lactobacillus, E. coli3, group B streptococci3 Less Important Organisms2 Haemophilus spp. and Streptococcus spp. S. aureus, Corynebacterium, various streptococci, Pseudomonas aeruginosa, anaerobes (e.g. Propionibacterium acnes), yeasts (e.g. Candida albicans) S. epidermidis, Corynebacterium, various streptococci Various streptococci, Eikenella corrodens Prevotella intermedia, Porphyromonas gingivalis Various streptococci (including S. pyogenes & S. pneumoniae), Neisseria species, Haemophilus influenzae, S. epidermidis Moraxella Helicobacter pylori, Lactobacillus spp. and Streptococcus spp. Bifidobacterium, Eubacterium, Fusobacterium, Lactobacillus, various Gram-negative rods, Enterococcus faecalis, streptococci, Clostridium Various streptococci, various Gram-negative rods, B. fragilis, Corynebacterium, Candida spp. S. epidermidis, coag-neg Staph., Corynebacterium, various streptococci, 26 various Gram-negative rods, e.g. E. coli3 Normal Flora is not found in some sites Deep tissues and organs CSF Blood Body fluids Implications for specimen collection, lab tests, and interpretation of results... 27 From: Petersen, C. & J.L. Round (2014) Defining dysbiosis and its influence on host immunity and disease. Cellular Microbiology 16(7) 1024-1033 Diseases Associated with Dysbiosis? Obesity and metabolic syndromes Inflammatory bowel disease Irritable bowel syndrome Asthma Antibiotic-associated diseases Autoimmune and inflammatory diseases Colon cancer Non-alcoholic fatty liver disease Atopy Hypertension 29 Prebiotics, Probiotics, Synbiotics Probiotics: live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Prebiotics: selectively fermented ingredients that stimulate specific changes in the colonic microbiota that benefit the health of the host. Synbiotics: combinations of pre- and probiotics that are designed to have synergistic or additive effect benefitting the host 30 31 32 Mechanisms of action of probiotics FIGURE 1. Inhibition of enteric bacteria and enhancement of barrier function by probiotic bacteria. Schematic representation of the crosstalk between probiotic bacteria and the intestinal mucosa. Antimicrobial activities of probiotics include the (1) production of bacteriocins/defensins, (2) competitive inhibition with pathogenic bacteria, (3) inhibition of bacterial adherence or translocation, and (4) reduction of luminal pH. Probiotic bacteria can also enhance intestinal barrier function by (5) increasing mucus production. 33 (Inflamm Bowel Dis 2009;15:300 -310) Mechanisms of action of probiotics FIGURE 2. Modulation of mucosal immune response by probiotic bacteria. Pathogen-associated molecular patterns (PAMPs) derived from probiotic bacteria are recognized by pattern recognition receptors, such as Toll-like receptors (TLRs) on DC in the epithelium or lamina propria. Probiotic bacteria can shape the mucosal immune system toward a noninflammatory, tolerogenic pattern through the induction of T cells with regulatory properties. Probiotics can also downregulate Th1 response and inhibit the production of proinflammatory cytokines, IL-12, TNF-, and IFN- by DC. The predominant cytokine profile depends on the nature of the stimulus and the types of probiotic bacteria. The IL-23/IL-17-mediated inflammatory axis has recently been implicated in the pathogenesis of IBD but there remain gaps in our knowledge on how probiotics influence the 34 differentiation of Th17 cells. (Inflamm Bowel Dis 2009;15:300 -310) 35 Nature Reviews Microbiology Vol 9, Jan. 2011 Fecal matters 36 Volume17 number 2 | february 2011 Nature Medicine Fecal transplants??? 37 From: Improper Bostonian 8/5-18/15

Opioid Pharmacology Opiate Opioid Narcotic

Opiod receptor ligans: Agonist: Morphine, Met-Enkephalin Antagonist: Naloxon, Naltrexone Agonist-Antagonist: Nalorphine, Buprenorphine Alkaloids: Natural (miorphine, codeine), Semisynthetic (heroin, hydromorphone), sunthetic (meperidine, methadone, fentanyl). Pro-Opiomelanocortin (POMC): B-Endorphin, MSH, ACTH Pro-Enkephalin A: Met-Enkephalin, Leu-Enkephalin Pro-Enkrephalin B: Dynorphin A Pro-Orphanin: Orphanin FQ Nociceptin Endomrophins 1 and 2 Choosing Opioids via Pharmacokinetics; Long: Methadone, Levorphanol Medium: Morphine, Hydromorphone Short: Meperidine, Fentanyl, Remifentanil Receptors: u1/2 MOP: Sufentanil, B-endorphin, Endomorphin-1 k1/2/3 KOP: Nalorphine, Dynorphin-A g1/2/3 DOP: Enkephalin, DPDPE ORL NOP: Nociceptin Opioid Receptor Structure: VII cross 3 intracellulat loop receptor Acute opiod effects: Analgesia • Respiratory Depression • Sedation • Vasodilation • Tolerance • Bradycardia • Nausea, Vomiting • Miosis • Smooth Muscle Spasm • Skeletal Muscle Hypertonus Chronic: tolerance + dependence presynaptic u,k,g cause low transmitter release u recptor on post synaptic cause hyperpolarization Imacts limbic system, descending modulation, and dorsal horn (inhibit SG cell) Fentanyl Hepatic CYP3A4,5 clearance Codeine CYP2D6 clearance Codeine O-Demethylation to Morphine • A minor pathway (<10%) of codeine metabolism, but it accounts for nearly all opioid activity • CYP3A4 and glucuronide metabolites (80%) are mostly inactive Mrophine GlucuronDemethyl clearance Morphine-6-Glucuronide: pain tollerance high then leaves Remifentanil Esterase extrahepatic clearance - pethidine 2D6 Phenotype: -Codeine: Inactive parent + active metabolite • Oxycodone: Active parent + active metabolite • Tramadol: Active parent + inactive metabolite 1. Almost all opioids are cleared by the liver 2. Many opioids have polar active metabolites, so most likely to cause problems in renal failure 3. Polymorphisms in clearance may be bad or good, depending upon - Effect of metabolite - Activity of parent drug Analgeisa is always accompanied by some respiratory effect Morphine will depress CO2 response with higher doses N-Methylnaltrexone (MNTX) • Highly polar quaternary ammonium compound that does not enter the CNS • Reverses opioid-induced constipation, delayed gastric emptying, & urinary retention • Does not affect CNS actions like analgesia, respiration or pupil constriction Opioid Cardiovascular Effects • Vasodilation - Centrally-mediated - Venous and Arterial • Bradycardia • No Important Effects on - Myocardial Contractility - Vascular Response to Catecholamines - Sympathetic Reflexes Opioid-Induced Histamine Release Duration & intensity of effect ↓ • Change may be profound • Cross-tolerance to other opioids • Opioid receptor changes? - Phosphorylation & internalization - ↑MAP kinase, ↑adenylyl cyclase, ↑PLC • Non-opioid receptor mechanisms? - Hyperalgesic effect (NMDA, NO) - Neuroinflammation (immune responses) • Opioid receptor changes? - Phosphorylation & internalization - ↑MAP kinase, ↑adenylyl cyclase, ↑PLC • Non-opioid receptor mechanisms? - Hyperalgesic effect (NMDA, NO) - Neuroinflammation (immune responses) Tolerance Affects Analgesia and Respiratory Depression Physical Dependence Abstinence • Nausea, Vomiting • Restlessness • Yawning • Gooseflesh • Mydriasis • Rhinorrhea • Lacrimation • Diaphoresis • Anorexia • Weight Loss • Hyperthermia • Drug Seeking • Behavioral Pattern of Drug Use • Harm to Individual & Society • Overwhelming Involvement - Obtaining Drug - Using Drug • High Tendency to Relapse After Withdrawal Methadone Maintenance Advantages • Long-acting (once a day) • Oral (no needles) • Reduces drug seeking - Prevents abstinence - Cross-tolerance to heroin • Regular contact with caregivers • Agent of choice for detoxification Requires special license, highly regulated • Separate from medical care • Limited availability • Stable and unstable patients mixed • No privacy; Social stigma • Tied to clinic; Employment difficult • Can't "graduate" from program - Buprenorphine - u Partial Agonis (merge with morphine causes downward slope) Buprenorphine Maintenance for Heroin Abusers • Buprenorphine feels "morphine-like" • Potent agonist (~30 X morphine) • Strong antagonist - May cause withdrawal sxs. - Blocks effects of injected heroin • Low-moderate abuse liability - Marketed with naloxone (Suboxone®) - Treatment by primary care physicians -Morphine for postoperative pain: dose range 3-60mg Factors Affecting Opioid Requirement • Pharmacokinetic - CYP2D6 polymorphism - Glucuronidation? • Pharmacodynamic - Type and Location of Pain - Co-existing Disease - Concomitant Drug Treatment - Culture, Expectations, Setting - Age, Gender - Receptor polymorphism -OPRM-1Gene and Single Nucleotide Polymorphisms Most frequent mutation is A118G - Asn to Asp substitution at position 40 - Frequency 10-20% in Caucasian, Hispanic - May be as high as 35-50% in Asians! • Implications of A118G not clear - Greater miosis from M6G in man - Reduced analgesia from M6G in man Are Opioids Inappropriate for Chronic "Benign" Pain? • Poor Response? • Pain Behavior? • Tolerance, Dependence? • Drug Diversion? • Addiction? Heroin Use? Mortality?

Parasympathetic Nervous System CHOLINERGIC DRUGS ACh Nicotinic (N) Muscarinic (M) AChE: A + Ch = ACh

PS: 1. Ach N on skeletal muscles, Ach N on M3 exocrine glands, M2 heart, Ach onto sum nerves Ach on N adrenal medulla to release blood E. M: SM, Glands, Cardiac m Nicotinic Acetylcholine Receptor Top view Side view -Ligand gated ion channel - 5 subunits -Excitatory receptor that transports Na+, K+ and Ca+ -Activated in milliseconds -Prolonged exposure evokes refractory desensitized state in which agonist is bound but closes -Receptor will gradually "reset" after agonist dissociation (seconds to minutes) Nicotinic Acetylcholine Receptor: Antagonist: Muscle relaxants, ganglionic blocker, etc. Agonists: Carbachol, AcH, Nicotine. Nicotinic Acetylcholine Receptor Agonists *Acetylcholine -Endogenous neurotransmitter -Quaternary amine - does not penetrate cell membranes -Activates nicotinic and muscarinic receptors -Rapidly inactivated by acetylcholine esterase (AChE) at synapse and esterases in blood *Carbamylcholine -Quaternary amine -Activates nicotinic and muscarinic receptors -Esterase resistant analog of ACh - Tertiary amine, CNS penetrating, rapid acting, rapid clearance - Drug of abuse ACUTE TOXICITY - Via ingestion of nicotine-based insecticides or tobacco products by children or via dermal contact in tobacco workers 1- Rapid onset of abdominal pain, nausea, diarrhea, disturbed vision, weakness, mental confusion 2 - Stimulates parasympathetic and sympathetic nervous system 3 - Central simulation, can cause convulsion, coma and respiratory arrest 4 - Skeletal muscle depolarization via receptor desensitization ACUTE TOXICITY Treatment - Activated charcoal to reduce GI absorption - Benzodiazepine to control seizures - i.v. fluids to maintain/increase blood pressure - Atropine for bradycardia if needed Nicotine poisoning in infant: - Incidence increasing due to concentrated liquid forms - Patient vomiting, truncal ataxia and poor respiration - Ingested small amount of liquid containing 1.8% nicotine (18mg/ml) - 5ml of this solution could be lethal to a 90-kg adult - Patient recovered without antidote within 6 hours- Ingested 5 ml of liquid nicotine containing 10 mg/ml nicotine - Involuntary vomited after ingestion - Lost consciousness and was unresponsive - EMT found no pulse did 40min CPR - Needed mechanical ventilation - Pupils fixed and dilated and had fetal incontinence - Feet and hands clammy - Brain edema - Never regained consciousness - Died on 44th day in hospital Neuromuscular (NMJ) Blocking Agents Non-depolarizing blockers (competitive) e.g., tubocurarine - Competitive inhibition of ACh binding to nicotinic receptor at skeletal muscle endplate - When approx 70% bound prevent ACh evoking muscle contraction - Results in flaccid paralysis of all skeletal muscle - Indirectly antagonized by AChE inhibitors which increase endogenous ACh (used post surgically to speed recovery) USED IN SURGERY TO PROMOTE SKELETAL MUSCLE RELAXATION - Tracheal Intubation e.g., succinycholine - Maintain controlled ventilation - Paralysis of skeletal muscle in area of surgery - To set fractures e.g., succinycholine Depolarizing blockers (non-competitive) e.g., succinycholine Mechanism of action: -Binds to nicotinic ACh receptor (nicotinic agonist) -Evokes receptor desensitization and voltage-gated channel inactivation -Nicotinic ACh receptors enter a desensitized state upon prolonged exposure to agonist -Receptor will gradually "reset" after agonist dissociation -Rapid onset and short duration of action - recover within minutes of infusion termination -Rapidly hydrolyzed by plasma cholinesterase - Cholinesterase inhibitors prolong effect as increase endogenous ACh receptor activation Neuromuscular Blocking Agents Depolarizing blockers (non-competitive) e.g., succinycholine Stages of block: Fasciculation: Initial excitation and disorganized muscle fiber contractions due to non-synchronous muscle receptor activation Phase I block (depolarizing): Prolonged depolarization due to persistent nicotinic ACh receptor activation leads to voltage-gated sodium channel inactivation. Muscle exhibits flaccid paralysis. Phase II block (desensitizing): Nicotinic ACh receptor activation progresses to receptor desensitization and receptor-operated channels close. Muscle repolarizes, but continues to exhibit flaccid paralysis due to non-responsiveness of ACh receptors. Skeletal Muscle Relaxants: Toxicity • Nondepolarizing - Histamine release with tubocurarine and some related agents • Depolarizing (succinylcholine) - Malignant hyperthermia in patients with this genetic skeletal muscle disorder (features excess Ca2+ release in skeletal muscle) • Incidence: 1 in 4,500-60,000 surgical procedures • Treatment: dantrolene (prevents Ca2+ release) - Prolonged action in patients with atypical plasma cholinesterase Skeletal Muscle Relaxants: Toxicity • Depolarizing (succinylcholine) -Hyperkalemia - potentially life threatening Due to intracellular K+ release through nicotinic ACh receptor channels Increased risk in burn and immobilized patients (due to ACh receptor up regulation) -Cardiac dysrhythmias, primarily due to stimulation of cardiac muscarinic receptors (usually bradycardia, although tachycardia is sometimes seen, particularly with high doses) -Postoperative muscle pain (mechanism unclear) Hereditary deficiency of which of the following enzymes can lead to prolonged effects of succinylcholine? Plasma cholinesterase b. Plasma cholinesterase c. Heme oxygenase d. Cytochrome oxidase e. Liver transaminase Botulinum toxin: A denervating protein toxin • Bacterial protein toxin (m.w. ~150,000) • Binds specifically to cholinergic nerve terminals, internalized into cytoplasm • Enzymatically degrades protein factors required for vesicle fusion and ACh release • Produces prolonged presynaptic impairment of cholinergic neurotransmission resulting in localized skeletal muscle paralysis • Prolonged action (months) - recovery due to axonal sprouting • FDAapprovedfor - Cosmetics (reduction of facial wrinkles) - Treatment of cervical dystonia (spasmodic torticollis) - Treatment of spasticity in flexors of elbow, wrist, fingers - Treatment of strabismus (misalignment of the eyes) - Treatment of blepharospasm - Treatment of severe primary axillary hyperhydrosis (excessive underarm sweating) - Treatment of urinary incontinence associated with neurologic conditions - Treatment of localized muscle cramps • Off-label uses include - Treatment of spasticity - Treatment of nondystonic muscle activity disorders e.g., tics, tremor - Treatment of localized muscle cramps - Treatment of smooth-muscle hyperactive disorders • Muscle weakness (may be long-lasting) - Difficulty in eating/swallowing (particularly with use in head & neck) - Paralysis of respiratory muscles, can result in death • FDA Warning (2008) - botulinum toxin has "been linked in some cases to adverse reactions, including respiratory failure and death, following treatment of a variety of conditions using a wide range of doses." • Treatment of botulism: antitoxin (but does not reverse toxicity once botulinum toxin has entered the nerve terminal) Muscarinic Acetylcholine Receptor M1M3M5: PLC, DAG, IP3 M2M4: -cAMP + K -Ca2+ channel Non-selective (N+M): Ach carbachol M selece EFFECTS OF STIMULATING MUSCARINIC RECEPTORS Bradycardia, NO Vasodilation, Bronchoconstriction, Miosis, More urnination, more tears, more sweating MUSCARINIC AGONISTS (parasympathomimetics) ACh - no clinical use!!!! Pilocarpine -NO nicotinic effects -Some selectivity for salivary glands -Used orally to treat xerostomia -Used topically to treat glaucoma Bethanchol -NO nicotinic effects -Some selectivity for bladder -Used orally to treat post-op urinary retention -Stimulates GI activity Carbachol -Pupil contraction for ophthalmological surgery Muscarinic Agonists: Toxicity • Salivation • Lacrimation (tear secretion) • Urination • Diarrhea • GI upset • Emesis (vomiting) • Bronchoconstriction • Hypotension • Bradycardia Muscarinic Antagonists: BELLADONNA ALKALOIDS Atropine and Scopolamine Trycyclic antidepressants and antihistamines have anti muscarinic effects Antimuscarinic drugs cause: tachycardia, vasodilation from feedback, bronchdilation, MYDRIASIS, DECREASED GI MOTILITY, DECREASED SALIVATION, DECREASED TEARS, DECREASED SWEATNG Antimuscarinic Agents BELLADONNA ATROPINE ALKALOIDS SCOPOLAMINE TX: USED WHEN A REDUCTION OF PARASYMATHETIC TONE IS DESIRABLE • Reduce tremor • Preop to reduce salivations / bronchial secretions • Treat rhinitis (inflammation of nasal membrane) • Reduce intestinal motility • Prevent vagal stimulation of heart during MI (i.e., reduce HR) • Treat overactive bladder • Ophthalmological examinations • Prevent motion sickness (scopolamine, CNS action) • Treat bronchoconstriction (asthma) • Treat AChE inhibitor poisoning or muscarinic agonism Antimuscarinic side effects and toxicity DRYASABONE HOTASAPISTOL REDASABEET BLINDASABAT MAD AS A HATTER • e.g., Deadly nightshade • Dry mouth • Dry, hot skin • Dilated pupils • Blurred vision • Flush • Delirium • Tachycardia • Urinary retention, constipation • Exacerbation of glaucoma (esp. narrow-angle) • Hyperthermia (esp. in children) • Sedation (particularly with scopolamine) • Give pt Physostigmine (ACh-esterase inhibitor) Which one of the following agents, when applied topically to the eye, would induce mydriasis?: Atropine Carbachol (muscarinic agonist) causes pupil constriction (myosis). Atropine Atropine (muscarinic antagonist) causes mydriasis (pupil dilation) Acetylcholinesterase (AChE) Inhibitors ACh - normally removed from synapse via AChE AChE inhibitors reduce AChE activity Increase ACh concentrations ENHANCE TRANSMISSION AT CHOLINERGIC JUNCTIONS Cholinesterase (AChE) Inhibitors Carbamate esters Primary clinical class - form reversible covalent bond Physostigmine -Penetrates CNS, drug of choice to treat atropine poisoning Neostigmine -No CNS penetration (quaternary amine) •Treatment of Alzheimer's Disease •Myasthenis gravis •Accelerating recovery from nondepolarizing muscle relaxants •Treatment of postsurgical paralytic ileus •Treatment of urinary bladder atony •Antimuscarinic antidote (atropine = competitive blocker) Cholinesterase (AChE) Inhibitors Organophosphates - IrreversibleAChEinhibitorsvia phosphate bond Insecticides e.g., Parathion Nerve Gases e.g., Sarin • Muscarinic toxicity (SLUDGE, bronchoconstriction) • Neuromuscular blockade • Respiratory failure • Confusion, coma, convulsions Antidote for Muscarinic Responses Produced by Overdose of Cholinesterase Inhibitors: Atropine ENHANCE TRANSMISSION AT CHOLINERGIC JUNCTIONS -Evokes muscarinic toxicity (SLUDGE) -Atropine blocks action at Muscarinic NOT Nicotinic receptors Pralidoxime - cholinesterase regenerator - Atropine blocks action at Muscarinic NOT Nicotinic receptors - PRALIDOXIME - Oximes bind organophosphate inactivated AChE - treat organophosphate poisoning or toxic gas exposure - Effects N and M responses Esteric site blocked by organophosphate blocking activity Anionic site free - Pralidoxime binds here Pralidoxime then binds to organophosphate changing its conformation Organophosphate and pralidoxime complex unbinds AChE function restored Efficacy in organophosphate treatment remains controversial Jimson weed (belladonna alkaloids) (Atropine-like toxicity have the opposite effect. i.e. Mad as a hatter, dry as a bone, blind as a bat, hot as hell.) Parathion (Parathion is a cholinesterase inhibitor, which will lead to accumulation of Ach. Symptoms are signs of Ach excess). Poisoning with malathion insecticide (organophosphate) is best managed by administration of which one of the following agents? Atropine (Atropine blocks the muscarinic effects of increased Ach resulting from cholinesterase inhibition). Pilocarpine Pilocarpine is a direct muscarinic agonist. This will make the situation much worse . Bethanechol Bethanechol is a direct muscarinic agonist. This will make the situation much worse. Physostigmine Physostigmine is a inhibitor of cholinesterase, increase Ach effect. This will make the situation much worse

Regulation of Drug Development, Marketing, and Prescribing in the U.S.

Patent Medicines of the 1800s • Botanical extracts • Alcohol as diluent • No controls over content or claims of efficacy or safety • Lydia Pinkham's Vegetable Compound promoted for women's ailments (19% alcohol) 1902: federal controls over vaccines • 1906: Food and Drug Act - drugs shipped in interstate commerce must be labeled properly according to ingredients • 1912: amendments - prohibition of false advertising claims Sulfanilamide Disaster • 1936-7 • Elixir formulated with diethylene glycol (antifreeze) as solvent • Resulted in over 100 fatalities • Mislabeling conviction - Elixir means alcohol Food, Drug and Cosmetic Act of 1938: Proof of Safety • Drugs shipped in interstate commerce must be proven experimentally to be safe • Experimental data on safety must be submitted to FDA for marketing approval Prescription vs OTC • 1951 Durham-Humphrey Amendment - Distinction between the two categories of prescription and over-the-counter (OTC) - Standards of labeling and safety apply to both - Certain criteria of safety must be satisfied to permit OTC status Thalidomide Disaster • 1961: thalidomide, a sedative used for morning sickness, found to cause major limb malformation in babies born in Europe • FDA denied approval • Generated climate for U.S. reforms to improve evidence of safety and efficacy Proof of Efficacy • 1962: Kefauver-Harris Amendments - Efficacy for indication must be proven experimentally - Label and advertisements must list all ingredients and side effects - Subjects in clinical trials must give informed consent Safeguards of Subjects in Clinical Trials 1991 Federal Policy on Protection of Human Subjects • Approval of trial by Institutional Review Board (IRB) • Are subjects rights and welfare protected? • Are risks to subject outweighed by benefit or importance of new knowledge? • Has subject given informed consent without coercion? Types of Products Regulated by the FDA • New molecular entities, new drug combinations, new dosage forms or strengths, diagnostics - Center for Drug Evaluation and Research, CDER • Proteins such as vaccines and cell products - Center for Biologics Evaluation and Research, CBER • Medical devices and materials - Medical device amendment of 1976 • Dietary supplements - Dietary Supplement Health and Education Act of 1994, DSHEA, false advertising only The IND • Notice of Claimed Investigational Exemption of a New Drug - Composition, manufacturing, animal studies, clinical protocols, clinical investigators • Must be approved by FDA before clinical trials begin Phase 1 • Pilot study in 20-80 healthy volunteers • Determination of safety of several doses • Determination of pharmacokinetic properties • Non-blinded • About 20% of tested drugs are approved Phase 2 • Studies in 100-200 patients with target disease • Placebo and active drug controls • Single-blind • About 30% of tested drugs are approved Phase 3 • Studies in many (about 5000) patients with target disease • Determination of efficacy for indication and side effects • Usually double-blind and cross-over design with placebo or active control • About 70% of tested drugs are approved New Drug Application NDA • Data from all studies submitted to FDA - FDA summary and response available at www.fda.gov for approved drugs • FDA may require additional studies prior to approval The 'Label' • Approved by FDA as part of NDA process • Same as package insert • Available in The Physician's Desk Reference • Contains chemistry, pk, indication, side effects, toxicity, contraindications, dosage, information in special populations • NOT a critical guide! (see NEJM 36:1230,2009) Out-of-Pocket and Capitalized Cost per Approved New Compound Phase 4 • Additional trials, in some cases FDA requested • Post-marketing surveillance - Detection of low-incidence side effects <1 in 10,000 - Based on physician and hospital reports to FDA and manufacturer • FDA MedWatch reporting program • Data available through FDA's Adverse Event Reporting System - May result in black-box warning on label or recall of drug • Additional trials, in some cases FDA requested • Post-marketing surveillance - Detection of low-incidence side effects <1 in 10,000 - Based on physician and hospital reports to FDA and manufacturer • FDA MedWatch reporting program • Data available through FDA's Adverse Event Reporting System - May result in black-box warning on label or recall of drug Impact of Label 'Indication' • Illegal to ship interstate for other than label indication • Physician may use product for 'off- label' indication in individual patient • IND must be obtained for clinical trial for new indication, even of approved drug • Label 'indications' change, as new data on efficacy are obtained Regulations to Decrease Cost: Approval of Generics • Patents expire 20 years after filing of patent - Patent filing occurs generally early in development - Effective patent life lasts less than 14 years • Generic formulations, marketed after patent expires, are generally much less costly than original patented drug Approval Process for Non-patented Drug • Small MW drug: a 'generic' - Generic manufacturer must prove bioequivalence of generic formulation to the patented drug product • Same amount of active ingredient • Similar bioavailability measured in healthy human subjects - within + 20% of proprietary formulation - based on AUC, peak Cp, and time of peak - Data submitted to FDA as 'abbreviated' NDA - Low research expenditure, so product typically much less costly Approval Process for Non-patented Drug • Protein therapeutic: a 'biosimilar' - More complex studies required as manufacturing in cell culture can lead to subtle differences in composition and structure - May require studies of clinical efficacy and safety, not just study of bioavailability - Regulations initiated in 2009 and evolving Measures to Increase Access to New Drugs • Treatment IND (1987) - Promising investigational drugs in late Phase II or III can be available for patients with life-threatening conditions or serious chronic conditions - Regulatory safeguards apply to patients as to experimental subjects - Sped access to drugs for HIV Regulations to Decrease R&D Cost and Speed Access: Shortening FDA Review Times • Prioritizing NDAs (1991) - Priority: important therapeutic gain, new class NDA review time about 6 months - Standard: modest therapeutic gain, 'me-too' drugs NDA review time about 12 months • Prescription Drug User Fee Act (PDUFA, 1992) - Fees collected from manufacturer to cover NDA review - Incentive for improved quality of the NDA to shorten review times Food and Drug Administration Modernization Act (FDAMA) 1997 • Access to information on clinical trials for serious illnesses ClinicalTrials.gov • Incentives for clinical trials in children • 2007 reauthorization included plans to improve post-marketing drug safety surveillance and review of TV advertising Are the FDA and its advisory committees serving the public need? - Avorn, J. Keeping science on top in drug evaluation, NEJM 357: 633-5, 2007. • Are the labeling requirements inadequate? - Stafford et al. New, but not improved? Incorporating comparative-effectiveness information into FDA labeling, NEJM 361:1230-2, 2010. • Is supply-chain globalization overtaxing the inspection capabilities of the FDA? - Mullin, R. C&EN News 89: 11-15, 2011. Direct-to-Consumer Advertising of Prescription Drugs • Substantial expenditure since about 1997; 4.4 billion dollars in 2010 (Kornfield et al., PLoS One, 8(3): e55504, 2013) - 1.4% of sales - about 18% of promotion to providers - declining since 2006 • Effective marketing strategy - Increases consumer awareness of drug products - Increases request by consumer for heavily promoted proprietary drugs - Increases prescribing by physicians in response to request of more expensive therapeutics • Substantial percent of total marketing budget and much more than direct-to-consumer (estimated at 5.4 times greater in 2010) • Includes free samples, the largest cost category, distributed to physicians upon FDA approval to generate sales • The 2013 Physician Payment Sunshine Act now requires manufacturers to report to DHHS all 'transfers of value' (meals, gifts, travel) to individual physicians - Information posted on DHHS website • Regulatory authority: state boards authorized by state legislation • Prescribers - physicians and osteopaths - in some states with restrictions pharmacists, nurse practioners, physician's assistants, optometrists • Prescriber • Patient's name and address • Drug name • Dose and quantity dispensed • Instructions for use • Refills • Date • Physician's signature Shift to e-Prescribing • About 4 billion prescriptions filled annually in U.S. • e-prescribing used by about 70% of physicians in 2014 (10-fold increase from 2008) • Required by some health plans, insurers, and hospitals • e-prescribing may improve efficiency, reduce cost, reduce errors, increase safety; but has drawbacks such as no e-discontinuation • Physician prescribes by generic name metoprolol • Physician prescribes by proprietary name TOPROL • Physician prescribes to prohibit substitution TOPROL " Dispense as written" Formularies in hospitals, managed care organizations, and insurers are designed for cost-saving • In some managed care settings - prescriber chooses drug class - pharmacist dispenses drug in that class from the formulary Regulation of Drugs of Abuse • Opioidsrecognized as major medical problem by end of 19th c. • Manypatent medicines contained high levels of opioids Harrison Narcotic Act of 1914 • Regulation of production and use of narcotics - opioids, cocaine, marijuana (1937) • Enforced by Federal Bureau of Narcotics in Treasury Department - taxation of physician prescriber Controlled Substances Act of 1970 • Purpose - decrease availability of drugs to potential abusers • Enforcers - Drug Enforcement Agency located within the Department of Justice - DEA issues permits to physicians for a fee for right to prescribe controlled substances Schedules of Controlled Substances • Chemicals categorized into schedules based on: - abuse potential - pattern and significance of abuse - risk of dependence • Schedule affects: - registration of handlers, record-keeping, storage security, limits on dispensing - penalties for illegal trafficking I Research use only heroin, marijuana (in conflict with laws of many states!) II No prescription refills morphine, amphetamine, III Limit to refills codeine IV Limit to refills benzodiazepines V O-T-C with some limits antitussives with low codeine • Physician's DEA registration number • Physician must also register with state • e-prescribing allowed by DEA as of 2010 Keep informed about changes in drug regulations and their interpretation! - Federal level: refer to FDA Guidance website - State level: • MA Prescription Monitoring Program now covers data on prescribing of schedule II-V substances and requires review of patient prescription history before prescribing schedule II and III drugs. • MA March 2016, three measures to reduce opioid deaths including 7-day limit on new opioid prescriptions • Work to improve regulations on behalf of your patients and public health • Consider that some regulations may increase cost of drug use and reduce incentives for drug discovery

Principles of Pharmacology and Therapeutics • Botanical preparations ("herbal medicine") - Complex mixtures of plant substances • "Small molecule" drugs (m.w. < 1000) - Racemic mixtures (e.g. D- and L- epinephrine) - Pure stereoisomers • Polypeptides & small proteins - Insulin (m.w. 6000) • Protein therapeutics (biologics) - Botulinum toxin A (m.w. 900,000) - Infliximab (IgG monoclonal antibody vs. TNF-; m.w. 144,000) • Nucleic acids - Mipomersen (antisense oligonucleotide for ApoB mRNA; m.w. 7500) Drug Names • Chemical name - Specifies chemical structure ( e.g. ethyl 12-fluoro- 8-methyl- 9-oxo- 2,4,8- triazatricyclo [8.4.0.02,6] tetradeca- 1(10),3,5,11,13- pentaene- 5-carboxylate ) • Code name - Chosen by manufacturer during drug development ( e.g. Ro 15-1788 ) • Proprietary name - Also referred to as brand or trade name - Chosen by manufacturer for marketing purposes ( e.g. Anexate®, Lanexat® ) • Official name - Assigned by US Adopted Name Council ( e.g. flumazenil ) - Also referred to as generic or nonproprietary name - Name used throughout DRx, including exams

Pharmacokinetics • The time course of drug absorption, distribution, and elimination • Factors that affect pharmacokinetics Principles of Pharmacokinetics Dose absorption distribution elimination EFFECT Cp 3 key pharmacologic phenomena that describe the time course of the drug in the body and the therapeutic response Biotransport: transport of drugs into and through cells • Passive diffusion - by passage through membrane - by filtration • Carrier-mediated transport • Receptor-mediated endocytosis Biotransport Passive Diffusion • First-order kinetic process - Rate dependent on concentration gradient • Cellular structure of membranes - Tight junctions - Lack of porosity • Chemical properties of drugs - Lipophilicity - Charge: Ionizable residues and pKa - Molecular weight Biotransport\Passive Diffusion Lipophilicity • Solubility in lipid relative to water • Measured by oil/water partition coefficient - Drug concentration in oil versus water Oil Water Biotransport\Passive Diffusion Impact of Ionizable Groups • Ionization markedly decreases lipophilicity and increases hydrophilicity • Drugs with fixed negative or positive charge do NOT diffuse through cell membrane - Example of quaternary ammonium compounds Biotransport\Passive Diffusion Impact of Ionizable Groups Neuromuscular Blocker Succinylcholine has a quaternary ammonium fixed positive charge Biotransport\Passive Diffusion Lipophilic Drugs with an Ionizable Group • Drugs with carboxylate group act as acids and become negatively charged (and nonlipophilic) as pH increases. • Drugs with amine group act as bases and become positively charged (and nonlipophilic) as pH decreases. • Only the uncharged, unionized form of a drug is lipophilic enough to diffuse through cell membranes Biotransport\Passive Diffusion Influence of pKa on Acid A and Base B AH A- + H+ BH+ B + H+ • • Weak acid is uncharged at low pH; as the pH increases it ionizes; gives up a H+ ion Weak base is ionized and charged in low pH; as pH increases it looses the H+ ion and becomes uncharged low pH high pH Biotransport\Passive Diffusion Lipophilic Drugs with an Ionizable Group Aspirin • weak acid (pKa 3.5) • at physiologic pH it gives up a H+ and becomes charged • pKa 3.5 < pH7.4 Biotransport\Passive Diffusion Lipophilic Drugs with an Ionizable Group • tertiary amine • weak base (pKa 8) • at physiologic pH it picks up a H+ and becomes charged • pKa 8 > pH 7.4 Lidocaine Molecular Weight TPA Biotransport • TPA(tissueplasminogen activator) is large molecule drug • Protein with 527 aa and MW of 70,000 • Administered parenterally • Doesn't travel across membranes • Stays in the plasma to lyse clots Molecular Weight • Chimeric monoclonal antibody (mAbs) MW 144,000 • Biologic agent (Protein therapeutic) • Administered parenterally Infliximab Biotransport (Fc) (Fab) Which drug most readily diffuses through cell membranes?Base at high pH Biotransport Filtration • Transport of small molecule drugs in bulk flow of aqueous fluid • Rate dependent on - hydrostatic pressure - molecular weight, size, charge, and binding to excluded macromolecules - tissue porosity • glomerular capillaries in kidney • choroid plexus in brain • sinusoids in liver Biotransport Convective transport • Important mechanism of transport of mAb from lymph or blood into tissue spaces • Depends on hydrostatic pressure gradient, fluid flow rate into the tissue, and on the sieving effect of paracellular pores • Sieving depends on size & shape of pores, and size, shape, & charge of antibody. Biotransport Carrier-mediated Transport • Saturable kinetics • Substrate competition • Stereospecificity • Tissue differences in expression of carriers Biotransport Drug Transporters • Organic anion transporter 1 (OAT1) • Solute carrier family member - SLC22A6 gene product • Organicanionicsubstrates • Probenecidsensitive • ABCtransporter P-glycoprotein 1 (Pgp), multidrug resistance protein (MDR 1) - ABCB1 gene product • Amphipathic,cationic and neutral substrates • Verapamilsensitive Biotransport Drug Transporters • MDR p-Glycoprotein • Actively transports many chemically different agents across membranes throughout the body. • Expression is upregulated by drugs causing resistance to many antitumor agents. • MDR is found on normal cells as well as tumor cells Biotransport Drug Transporter Localization • Intestinal mucosa • Hepatocyte - into bile • Proximal renal tubule - plasma into nephron lumen • Brain - Choroid Plexus - Vascular endothelium - back into plasma and away from brain Biotransport Receptor-mediated Endocytosis • Importance in selective tissue uptake • Rate dependent on receptor expression and membrane insertion • Saturable kinetics • Mechanism for some protein therapeutics (biologic agents) - monoclonal antibodies (mAbs) Biotransport Endocytosis of mAbs Receptor-meditated endocytosis: • specific interaction of the Fab binding domains of the antibody with target epitopes on the cell surfaces • results in target-mediated disposition of drug Fab with its pharmacological target Fluid-phase endocytosis: • nonspecific process driven by the Fc concentration of mAb on the extracellular side; does not require ligand binding to cell surface • responsible for mAb entering into endothelial cells and peripheral tissues. Principles of Pharmacokinetics DOSE absorption distribution Cp elimination EFFECT Absorption Kinetics • Time-course of transport from site of administration to systemic circulation • Characterization by - Absorption half-life - Percent dose absorbed (Bioavailability) Routes of Administration Routes of Administration Intravenous (iv) • 100% bioavailability • Fastest possible input rate • Rate dependent on technique of administration (IV bolus or IV infusion), not physiological process • Importance of sterility, lack of particulates, aqueous solubility of agent • Route for high MW, polar molecules Biologic agents (therapeutic proteins) Routes of Administration Enteral Administration Bioavailability Bioavailability (F) is the fraction of the dose absorbed into the systemic circulation. Bioavailability is influenced by: •GI absorption •Metabolism in GI tract and/or liver (first pass effect) Brenner and Stevens, Pharmacology 2nd ed (2006) Routes of Administration Oral (p.o.) • Absorption half-life affected by - Formulation of drug • Dissolution from solid - Gastric emptying time - Interactions with diet or other drugs in gastrointestinal lumen Routes of Administration Oral (p.o.) Bioavailability may be low due to: • Mucosa as barrier for drugs with high MW low lipophilicity carrier-mediated extrusion from mucosa back into GI lumen • First-pass effect lumenal, mucosal and/or hepatic biotransformation during absorption process Routes of Administration Subcutaneous (s.c.) & Intramuscular (i.m.) • Low MW lipophilic drugs into blood - Blood flow may affect absorption rate • High MW compounds into lymphatics - Improved bioavailability over oral route for polar and high MW drugs (proteins, mAb) - Uptake of high mw compounds into the lymphatics provides slow absorption rate • Delivery devices for sustained release Inhalation • Rapid absorption of gases and vapors due to - high pulmonary blood flow - low diffusional distance from alveolus to blood • Route for localized delivery of drugs with actions in pulmonary tissue - Nebulizers - Metered dose inhalers Routes of Administration Topical • Absorption through skin generally slow due to surface layers of dead, keratinized cells (stratum corneum) • Drugs with sufficient lipophilicity and potency may have systemic clinical efficacy upon topical application in transdermal patch - Avoids first-pass effect Principles of Pharmacokinetics DOSE absorption distribution Cp elimination EFFECT Distribution Kinetics • Rate and extent of distribution from vascular fluid to tissue space • Determinants of equilibration rate - Blood flow Distribution Kinetics Tissue Perfusion Rates (ml/min-100g tissue ) High Lung 400 Kidney 350 Liver 85 Brain 55 Intermediate/variable Skeletal muscle 5 Low Fat 3 Distribution Kinetics Determinants of equilibrium gradient -Tissue factors Vascular permeability Macromolecular binding in tissue Plasma protein binding P + D PD Dissolution of lipid-soluble drugs in adipose tissue -Physicochemical factors of drug mw, lipophilicity, affinity of carriers Distribution Kinetics Vascular permeability: The Blood-Brain Barrier • Tight junctions between vascular endothelial cells - exclusion of high MW, non-lipophilic drugs • Transporters which actively transport some drugs from endothelium back into plasma PROTEIN Albumin 1-Acid glycoprotein MW CONCENTRATION 67,000 500-700 mM 42,000 9-23 mM Distribution Kinetics Plasma Protein Binding Plasma protein binding P + D PD Drug Warfarin Codeine % Bound in plasma 99% 7% Distribution Kinetics Plasma Protein Binding Apparent Volume of Distribution (Vd) Vd is the volume of the body into which the drug appears to have distributed Drugs will distribute into their apparent volume of distribution dependent on their chemical characteristics Vd governs the concentration of drug in the plasma (Cp) The amount of drug in the body can be related to it's plasma concentration by a proportionality constant that reflects the apparent fluid volume into which the drug distributes (Vd) Apparent Volume of Distribution (Vd) Plasma Concentration = Amount in Body (Cp) Volume Initial Plasma Concentration = Dose (g) (C0) Vd (liters) Vd does not change with dose Distribution Kinetics Apparent Volume of Distribution Fluid compartments of 70 kg subject in liters and % body weight: plasma 3-4L (4%) extracellular 12-14L (17%) total body water 40-42L (58%) Whalen, Lippincott's Illustrated reviews: Pharmacology 6th ed (2015) Fluid Compartments 70-kg Adult Compartment Liters %BW plasma 3 4 extracellular water 12 17 total body water 41 58 Indicator 131I-albumin sucrose D2O Distribution Kinetics Apparent volume of distribution (Vd) depends on the chemical characteristics of the drug: • High MW such as therapeutic proteins - Vd ~ plasma volume, 4% BW • Small MW and polar - Vd ~ extracellular fluid volume, 17% BW • Small MW and lipophilic - Vd ~ total body water or even larger if highly lipophilic, > 58% Which drug is most likely to have a Vd greater than the volume of total body water? Highly lipophilic drug Vd decreases in obesity Elimination Kinetics • Rate and extent of elimination by excretion or biotransformation • Quantitation by total clearance (ClT) - Volume of plasma cleared of compound per unit time by all routes and mechanisms - Expressed as ml/min or ml/hr Elimination Kinetics Clearance depends on organ plasma flow Clorgan = (plasma flow) (Cpa-Cpv) Cparterial Cpa Cpvenous Organ Clearance Elimination Kinetics Total Clearance ClT determines the elimination rate of a drug from the body (dA / dt) (ClT)(Cp)= dA/dt Note: A is amount of drug in the body Elimination Kinetics Total Clearance • Is sum of renal and nonrenal clearances ClT = ClR + ClNR • Renal refers to urinary excretion of unchanged drug • Nonrenal refers to all other routes and mechanisms Elimination Kinetics Monoclonal Antibodies (mAb) Mechanisms of Elimination • Nonspecific endocytosis • Degradation by proteolysis • Target-mediated clearance • FcRn-mediated clearance • FcgR-mediated clearance • Formation of immune complexes (IC) Elimination Kinetics\ mAbs Proteolytic degradation: • mAbs undergo intracellular lysosomal proteolytic degradation • May occur widely in the body or limited to specific organs (liver, kidneys, spleen) • Liver may metabolize mAb by the same catabolic pathways as endogenous proteins • Kidneys may metabolize biologics small enough (<60kDa) to undergo glomerular filtration • The reticuloendothelial system (RES) predominantly phagocytes (monocytes, macrophages) in the liver, spleen, and lymph nodes eliminates biologics, and endogenous IgG molecules Elimination Kinetics\ mAbs Fab-mediated elimination • The specific interaction between the Fab region of the antibody and target epitopes on cell surfaces Fab • A saturable, target-mediated clearance pathway • mAb enters the target cell by receptor-mediated endocytosis and undergoes intracellular catabolism Fc Elimination Kinetics\ mAbs Fc-mediated elimination • A nonspecific elimination pathway for both endogenous IgGs and therapeutic mAbs Fc Fab • Involves either FcRn or Fcγ receptors. Elimination Kinetics\ mAbs FcRn-mediated elimination/ recycling • FcRn, Brambell receptor, binds the Fc domain of mAb • A FcRn-mAb complex forms due to high affinity of FcRn for mAb in the acid environment of the early endosome. • A large fraction of the endosomes are not delivered to the lysosome but are sorted to the cell surface for fusion with the cell membrane. • Upon fusion, the IgG-FcRn complex dissociates in the physiological pH and free IgG is released back into extracellular fluid. Elimination Kinetics\ mAbs FcRn-mediated elimination/ recycling • FcRn is expressed in a wide variety of cells • FcRn-mediated recycling results in the long elimination half-life of IgG and many mAbs (3 weeks) Elimination Kinetics\ mAbs Fcg-mediated elimination • Many cell types (monocytes, macrophages, dendritic cells) express Fc receptors that bind to the Fc domain of biologic agents (mAbs and fusion proteins) • Biologic agents are internalized and degraded by lysosomes in RES • FcgR binding may also be responsible for the elimination of mAb-antigen immune complexes (IC) or cells opsonized by the mAb. • The exact mechanism of action of FcgRs in antibody clearance is not fully understood Elimination Kinetics\ mAbs Formation of immune complexes (IC) • Administration of an exogenous protein therapeutic agent (mAb), often triggers an immune response resulting in generation of endogenous antibodies directed against the protein drug (antidrug antibody; ADA). • The biologic-ADA immune complexes (ICs) are then elimination via the RES Elimination Kinetics\Total Clearance (small molecules) Parent Compound ClR ClNR Metabolite Excretion ClR = renal clearance Excretion Further metabolism ClNR = nonrenal clearance (hepatic) Elimination Kinetics Nonrenal Clearance • Excretion of unchanged drug - Lungs (if high vapor pressure) - Bile • Biotransformation - Phase I oxidation, reduction, hydrolysis - Phase II conjugations Elimination Kinetics Significance of Biotransformation Prodrug Metabolite 1 (inactive) Active Drug Metabolite 2 Metabolite 3 (active) (toxic) Elimination Kinetics Kinetics of Biotransformation • Enzymatic reactions that obey Michaelis-Menton kinetics • Generally therapeutic levels << Km - FIRST ORDER kinetics - Rate of biotransformation proportional to concentration - Described by a half-life, t1/2 Elimination Kinetics\ Biotransformation Exceptions to First-Order Kinetics • Drugs where therapeutic levels >> Km - Ethanol - Aspirin • Drugs where overdose levels >> Km - Acetaminophen Elimination Kinetics\ Biotransformation Causes of Patient Variability in Clearance by Biotransformation: • Dietary and drug interactions - Inhibition: substrate competition, suicide inhibition • Grapefruit juice - Induction: promoters of enzyme expression • Cigarette smoke • Disease - Cytokine-induced inhibition of hepatic enzyme synthesis - Reduction in hepatic blood flow Elimination Kinetics\ Biotransformation Causes of Patient Variability in Clearance by Biotransformation: • Genetic polymorphisms in enzymes of biotransformation - Generally result in prolonged half-life - Frequency depends on enzyme and ethnicity • Examples - CYP450s - N-acetyltransferase - pseudocholinesterase Elimination Kinetics\ Biotransformation Pathways of Biotransformation Drug phase 1 Metabolite phase 2 Conjugated Metabolite (usually inactive & more polar than parent compound) Elimination Kinetics\ Biotransformation Phase 1 Cytochrome P450 Enzymes • Heme-containing • Imbedded in membrane of smooth endoplasmic reticulum (microsomes) • Highest activity in liver • Catalyze addition of molecular oxygen Elimination Kinetics\ Biotransformation Phase 1 Cytochrome P450 Enzymes • About 17 human genes • 3A4 and 2D6 important for many drugs • Isozymes differ in substrate specificity, inducers, and inhibitors • Inducers and inhibitors include drugs, herbal medicines, and environmental chemicals Elimination Kinetics\ Biotransformation Phase II Conjugating Moieties • Glucuronic Acid • Sulfate • Methyl Groups • Glutathione • Acetyl Groups • Glycine Elimination Kinetics\ Biotransformation Sites of Morphine Biotransformation Elimination Kinetics Renal Clearance • Volume of plasma cleared by excretion unchanged into urine • Mechanisms - Glomerular filtration - Proximal tubular carrier-mediated secretion - Passive reabsorption Renal Clearance Elimination Kinetics\Renal Clearance Glomerular Filtration • 20 % renal blood flow filtered • Protein-bound drugs not filtered Elimination Kinetics\Renal Clearance Proximal Tubule Secretion • • • • • Organic anion transporter (oatp) for acid drugs Organic cation transporter for basic drugs Competition between 2 acid drugs for organic anion transporter Competition between 2 basic drugs for organic cation transporter Protein binding does not affect secretion Elimination Kinetics\Renal Clearance Distal Tubule Reabsorption • Passive reabsorption of lipid-soluble, unionized drug • % Ionization depends on urine pH and drug pKa • Acid drug in alkaline urine has increased excretion • Basic drug in acidic urine has increased excretion Elimination Kinetics\Renal Clearance Distal Tubule Reabsorption: % Ionization Compartment in which the drug is more ionized will contain a greater concentration of drug (trapping) Figure shows a basic drug trapped in an acid environment Katzung et al., Basic and Clinical Pharmacology 11th ed (2009) Elimination Kinetics\Renal Clearance Determination of Renal Clearance (ClR) ClR = Urinary excretion rate Cp Amt excreted during collection interval Interval length ClR = ----------------------------------------------------- Cp at midpoint of interval Elimination Kinetics\Renal Clearance Relationship of ClR to Mechanism of Renal Excretion ClR ml/min/70 kg ------------------------------ 0-120 120 (GFR) 120-640(RPF) glomerularfiltration,tubular secretion, no net reabsorption Mechanism -------------------------------------------------------------- exclusion from filtration and/or extensive reabsorption glomerular filtration and no reabsorption Which is most likely to have a renal clearance > 120 ml/min in a 70 kg man? Acidicdrugwith affinity for oatp, high urine pH Elimination Kinetics\Renal Clearance Causes of Patient Variability in ClR • Proportional to body weight 0.7 • Decreases with age - Linear decline in creatinine Cl to 50% by age 90 • Altered by drug interactions - Reduced by carrier competition - Affected by pH if ionizable drug • Reduced by renal disease

Evaluating Drug Efficacy and Toxicity: NSAIDs for Osteoarthritis •Why the study was done, and who sponsored it? •What was the study design? •What were the end-points, and how were they defined? •What statistical analysis was applied, and was it appropriate? •Are there problems that limit the validity of the study, either internally or with respect to generalizability to the case (e.g., choice of subjects, dropout rates, duration of drug treatment)? •Whether the data provide evidence of drug efficacy and/or toxicity that relate to this case?

Randomized Controlled Trial • Advantage: estimation of absolute and relative risk of efficacy or toxicity, determination of causality • Disadvantage: expensive, not feasible for outcomes with low incidence due to need for recruitment of large numbers of subjects • Limitations: choice of study population, drug regimen, end points, duration of study; comparator treatment; subject drop-outs; need for monitoring of drug use; generalizability of findings Interpretation of Data • Rate or proportion data - percent of subjects with outcome of interest compared with chi-square analysis - absolute risks, p, compared in treatment groups • Ratio provides 'relative risk', p1/p2 • Difference, p1-p2, provides 'absolute risk reduction' (ARR) • Number needed to treat NNT = 1/ARR • Continuous measurement data - Evaluated with analysis of variance 1.4%/2.90 = RR Greater mean effect of diclofenac than acetaminophen after 6 weeks in reduction of primary OA outcome measure (WOMAC Score) based on ANOVA Stratification of Patients by Baseline Disease Severity Efficacy of diclofenac and acetaminophen after 6 weeks did not differ in patients with mildest baseline OA (p>0.05) Greater efficacy of diclofenac than acetaminophen observed in patients with more severe disease (p<0.05) Potential Pitfalls - bias for publication of 'positive' findings, study search process, and selection for inclusion - heterogeneity of outcome - lack of detailed study information - method of data analysis • Advantage - greatly increased sample size Key Features of Valid Meta-Analysis (Cleveland Clinic Journal of Medicine, CME) • Well-defined objectives, including precise definitions of clinical variables and outcomes • An appropriate and well-documented study identification and selection strategy • Evaluation of bias in the identification and selection of studies • Description and evaluation of heterogeneity • Justification of data analytic techniques • Use of sensitivity analysis. • Advantages - study of rare outcomes - low cost and short study time relative to randomized control trial • Disadvantages - inability to establish causality, only an association - many potential biases - inability to determine absolute risk of the outcome from drug use, because subjects with the outcome are selected for the study Recall bias - Problem with self-report or interview - Reduced by use of data from automated collection sources or information recorded prior to outcome • Informationbias - Due to measurement error or misclassification - Differing assessment of outcome in case vs control group • Indicationbias - Drug given to those at higher risk of outcome, so appears drug causes outcome Case control: odds ratio Odds ratio = ad/bc, the 'cross-product' ratio 95% Confidence Interval of Odds Ratio • Determined by sample size in each cell - Larger the size, the smaller the confidence interval • Interpretation of 95% confidence interval - If includes 1.0, then no association of drug use with effect - If greater than 1, effect more likely with drug use - If less than 1, effect less likely with drug use E N E DU. A. B NDU C. D Odds ratio = ad/bc Relative risk = a/(a+b) / c/(c+d) first row over second row If b>>a and d>>c, then OR approximates RR Based on OR of 6.3 (2.4-16.4), daily acetaminophen use was associated with...Increased risk of ESRD, as 95% confidence interval of OR exceeds 1.0 Potential flaws to many case-control studies, including confounding by indication - Recommendation for use of acetaminophen rather than NSAID in patients with renal impairment - Association in case-control study does not prove causation! How does diclofenac compare to other NSAIDs with respect to GI toxicity? • Garcia Rodriquez, Gastroenterology, 132:498, 2007, Risk of upper gastrointestinal complications among users of traditional NSAIDs and COXIBs in the general population. • Astra Zeneca supported study • About 1500 cases of UGIC selected from UK The Health Improvement Network database of patient records on GP office computers; well defined criteria for UGIC identification; 10,000 controls matched for age, gender and calendar year • Characterization of drug exposure as 'current', 'recent' or nonuse and duration of use by defined criteria, based on prescription records to avoid recall bias • Strategies to avoid information bias (by validation of UGIC diagnosis), confounding by indication, and misclassification of drug use (computer-based records of prescriptions for NSAIDs) Major findings of Rodriguez 2007 case-control study • Current use of tNSAID associated with 3-4 fold increase of UGIC with lesser risk from COXIB • Concomitant use of aspirin with the COXIB eliminated superior GI safety • Risk shown to be dose dependent • Risk greater in RA than OA patients, partly due to higher dose used in RA • Is acetaminophen likely to be effective? • If not, can she tolerate oral NSAIDs? - Is there a history of 'aspirin intolerance syndrome'? - What are GI, CV, renal risks in the patient? • Use PPI to reduce GI risk, if GI history • Consider CV risks of tNSAIDs and COX2 inhibitors, if CV history • Does the patient achieve a therapeutic effect after titration up to the maximum NSAID dose? • If not, consider other approaches - Combination of acetaminophen and NSAID, topical NSAID, steroids (intra-articular injections), opioids, duloxetine (5HT/NE reuptake inhibitor)

The Time-Course of Plasma Concentrations: Single Dose and Multiple Dose Regimens

Relationship of Drug Dose to Plasma Concentration (Cp) and Therapeutic Effect • Single compartment distribution - Drug rapidly equilibrates into volume of distribution • First-order elimination - Drug is cleared at constant fractional rate - Kel is fraction eliminated per unit time Cp = Cpo e ^ -kelt Cp as a function of time Cp(t): Single Compartment with First-order Elimination Linearization of Cp(t) ln Cp = ln Cp0 - kel t y int: lnCpo slope: kel Elimination Half-life Time to eliminate 50% of drug from plasma Dose-independent under first-order conditions of elimination kel= .693/t1/2 Dependent on both Vd and ClT t1/2 = 0.693*Vd/Clt Estimation of Vd single compartment analysis - Administer dose iv • Collect plasma at various times • Measure drug concentrations (Cp) • Estimate Cp at time 0 (Cp0) by extrapolation • Compute Vd = Dose / Cp0 Determined from Cp vs time data after iv administration (see lecture notes for derivation) ClT = ( kel ) (Vd) Clt = .693/t1/2 * Vd How do ClT and Vd in a specific patient affect Cp vs t? Duration of Drug Action • When drug readily distributes to site of action to generate its effect, how is duration of effect related to drug dose? • Note that usually Cp increases with dose, but Vd, ClT and half-life do not change. ln Cp vs time for dose Y, 2Y, 4Y iv administration, 1st order elimination, one compartment distribution, rapidly reversible effect, no active metabolites Exponential increase in dose results in arithmetic increase in duration Doubling the dose increases the duration of action by one t1/2 3* t1/2 Nonlinear Kinetics • Dose-dependence in PK parameters • Saturation of enzymatically mediated clearance • Saturation of plasma or tissue binding site Exceptions to First-Order Elimination • ZEROORDERkinetics - constant reaction rate due to enzyme saturation - 50% biotransformation time increases with concentration - Cp vs time is linear without ln transformation • MIXED ORDER kinetics - Multiple elimination pathways with differing kinetics Distribution of Drugs Drug is administered into the body IV; rapidly distributes entirely into its Vd Drug concentration is calculated Some drugs behave as if the body is made up of two compartments Drug is administered IV and one compartment (V1; plasma and well perfused tissues) has a high concentration quickly, and then the drug distributes slowly into another compartment (V2) Cp (t) of Two-Compartment Model Cp = Ae - (at) + Be - (bt) Cp and Ctissue of Two-Compartment Model ln Concentration concentration in highly perfused tissue (V1) ln Cp concentration in poorly perfused tissue (V2) Duration of Action Dependent on Redistribution • For drugs with multi-compartment kinetics, duration of action depends on distribution into and out of compartment where target is located. • For lipophilic drugs that act in the brain, duration of action depends on redistribution out of brain to slowly perfused tissues. • Duration of action may be short, although elimination half-life is long. Cp is determines Cp?...BOTH absorption and elimination kinetics and the bioavailability Effect of absorption rate on Cp The faster the absorption rate: • the higher Cpmax • the earlier tmax Determination of Bioavailability (F) • Bioavailability (F) is the fraction of the Dose absorbed into the systemic circulation • The bioavailability by a specific route of administration of a specific formulation of a drug, made by a specific manufacturer, is determined by a clinical study of the drug's plasma concentrations • Endpoint data: the AREA under the plasma concentration vs time curve (AUC) Bioavailability (F), the fraction of the dose absorbed into the systemic circulation. Bioavailabilty = AUCpo / AUCiv x 100 Integral Cp = AUC = F (D / ClT) • ClT must be constant during study, so cross-over design used - each subject receives same drug dose in two arms of the experiment Absolute bioavailability: determined for new drug = AUC of noniv dose / AUC of iv dose • Relative bioavailability: determined for generic in comparison to proprietary formulation of drug = AUC of formulation1 / AUC of formulation2 Effect of ka and F on Cp via time above TC Comparison of iv and po Relative Magnitude of ka and kel • t1/2 elimination longer than t1/2 absorption • kel<ka • terminal slope of Cp(t) is same for iv and noniv route and reflects kel PO: elimionation longer than absorption halflife terminal slope of Cp(t) is shallower for noniv than iv route and reflects ka

Adrenergic Drugs Norepinephrine - NE (adrenergic receptors) NE a1, a2, b1, b2 Dopamine D1

Sym: B1 renal, B1 heart, a1 b2 smooth muscle NE Synthesis is from tyrosine -> catechol -> DA -> NE/E : Tyrosine LDOPA DA is Presynaptic, NE reuptake into MAO or by COMT a2 is on presynaptic rec. -Agonist Potency Defines Receptor Subtypes -α Receptor EPI > NE > PE > ISO α1&α2-adrenoceptors β1 & β2-adrenoceptors β Receptor ISO > EPI/ NE > PE B1: Cardiac Force B2: Tracheal Relaxation Alpha 1 Receptor Signal Transduction via PLC Beta Adrenergic Receptor Signal Transduction via Adenylyl Cyclase a1 agonist phenylephine: PLC+ DAG+ IP3+, vascular contraction, smooth muscle contraction, arterial resistance, less venous capacitance, intestinal smooth msucle relaxation,. mydriasis, glycogenolysis, glucongegensis a2 agonist clonidine: -cAMP: low insulin, low NE, low sym tone in CNS. platelet aggregation b1 agonist dobutamine: + camp = HR, RR, AV, Renin b2 agonist: albuteral +CAMP sm relax, glycogenolysis, D21 agonist dopamine cAMP dilated renal vcas Endogenous Adrenergic Agonists E: α, β non-selective, released from adrenal medulla into blood N: α1 = α2, β1 > β2, endogenous sympathetic neurotransmitter D: D1 specific, endogenous activator of β1 > α1 Selectivity refers to a drug's ability to preferentially produce a given effect • Selectivity arises from differences in potency at different receptors or tissues NE: a1a2>B1>b2 Isoproterenol:B1/B2 Adrenergic Agonists α1 selective: Phenylephrine α2 selective: Clonidine b1+ b2: Isoproterenol β1 selective: Dobutamien β2 selective: albuterol Selectivity is not usually absolute (nearly absolute selectivity has been termed "specificity"), and at higher agonist concentrations, a drug may interact with related classes of receptors. Low Conc: E= Relaxation High: A1 occupied bc more receptors exist Adrenergic agonist specificity is relative, not absolute. Agonist ability to selectively activate specific receptors depends on dosage Low doses, selectivity is maximal High doses, selectivity declines Major Cardiovascular Effects of Adrenergic Agonists B1: CF,HR,ReninR B2: Vasodilation, low TPR A1: Vasoconstriction, high TPR Physiological Effects Dependent on - Relative potency for receptor subtype - Agonist dose - Receptor distribution determines balance between α1 vasoconstriction and β2 vasodilation - Impact of baroreflex Norepinephrine - α1 vasoconstriction = increased MAP = increased resistance - β1 effect should increases HR - Increased MAP triggers vagal-mediated decrease in HR - β2 effect on blood vessels not observed as α1 receptors predominate - Sympathoinhibition requires CNS activation, when given peripherally poor CNS penetration due to polarity also short half life/rapid metabolization E: Moderate dose - α1 vasoconstriction + β2 vasodilation almost equal = stable MAP - β1 effect increases HR - β2 vasodilation reduces resistance NE: - α1 vasoconstriction + β1 cardiac effects increase MAP - Baroreflex decreases HR - α1 vasoconstriction increases resistance Isoproterenol:- β2 vasodilation outweighs β1 cardiac effects to reduce MAP - β1 increases HR - β2 vasodilation reduces resistance Therapeutic Uses of Adrenergic Agonists • Nasal decongestants (a1; phenylephrine: counters dilation and engorgement of nasal mucosa blood vessels) • Cardiovascular - Extend local anesthetic action (a1; epinephrine: vasoconstriction reduces drug absorption) - Resuscitation after cardiac arrest (mainly a1, b1; epinephrine) - Antidote to hypotensive agent overdose (a1: vasoconstriction) - Maintaining blood pressure in spinal damage or anesthesia (a1: vasoconstriction, usually in combination with fluids) - Restoring blood pressure in cardiogenic shock (DA, a1) - Treatment of hypertension (a2; clonidine, not a first-line therapy) • Ophthamology - Mydriasis (a1 dilation: epi, phenylepinephrine) - Glaucoma (a2, b1 reduce fluid generation, a2 increase fluid outflow) • Allergic - Asthma (b2) mediated bronchial smooth muscle relaxation e.g. albuterol - Anaphylactic shock (a1, b1, b2) β2 relaxes airway smooth muscle and inhibits mast cell release of anaphylaxis mediators α1 maintains MAP • Other uses - Obstetrics: Delay premature labor (b2 relaxation of uterine smooth muscle) - Treatment of opioid withdrawal (a2) - TreatmentofAttentionDeficitDisorder(amphetamine, methylphenidate) Anaphylactic shock Anaphylaxis, a life-threatening allergic reaction caused by severe hypersensitivity to insect venoms (e.g., bee), certain foods (e.g., peanuts), and certain drugs (especially penicillin). Acute Tx - Epinephrine as activates α & β receptors α1 receptor vasoconstriction increases blood pressure and suppresses glottal edema β1 receptors increases cardiac output, thereby helping elevate blood pressure β2 receptors counteract bronchoconstriction Toxicity & Side Effects of Adrenergic Agonists • Vasoconstriction,ischemia(a1) • Hypertensive reactions, can cause cerebral hemorrhage (a1) • Reboundnasalcongestion(a1) • Withdrawalsyndrome(a2agonists) • Tachycardia(b1) • Myocardialischemia(b1) • Cardiacventriculararrhythmias(b1) • Increased risk of asthma exacerbation with extended use of long-acting b-agonists (b2) • Hyperglycemia (Liver + skeletal b2 activation converts glycogen to glucose) • CNSstimulation(mechanismunknown) Indirect-acting Sympathomimetics : increase availability of endogenous adrenergic transmitters: Octopamine, Amphetamine, Methylphenidate, Cocaine. Indirectly acting adrenergic agonists Tyramine, ephedrine, amphetamine (increase NE release) Tyramine (increase NE release) Present in food e.g. cheese, red wine Degraded by MAO (monoanimine oxidase) - when MAO is inhibited can trigger hypertensive crisis by release of NE from nerve terminals LONG TERM Tyramine converted to octopamine and stored in synaptic vesicles replacing NE. Octopamine is released but has little action at adrenergic receptors. Long term MAO therapy can impair function of sympathetic nervous system Indirectly acting adrenergic agonists Tyramine, ephedrine, amphetamine (increase NE release) • ADHD medications e.g. dextroamphetamine, methylphenidate - Evoke NE and DA synaptic release following oral administration - Also directly stimulate α + β1 • Ephedrine - Releases NE and directly activates α + β receptors, present in many "nutritional supplements" - FDA restricted use due to adverse effects Pseudoephedrine - widely used over counter decongestant, similar to ephedrine Indirectly acting adrenergic agonists Tricyclic antidepressants, Cocaine (prevent NE and DA reuptake) both centrally active Direct neuronal blockers Reserpine - rarely used - Depletes NE stores by inhibiting vesicular monoamine transport (VMAT) within synapse. NE not transported into pre-synaptic vesicle for release, NE metabolized by intra-neuronal MAO Alpha Adrenergic Antagonists a1 a2 phentolamine a1 a2 irreversible phenoxybezamine a1 selective prazosin a2 selectibve yohimbine Therapeutic Uses of a-Antagonists • Treatment of hypertension • Treatment of pheochromocytoma (b-blockers may also be used, but only after blocking a1-receptors) • Treatment of Raynaud's Disease (reduce digital vasospasm) • Treatment of heart failure (reduce vascular resistance) • Treatment of benign prostatic hyperplasia (BPH) - decreases bladder smooth muscle tone • Antidote to a-agonist vasoconstriction agonists • Postural hypotension - due to inhibition of α-mediated vasoconstriction - Reflex tachycardia - Myocardial ischemia (increased O2 demand from reflex tachycardia) - Salt & water retention (reflex β1 stimulation of renal RAS system) - Peripheral edema • GI stimulation (abdominal pain, nausea) • Inhibition of ejaculation Beta Adrenergic Antagonists b1 b2 :( proranol b1 selective :( metorpolol b1 b2 a1 carvedilol b1 and b2 pinodolol b1 short esmolol b1 b2 a1 labetalol b b b 1& 2 - propranalol 1 - Atenolol • Treatment of cardiac arrhythmias • Reducing mortality after myocardial infarction • Shock - maintains cardiac output and tissue perfusion • Treatment of heart failure, increases cardiac output (carvedilol FDA approved) • Treatment of hypertension (reduce cardiac output, inhibit renin production) • Treatment of angina (to control O2 demand) • Treatment of hyperthyroidism (propranolol) • Reducing anxiety ("stage fright") • Migraine prophylaxis • Treatment of open-angle glaucoma Protective Effect of b-Blockers after MI Protective effect of carvedilol in heart failure Toxicity & Side Effects of b-antagonists • Bronchoconstriction • Bradycardia • Withdrawal:exacerbationofangina,riskofsudden death • Fatigue • Coldextremities • Potentiation of epinephrine vasoconstriction • Precipitation of congestive heart failure (via inhibition of adrenergic compensation) • Possible predisposition toward new-onset diabetes

Environmental Pathology Definition: Study of poisons (distribution, effects, mechanisms of action) Toxic agents enter through skin/lung/GI tract → bloodstream Chemical Agents (Xenobiotics) Air Pollutants Heavy Metals Organic Chemicals Tobacco Alcohol Physical Agents - Type of Injury Mechanical Thermal Electrical Radiation Not included: infectious agents, firearms, drugs, nutrition Chemical Agents (Xenobiotics) Air Pollutants o Outdoor: sulfur dioxide, carbon monoxide, ozone, nitrogen dioxide, particulates o Indoor: formaldehyde, asbestos fibers, radon Heavy Metals - lead, mercury, arsenic, cadmium Organic Chemicals o Volatile: chloroform & carbon tetrachloride, benzene & 1,3-butadiene o Manufactured: organochlorines (polychlorinated biphenyls, dioxin, DDT, lindane), vinyl chloride monomer, phthalate esters, bisphenol A Tobacco - nicotine, polycyclic hydrocarbons, nitrosamines, tar, formaldehyde, nitrogen oxides Alcohol aldehyde, NAD Physical Agents - Type of Injury Mechanical - abrasion, contusion, laceration, incised wound, puncture wound Thermal - burns (superficial, partial thickness, full thickness), hyperthermia (heat cramps, heat exhaustion, heat stroke), hypothermia [systemic (mild, moderate, severe), local (frost bite, trench foot)] Electrical - burns, ventricular fibrillation or cardiac & respiratory center failure, paralysis of medullary centers Radiation o Ionizing (X-rays, gamma rays, particulate) mutations carcinogenes., teratogenesis o UV - sunburn ( wrinkles, cataracts), mutations (pyrimidine dimers melanoma) o Electromagnetic field (radiowaves, microwaves) burns, cancer?

XENOBIOTICS : CYPs = cytochrome P-450 enzymes (ROS byproducts) TO ELIMINATION OR REACTIVE METABOLITE CAUSING TOXICITIY Outdoor air pollutants Smog = smoke + fog Major constituents (in US, upper safety limits set by the Environmental Protection Agency): Sulfur dioxide Carbon monoxide (CO) Ozone (O3, ground-level) Nitrogen dioxide Particulates Major source - combustion of fossil fuels (gasoline, oil, coal, wood, natural gas) by motor vehicles, power plants, factories, homes Respiratory irritants, cause lung damage/inflammation, especially in asthmatics Toxicity mechanisms of some air pollutants Ozone (O3, highly-reactive oxidant) → free radicals → damage to lung epithelium, inflammation) Carbon monoxide (CO) - colorless, tasteless, odorless gas (from burning carbon-containing materials), rapidly oxidized to carbon dioxide (CO2). CO is highly dangerous in close proximity, e.g., automotive fumes in closed garage) → HbCO (carboxyhemoglobin, blocks binding of oxygen) → hypoxia → CNS depression, heart damage, death (mild CO poisoning - dizziness, confusion, headache) Particulates - fine and ultrafine (< 10 μm diam.) → phagocytosed by alveolar macrophages & neutrophils → release of inflamm. mediators (> 10 μm diam. removed by nose) → heart rate irregularities (in severe cases) Formaldehyde (a gas) Foam insulation, glues, wood products Asthma, eye/nose/throat irritation, contact dermatitis, nasopharyngeal cancer (rarely) Asbestos fibers (silicate minerals) Insulation, floor & ceiling tiles, before banned in 1970 Mesothelioma (cancer of lining of pleural or abdominal cavity), lung fibrosis, lung cancer Radon (radioactive gas derived from uranium, greatest single source of background radiation) Soil (high conc. in some basements), uranium mines Lung cancer Lead Water, lead paints (banned in US 1978), leaded gasoline (banned in US 1996) Hematologic, skeletal, neurologic, GI, renal; children > adults (>50% vs ≤15% absorption) Binds to sulfhydryl groups in proteins & blocks Ca metabolism Mercury Contaminated fish (methyl mercury), dental amalgams (metallic mercury vapor) Tremors, confusion, mental retardation (if in utero exposure), death (if high doses) Binds to sulfhydryl groups in some proteins, especially in CNS & kidney Arsenic (historical murder weapon) Soil, water, wood preservers, herbicides Acute GI, cardiovasc. & CNS damage, death (if high amt. ingested); hyperpigmentation & hyperkeratosis; lung, bladder & skin cancers Trivalent arsenic replaces phosphates in ATP → inhibits mitoch. oxid. phophoryl'n Cadmium Nickel-cadmium batteries → water, soil, food Obstructive lung disease, kidney damage, lung cancer (from occup'l exposure) Increased ROS production? Leadlines, death 150, less blood protein, less iq at 10 ug/ml Ground water arsenic poisoning Organic chemicals - occupational & consumer 1) Volatile organic compounds Examples - chloroform, carbon tetrachloride, found in degreasing agents, e.g., dry-cleaning agents, paint removers - benzene, 1,3-butadiene, used in manufacturing of plastics, lubricants, rubbers, dyes Route of exposure - mainly lung exposure Toxic effects - headache, dizziness, impaired liver & kidney function Organic chemicals - occupational & consumer 2) Manufactured products Route of exposure - mainly through skin, GI or respiratory tracts Organochlorines 1. Polychlorinated biphenyls (PCBs) 2. Dioxin (TCDD) 3. DDT*, Lindane 1. Old capacitors, transformers (banned 1977) 2. Incomplete waste inciner. 3. Pesticides (*banned1973) Chloracne, rashes, liver damage; endocrine disrupter, probable carcinogens / → binding to aryl hydrocarbon receptor on cells → transcriptional changes Vinyl chloride monomer (VCM) - gas with sweet odor) Manufacturing (pre- regulation) of polyvinylchloride (PVC) products (pipes, flooring); improper PVC incineration Liver angiosarcoma / ? Processing of metabolites in liver Phthalate esters (used as plasticizers) Flexible plastics (shower curtains, toys, adhesives, catheters) Reproductive toxin in animals ? Human effect Bisphenol A (BPA)? Polycarbonate bottles, coatings of food cans Estrogen mimic (proliferative effect) Chloracne An acne-like eruption, with cysts, hyperpigmentation, and hyperkeratosis; from exposure to chlorinated organics (a.k.a organochlorines). Two incidents (of ~2000 people each) of PCB poisoning in late 1960s in Japan and China, from exposure to contaminated cooking oil → chloracne (Yusho disease in Japan; Yu-Cheng disease in China) 14 Tobacco Cigarette (& cigar & tobacco pipe) smoking causes 1 in 5 deaths/year in the US & 90% of lung cancers (including second hand smoke). Life expectancy for smokers is 10 years less than for nonsmokers (lung cancer, cardiovascular disease, chronic respir. disease). Quitting smoking before the age of 40 reduces the risk of dying from smoking-related disease by ~90%. Smoking during pregnancy → spont. abortion, preterm births↑ birth weight↓ Tobacco cigarette smoke contains > 4000 chemicals, including many toxic agents Toxicity mechanisms of tobacco Nicotine [an alkaloid (organic nitrogen-containing base)] - addictive → nicotinic acetylcholine receptor in brain → catecholamines↑ → heart rate, blood pressure, cardiac output↑ Polycyclic hydrocarbons, nitrosamines - carcinogens Many smoke components (tar, formaldehyde, nitrogen oxides) → inflammation, mucus production (bronchitis), neutrophil recruitment to lung↑ → elastase↑ → tissue injury → emphysemaEmphysema, an irreversible lung disease, results from destruction of elastin in alveolar walls; patients have difficulty breathing; Chronic obstructive pulmonary disease (COPD) = chronic bronchitis, emphysema Alcohol (ethanol) Mechanism of alcohol toxicity Alcohol is metabolized mainly in the liver, but also in brain and GI tract. Alcohol metabolism decreases NAD and generates toxic acetaldehyde: NAD is required for fatty acid oxidation fat accumulation; acetaldehyde is a carcinogen (interferes with DNA replication and repair), contributes to incoordination, memory impairment, sleepiness, and can cause facial flushing, nausea, rapid heart beat. Genetic variations: variants of the ADH and ALDH enzymes in different individuals determine the acetaldehyde level and hence the level of 'tolerance' to alcohol. Fast ADH and/or slow ALDH protect against alcoholism (e.g., slow ALDH variant in 50% of Asians). Health effects of heavy alcohol consumption Exhaled alcohol is proportional to blood level Breath Test - 80 mg/dL blood = drunk driving in the U.S. 200 mg/dL - drowsiness 300 mg/dL - stupor (greatly diminished responsiveness) Higher levels - coma with possible respiratory arrest Heavy alcohol consumption accidents, homicides, suicides, personal violence. Acute alcoholism: Mainly CNS effects [depression of neuronal centers, including those that regulate respiration (respiratory arrest at very high alcohol levels)] Sometimes reversible liver and stomach damage, e.g., fat accumulation in hepatocytes (fatty change), acute gastritis & ulceration Chronic alcoholism: ~15 million chronic alcoholics in the U.S. Liver and GI tract: Fatty change, alcoholic hepatitis, cirrhosis (loss of hepatocytes and irreversible scarring) portal hypertension large swollen veins (varices) in GI tract, which may rupture life-threatening complications; risk of liver cancer; cognitive decline due to poor liver function; also bleeding from gastritis or gastric ulcers. Cardiovascular system: injury to myocardium alcoholic cardiomyopathy Fetal alcohol syndrome (from consumption of even small amounts during pregnancy, esp. in 1st trimester): microcephally, facial abnormalities, growth retardation, reduced mental functioning; affects ~3 per 1000 children born in US. Mechanical trauma (not included: head & bone injuries) Leading cause of death in the 1- 44 yr. age group Trauma occurs when a force of sufficient magnitude is applied to the body. The force can be: accelerating or decelerating blunt or sharp accidental or deliberate Soft tissue injury types: abrasion (scraping or rubbing) removal of superficial layer contusion (bruise from blunt object) extravasation of blood into tissue laceration (tear or disruptive stretching from blunt object) intact bridging blood vessels incised wound (from sharp object) severed bridging blood vessels puncture wound (pierced tissue from penetration of long & narrow sharp object) - penetrating or perforating (if exit wound also formed) Determinants of the clinical significance of a thermal burn Depth of burn Percent of body surface involved Internal injuries from inhalation of hot & toxic fumes Promptness & efficacy of therapy Classification of Burns by Depth Superficial (formerly 1st degree) Limited to epidermis Red, dry, painful Partial Thickness (formerly 2nd degree) Epidermis + part of dermis destroyed Red, moist (blisters), painful Full Thickness (formerly 3rd and 4th degree) Epidermis + dermis destroyed Anesthetic Not capable of regeneration High risk of infection Thermal injury - hyperthermia Heat Cramps - from loss of electrolytes via sweating Heat Exhaustion (most common) - prostration (extreme physical weakness) and brief period of collapse (due to hypovolemia caused by dehydration); rehydration recovery Heat Stroke (most serious) • Due to high ambient temp/humidity cessation of sweating • Advanced age, physical stress are major risk factors Prolonged core temp > 40oC (104oF) Peripheral vasodilation Reduced blood flow to brain/heart Confusion/coma/death Thermal injury - hypothermia • Prolonged exposure to low ambient temp • Homelessness is major risk factor Effects of systemic hypothermia (drop in core temp) Mild 32-35oC (90-95oF) Hyperventilation Lethargy Vigorous shivering Moderate 28-32oC (82-90oF) Hypoventilation Confusion Loss of coordination Severe <28oC (<82oF) Unconsciousness Slow or no breathing Weak or no pulse Effects of local hypothermia (e.g, frostbite, trench foot) - Suppression of vital metabolism - Crystallization of intra- & extracellular water - Increased permeability of vessels - Ischemia, hypoxia, infarction "Trench foot" - gangrene Electrical injury From contact with low-voltage current (home, workplace = 120 or 220v, alternating; wet skin lowers resistance 100-fold) or high-voltage currents (high power lines, lightning) Severity of injury is determined by: type and intensity of current path of current resistance of tissues duration of exposure Types of injuries: Burns - at entry & exit sites & internal organs ventricular fibrillation or cardiac & respir. center failure (due to disruption of normal electrical impulses) Alt. current tetanic muscle spasm irreversible clutching; severe spasm of chest wall muscle asphyxia High-voltage current paralysis of medullary centers & extensive burns 29 Radiation injury types of radiant energy - sources / uses Electromagnetic radiation (wave-propagated) Radio (RF - radio, TV, cell phone, WiFi, radar), microwave Infrared (IR, felt as heat, most encountered), visible Ultraviolet ( - UVR from sun) X-rays [from firing electrons at a metal target - X-ray imaging, computed tomography (CT) scans (computer-processed combination of many X-ray images)] Gamma rays ( - cancer cell killing, sterilization) Particulate radiation Alpha particles (2 protons & 2 neutrons - smoke alarms) Beta particles (electrons - paper & aluminum foil production) Neutrons (react with nuclei of other atoms new isotopes) EM spectrum Sensitivity of Cells/Tissues to Ionizing Radiation Sensitivity level High (< 25 Sv *) Lymphoid and hematopoietic cells, germ cells, GI epithelium Moderate Skin, blood vessels, squamous epithelium, growing bone and cartilage Low (> 50 Sv) Kidney, muscle, brain, endocrine organs, adult bone and cartilage * Sievert (Sv) = unit of equivalent biologic dose of radiation = Gray (Gy, energy absorbed by a tissue per unit mass) x the relative biologic effectiveness of the radiation. Injuries Caused by Ionizing Radiation Vascular damage Inflammation Fibrosis (especially lungs radiopneumonitis) Acute - short-lived, reversible • erythema • pigmentation • depletion of Langerhans cells Long-term - cumulative, largely irreversible • degeneration of elastin and collagen wrinkled and leathery skin • cataracts Melanoma: neoplastic: UVB pyrimidine dimers in DNA mutations or transcriptional errors cancer Research: Dose-related risk: high levels cause burns and possibly cancer; main global concern is with long-term low level exposure Based on > 25,000 published studies, consensus is little or no risk except for "prolonged and heavy" mobile phone use (? brain cancer) The World Health Organization has classified cell phones as "possibly carcinogenic to humans" - long term studies needed.