Block 6 SFM (+HSS) - Marcus

How is (3'-5') cAMP generated?

*Adenylyl Cyclase* synthesizes cAMP from Mg-ATP (3'-5' bond formed); terminal 2 P leave (PPi), and a link is formed between the 5' hydroxymethyl group and the 3' phosphate.

Current Breast Cancer Guidelines for the Woman at *Average Risk*

*Age 40-44:* women have the choice to start annual screening if they like *Age 45-54:* mammograms every year *Age 55+:* mammograms every 2 years Screening should occur as long as a woman is in good health (and is expected to live 10+ years). All women should be familiar with the known benefits, limitations, and potential harms associated with breast cancer screening. They should also be familiar with how their breasts normally look and feel and report any changes immediately.

Heterochronic Parabiosis Experiments

*Aging can be modified by physiologic factors.* *Experiment:* Two animals are joined together -- shared circulation *Result:* -Old rats lived longer when joined to young rats -Young rats did not have reduced longevity *Interpretation:* Result is mediated by the transfer of stem cells (which become senescent in old age).

Checkpoint Blockage Immunotherapy

*Antibodies that Block Inhibition:* -Anti-PD-1 (T cell) -Anti-PD-L1 (tumor cell) -Anti-CTLA-4 (T cell) *Result:* -Allows for activation of CTLs (TCRs are able to bind MHC-I-presented antigens from tumor cells) -Prevents T cells from becoming anergic -Significant increase in antigen-specific immune responses in the periphery and CD8+ T cell infiltration into the tumor

Cyclin Dependent Kinase Inhibitors

*CDKIs are key regulators of CDKs, apoptosis, and cell differentiation.* -CDKIs usually bind to cyclin/CDK complexes, regulating CDK activity. -CKIs are necessary for CDK/cyclin complex formation (inhibit at high concentrations) Two classes of CKIs: KIP and INK *1. KIP (G1/G2/M)* *p21 targets: D, E, A, B (G1/G2/M)* ← p53-activated p27 targets: D, E (G1/G2) p57 targets: D, E (G1/G2) *2. INK (G1)* p15 targets: D/4,6 (G1) *p16 targets: D/4,6 (G1)* p18 targets: D/4 (G1) p19 targets: D/4 (G1)

*Anti*cholinergic Poisoning *ANTI* = "can't" -*Can't* see (dilation) -*Can't* spit (dry mouth) -*Can't* pee (bladder sphincter not relaxed) -*Can't* shit (sphnicter not relaxed) -Hot (hyperpyrexia) -Dry (reduced salivation) -Tachycardia: ACh block of parasympathetic -Wacky (agitated; seizures; coma = CNS) -BP not affected (muscarinic)

*Can't* see, spit, pee, shit (rhyme); hot, dry, tachy, wachy. *Causes:* -Anti-histamines (Benadryl, H1 agonist) -Jimsonweed -Atropa Belladonna (atropine) (Olive Oil's eyes widen, dilation). *Mechanism:* Blocks muscarinic receptors *Treatment:* Physostigmine. -*Blocks ACh-ase to increase ACh present.* -Contraindicated for wide QRS → flatline.

Acute Rejection (weeks to months)

*Cell-Mediated* -Inflammation caused by CD4+ cytokines and CTL killing of graft parenchymal and endothelial cells -Infiltration and accumulation of lymphocytes and MØ *Antibody-Mediated* Alloantibodies against HLA formed after graft contribute to parenchymal and vascular injury (complement activation)

Poisoning Treatment Overview

*Cholinergic* -Pralidoxime (paralysis): reverses Ach-ase phosphorylation -Atropine (all others): block muscarinic receptors *Anti-cholinergic* -Physostigmine: blocks ACh-ase to increase ACh present *Amphetamines* -Benzodiazepines (e.g., Valium) *Beta blockers* -"-olol" drugs for hypertension -Glucagon: binds glucagon receptors on heart and triggers cAMP production to open Ca2+ channels → Ca2+ cross links actin/myosin to increase heart rate (muscle contraction).

Mutation Accumulation Theory

-Mutations that kill early in life are selected against -Mutations that kill late in life will not be selected against because they exert an effect after procreation (e.g., Huntington's)

Somatic Neuron Pathways

-One neuron pathway -Cell body in CNS, axons in PNS -All somatic motor neurons are cholinergic (secrete ACh as their neurotransmitter). -Ach binds to cholinoreceptors on the effector organ (which can be *nicotinic* or muscarinic) -In *somatic* pathways, the effector organ is *skeletal muscle*, so the receptor is *nicotinic*.

Poisoning Overview

*Cholinergic:* inhibit ACh-ase (too much ACh on muscarinic receptors); *SLUDGE, twitchy * *Anti-cholinergic:* blocks muscarinic receptors; *can't see, spit, pee, shit; hot, dry, tachy, wacky* *Amphetamines:* stimulates NE release (acts on a1/b1); *high everything (↑BP/HR)* *Beta blockers:* block beta receptors; *low everything (↓BP/HR)*

Chronic Rejection (months to years)

*Chronic allograft vasculopathy*: arterial occlusion or arteriosclerosis → fibrosis and atrophy → intimal smooth muscle thickening/proliferation

Cocoon vs. Grandmother Hypothesis -Both suggest that aging is non-adaptive (it's good that aging exists)

*Cocoon:* older organisms are less locomotive, so less subject to hazards that would account for selection *Grandmother:* older organisms have value in promoting the survival of younger organisms

Mechanisms of Alloantigen Recognition by T cells

*Direct Allorecognition:* -T cells of the transplant recipient recognize *intact, unprocessed donor MHC molecules plus peptide* on the surface of DCs in the graft -CD8+ T cells recognize MHC-I → activate CTLs → destroy graft tissue -CD4+ T cells recognize MHC-II → Th1 cells → IFNg → MØ damages tissue via inflammation *Indirect Allorecognition:* -T cells of a transplant recipient recognize donor *MHC molecules that have been processed into peptides by recipient DCs* in the graft. -CD4+ T cells recognize graft antigens on host APCs that entered the graft → activate B cells to produce antibodies against that antigen → endothelial injury delayed hypersensitivity -CD4+ cells → Th1 → IFNg → MØ damages tissue via inflammation

Dosing Rate Equation

*Dosing/Infusion Rate = (CLt)(Css)* CLt = total clearance rate Css = desired steady state concentration

Ganglion blockers Effects: 1. Heart → tachycardia (sympathetic) 2. GI Tract → constipation/dry mouth (sympathetic) 3. Vascular Smooth Muscle → orthostatic hypotension (dilation to accommodate ↑blood pumping → drop in BP)

*Drugs that block all of the nicotinic receptors of the autonomic system (of post-ganglionic neurons).* Blocks ACh from binding for depolarization (hypopolarization). All post-ganglionic neurons and Chromaffin cells of the adrenal medulla have nicotinic receptors, when the receptors are blocked *the entire autonomic nervous system is blocked*. The result of administering a ganglion-blocking drug is the same as blocking the dominant system (below)

Graft vs. Host Reactions *Destroyed Epithelia:* -Skin -Liver -Intestines *This can be avoided by HLA matching,* *T cell depletion before the graft, and immunosuppression.*

*GVHD:* major complication in hematopoietic stem cell transplantation 1. *Acute: occurs within 60 days of transplant* -*Destruction of epithelial cells* in the skin (rash), liver (jaundice), and GI tract (diarrhea) by CD8+ CTLs, NK cells, and cytokines. -Extensive epithelial cell death = mortality in recipient (15-40%) 2. *Chronic: occurs >100 days after transplant* -Increased fibrosis (not as fatal as acute) *Cause:* Donor T cell recognition of recipient alloantigens (donor cells attack graft recipient who is usually immunocompromised) *Mechanism:* 1. Immunocompetent lymphoid cells are transplanted into individuals who are immunologically compromised (e.g., following high-dose chemotherapy). 2. Mature T cells (donor) may recognize MHC molecules or minor histocompatibility antigens of the recipient as foreign 3. Mature T cells (donor) proliferate and progressively cause damage to host tissues and cells

IGF-1 -Protein + calories → ↑IGF-1 → shorter "doubling" times of chondrocytes in epiphyseal plate

*Growth factor responsible for chondrocyte proliferation and hypertrophy in the epiphyseal plate.* Knockout studies showed that IGF KO leads to a longer time in G1. This *reduces proliferation (longer doubling time)*, leading to short stature due to *decreased cell number* rather than decreased cell size. Influenced by caloric and *protein content* of diet (poor diet leads to less IGF-1 and chronic stunting) Small dogs have IGF-1 SNP mutation; absent in giant breeds.

HER Family Receptors LBD: ligand binding domain TKD: tyrosine kinase domain

*HER1:* has LBD; has TKD (EGFR) *HER2:* no LBD; has TKD (preferred dimerization partner) *HER3:* has LBD; no TKD *HER4:* has LBD; has TKD

Types of Graft Rejection

*Host vs. Graft* *1. Hyperacute: Type II Hypersensitivity* -Preformed antibodies against donor tissue (e.g., ABO) -Immediate *2. Acute: Type IV Hypersensitivity* -T cell-mediated response against foreign MHC -Antibody-mediated response against foreign MHC (takes time to produce antibodies against the foreign MHC) -Occurs within weeks to months *3. Chronic: Type III/IV Hypersensitivity* -T cell-mediated process resulting from foreign MHC looking like self MHC carrying an antigen -*Intimal thickening and fibrosis of graft vessels* -Graft atrophy -Occurs within months to years *Graft vs. Host* (Type IV Hypersensitivity) -Donor T cells in graft proliferate and attack the recipient's tissues -Common in bone marrow transplants (immunocompromised patient) -Diarrhea, rash, jaundice

Control of Vascular Smooth Muscle of Skeletal Muscles

*Initial:* a1: contraction of smooth muscle to maintain BP via baroreceptor reflex (NE) *Fight-or-Flight* b2: vasodilation of the vasculature that feeds skeletal muscle (E) (overrides a1)

Routes of Drug Administration

*Intravenous (IV)* -Immediate entry into vascular compartment -*Rapid effects* -Not suitable for oils and substances with poor solubility *Intramuscular* -Rapid absorption for aqueous forms -Slow and sustained absorption for repository drugs -Some substances can cause muscle injury or abscess *Subcutaneous* -Rapid absorption for aqueous forms -Slow and sustained for repository drugs -Can only be given in small volumes (in a bolus under the epidermis/dermis) *Oral* -Variable absorption -Bioavailability can be low and erratic (might need to give extra for intended effect)

The principal mechanism of tumor elimination is...

*Killing by CTLs specific for the tumor antigens* CD8+ T cell responses to tumors may be induced by antigen cross-presentation, in which the tumor cells or antigens are taken up by DCs, processed, and presented to CD8+ T cells, which become activated, migrate to the site of the tumor, and kill.

Pharmacokinetics *LADME*

*Liberation*: drug leaving the form in which it was administered (e.g., pill) *Absorption*: across skin / GI tract to enter blood *Distribution*: to various compartments (e.g., blood, ECF, across BBB) *Metabolism*: how is the drug broken down? *Excretion*: bile/poop or urine

Disposable Soma Theory -Why commit resources to something if its duration of usefulness is limited?

-Sexual reproduction comes at the expense of longevity -*Antagonistic pleiotropy:* a gene or gene having 2 effects: 1 beneficial; 1 detrimental (e.g., *testosterone* increases fitness and reproductive ability in early life, but predisposes to prostate hypertrophy and cancer later in life) Lack of evidence: calorie restriction is helpful

Hyperacute Rejection (immediate)

-Thrombosis and occlusion (by complement and blood clotting cascades) of graft vessels occurs within minutes-hours (during surgery) -*Preformed antibodies* to incompatible MHC antigens (or ABO carbohydrates, etc.) result in the destruction of the transplant. -Preformed antibodies can be the result of previous transplants, blood transfusions, or pregnancies -*Cytotoxic antibodies activate complement and clotting cascades, occluding vessels*

Transplantation

-Treatment for end-stage organ failure -Immune response against transplant is the major barrier to successful transplantation -Treatment: *Suppress immunity against transplant*

Erb-b -Non-receptor tyrosine kinase (not regulated)

-Truncated (non-regulated) EGFR with *no hormone-binding domain*. -Has intact tyrosine kinase function (intracellular). -Constitutively active (doesn't require EGF for activity). *Unregulated.* -Mutations in Erb B family are found in high frequency in many breast cancers.

Components of Aging

-Universal -Depends on intrinsic factors -Progressive -Deleterious

Relationship between donor and recipient in transplant

1. *Autograft*: graft or transplant from one area to another on the *same individual* -The graft is *autologous* (self) and no immune response is induced against it -The graft will survive 2. *Isograft*: graft or transplant from one individual to another individual who is *synergistic* (genetically identical) to the donor. -Synergistic at MHC and all other loci (*histocompatible*) -Donor's tissue is recognized as "self" 3. *Allograft*: graft or transplant from one individual to an *MHC-disparate* individual of the *same species* -Allogenic transplant will result in rejection due to histoincompatibility 4. *Xenograft*: graft between a donor and a recipient from a different species (histoincompatible) -Transplant is recognized as foreign, and an immune response will be mounted to destroy it

Chemoprevention in BRCA2

1. *Tamoxifen:* modulates *estrogen receptor* and prevents estrogen gene transcription (reduces risk for second breast cancer in BRCA+ women) 2. Raloxifene: reduced risk of breast cancer in post-menopausal women 3. *Herceptin (Trastuzumab):* Her-2/neu positive cancers (tyrosine kinase growth factor receptor)

Graft Rejection Process

1. *Transport of alloantigens to lymph node (via APCs from graft site)* -Direct: donor APC with MHC and antigen -Indirect: host APC with donor alloantigen presented 2. *Activation of host T cells*; generation of effector T cells by direct and indirect antigen presentation 3. *Migration of effector T cells to allograft (both CD4+ and CD8+)* 4. *Once in the graft:* -CD8+ T cells: *CTLs kill graft tissue cells* -CD4+ T cells: damage by *cytokine-mediated inflammation* (IFN→ MØ)

Strategies for enhancing anti-tumor immune responses

1. Active immunization 2. Passive therapy with antibodies 3. Antibody-drug conjugates (chemo drug, radioactive particle, or toxin) 4. Adoptive transfer of effector cells

Drug Behavior

1. Agonists: mimic things that normally activate the receptor 2. Primary agonists: bind to the same site on the receptor as its endogenous agonist 3. Allosteric agonists: bind to a different site as the endogenous agonist 4. Antagonists: may bind to the agonist site or an allosteric site 5. Chemical antagonist: interacts with the endogenous agonist to block its action (e.g., antibodies that bind adrenaline, blocking its function)

Introduction to Signal Transduction (Tyrosine Kinases)

1. Cancer can be transmitted by viruses 2. This occurs via introduction of viral genes (oncogenes) into host cells 3. Oncogene protein products can induce malignant transformation 4. Oncogenes have normal cellular homologues (proto-oncogenes)

CDK activity regulated by

1. Cyclin content (needs a binding site) 2. CDKI content (too much can inhibit) 3. Phosphorylation status (needs P for activity)

RTK Signalling Mechanisms (important!) RTK = receptor tyrosine kinase Mechanisms Dissected below: know the mechanism, not necessarily the specific examples of each

1. Direct activation of signaling enzyme by tyrosine phosphorylation. (e.g., Phospholipase Cγ) 2. Conformational change of an enzyme's regulatory subunit leads to localization and activation of catalytic subunit. (e.g., PI3K) 3. Formation of a multisubunit signaling particles that activate small G-Proteins. (e.g., SOS/Grb-2 and Ras) 4. Direct activation of transcription factors (e.g., JAK/STAT)

Likelihood Ratios *Positive*: how much *more likely* a condition is given a *positive test* *Negative*: how much *less likely* a condition is given a *negative test* Reminder: Sensitivity = TP/TP+FN Specificity = TN/TN+FP

*Likelihood Ratios are FIXED and do not change with prevalence. You must start with an estimation in mind for the ratio to be of use.* *LR+* = sensitivity/(1-specificity); LR+ >10 (good test) *LR-* = (1-sensitivity)/specificity; LR- <0.1 (good test)

BLOOD PRESSURE IS NOT AFFECTED BY

*MUSCARINIC ACTIVITY* (parasympathetic stimulation) (Ach) Blood pressure is *affected by a1 adrenergic receptors* (vasoconstriction).

RTK Mechanism 4: Direct activation of transcription factors (JAK/STAT pathway)

*Mechanism:* 1. Activation of a receptor-associated kinase (JAK). 2. JAK phosphorylates STATs (transcription factors) 3. Phosphorylated STATs dimerize and travel directly to nucleus to activate transcription. 4. This system is the predominant manner in which immune cells respond to cytokines. It's also responsible for IFNg, GH, prolactin, and EGF.

Cholinoreceptors

*Nicotinic Receptors* -Found on all post-ganglionic neurons (both sympathetic and parasympathetic) -Found on Chromaffin cells of the adrenal medulla -Found at motor end plates of skeletal muscle (which are somatic, not autonomic) -Ganglion blockers (e.g., curare) do not block nicotinic receptors on motor end plates, but block nicotinic receptors of post-ganglionic autonomic neurons and Chromaffin cells *Muscarinic Receptors* -Located on all effectors of the parasympathetic system -Located on thermoregulatory sweat glands innervated by the sympathetic system.

Ovarian Cancer Screening/Prophylactic

*Ovarian cancer screening hasn't been clearly shown to be efficacious.* *Prophylactic Tx:* salpingo-oophorectomy -Reduces ovarian cancer risk by >90% -Reduces the risk of *breast* cancer by 50% in pre-menopausal women

Movement of Drugs Across Biological Barriers *Affected by:* -Molecular size -Structure -Charge -Lipid solubility -Ability to bind to serum, tissue, and cell surface proteins

*Passive Transport*: with a gradient -*Paracellular transport*: transfer of substances across an epithelium by passing through the *intercellular* space between the cells -*Simple diffusion*: substances pass through a membrane without the aid of an intermediary (e.g., an integral membrane protein) -*Facilitated diffusion*: the process of spontaneous passive transport of molecules or ions across a biological membrane via specific transmembrane integral proteins (down a gradient, no ATP). *Active Transport*: against a gradient -*Drug transporters*: specific carrier proteins carry drugs that closely resemble the structure of the naturally-occurring metabolites specific for the carrier across a membrane.

Subdivisions of Pharmacology

*Pharmacokinetics*: what the body does to the drug *Pharmacodynamics*: what the drug does to the body (mechanism of action on receptors/effectors) *Pharmacognosy*: Study of drugs from natural sources, medicinal herbs, etc.

Clinical Trial Phases Years 0-3: Basic Science: synthesis, examination, screening 3-5: Pre-clinical testing (animals) 5-10: Clinical Trials Note: patents last 20 years -- the average new drug enjoys patent protection for about 10-12 years

*Phase 1*: First in Human -10-100 volunteers -Open label (indications, dosage, side effects, special warnings like pregnancy) -*Safety, side effects, delivery* -70% success rate *Phase 2*: First in Patient -50-500 patient subjects -*Randomized and controlled* -*Efficacy, dosing, adverse effects* -33% success rate *Phase 3*: Multi-Site Trial -Few thousand patient subjects -Randomized and controlled or uncontrolled (in cases where it would be unethical to give a placebo) -*Confirmation of efficacy, dosing that doesn't cause side-effects* -25% success rate *Phase 4*: Post-Marketing -Many thousands of patients -Open label -*Monitoring for adverse events, comparative efficacy, marketing* -80% success rate

Polymorphisms associated with altered drug metabolism -SNPs alter enzyme function, which can affect drug metabolism

*Phase I Reactions* CYP Enzymes: 2C9: NSAIDs, phenytoin, warfarin 2D6: antidepressants, β-agonists, codeine 3A5: Ca2+ channel blockers, lidocaine, steroids *Phase II Reactions* N-acetyltransferase-1: sulfamethoxazole Sulfotransferases: acetaminophen, dopamine, estrogens Catechol-0-methyltransferase: catecholamines, L-dopa, M-dopa

Phase I and Phase II Metabolism *Phase I: Modification* Drug + O2 + NADPH + H+ → Modified Drug + H2O + NADP+ -Polar group added: *oxidation*, reduction, hydrolysis *Phase II: Conjugation* -Glutathione: R=O→R-SG -Sulfation: R-OH/SH/NH2→R-SO3H -Acetylation: R-OH/SH/NH2→R-Ac -Glucorinidation: R-OH/SH/NH2→R-GI

*Phase I: Modification* -*Convert a lipophilic drug to a more polar molecule (add -OH or -NH2)*. -Frequently catalyzed by *CYP450 enzymes* -Gene variability in CYP450 enzymes is a major contributor to person-to-person differences in drug metabolism -CYP inhibition is usually a result of competitive inhibition → 2 drugs compete for the same enzyme *Phase II: Conjugation for Solubility* -Involves the *conjugation of drugs that have been modified by Phase I reactions* -Usually reduces drug activity -If the Phase I product is polar enough, it can be excreted directly through the kidney without a Phase II reaction. -*Phase II reactions make Phase I products MORE polar* -Drugs with -OH, -SH, -NH2, or -COOH can go directly to Phase II metabolism without getting modified in Phase I.

Breast Cancer Testing Results

*Positive:* pathogenic variant is identified *VUS:* missense mutation with unknown significance *Negative:* -True negative if there is a known pathogenic variant in the family -Uninformative if there is not a known variant in the family

Neurotransmitter and Receptors of the Peripheral ANS, *Summary*

*Pre-Ganglia:* 1. All pre-ganglionic neurons are *cholinergic*, therefore 2. All post-ganglionic neurons are *cholinoreceptors*, and more specifically, 3. All post-ganglionic neurons have *nicotinic* receptors. *Post-Ganglia:* 4. Post-ganglionic neurons may be *cholinergic* or *adrenergic*. 5. Post-ganglionic sympathetic neurons are mostly *adrenergic*, with the exception of the *cholinergic (muscarinic)* neurons that innervate sweat glands. 6. *Most* post-ganglionic parasympathetic neurons are *cholinergic*.

Tumor Cell PD-L1 Expression

*Protective mechanism leading to suppression of tumor-infiltrating lymphocytes (CTLs) in the tumor microenvironment.* PD-L1 on tumor cells binds PD-1 on activated T-cells. -Inhibits CTLs, inducing tolerance. -Makes CTLs anergic and does not allow for further activation

Receptor Tyrosine Kinase Structure

*RTK Components:* 1. Extracellular ligand binding domain 2. Transmembrane Region 3. Tyrosine kinase domain with ATP- and substrate-binding (required for biological activity) 4. Intracellular domain contains Tyrosine autophosphorylation sites and Serine/Threonine sites.

Calculation of Required Dose (in mg) equation

*Required Dose = Vd(C2-C1)* Vd = volume of distribution (ml) C2 = *desired* drug concentration (mg/ml) C1 = *known* drug concentration (mg/ml) *Multi-comparment Model* *-↑Vd → ↑t(1/2): A drug with a large Vd is distributed in multiple compartments, so it is less readily excreted or metabolized.* -Vd > plasma volume when the drug is distributed to multiple compartments

Breast Cancer Lifestyle Risk and Protective Factors

*Risk Factors* 1. Nullparity (not having children) 2. Hormone Replacement Therapy (HRT) after menopause 3. Early periods, late menopause 4. Alcohol consumption *Protective Factors* 1. Exercise a few hours per week (↓18%) 2. Breastfeeding 3. Ideal body weight (especially after menopause; adipose tissue secretes estrogen) 4. Having children before 30 5. Having multiple children

Cholinergic Poisoning *Symptoms:* -*S*alivation -*L*acrimation -*U*rination -*D*efecation -*G*astrointestinal cramping -*E*mesis (vomit) -Twitch: ACh on nicotinic (skeletal muscle) -Bradycardia: reduced heart rate (SA/AV nodes) -Small pupils (miosis) -Blood pressure NOT affected (no muscarinic receptors) -BP not affected (muscarinic)

*SLUDGE, Drippy, and Twitchy.* *Causes:* -Organophosphates (patient smells like garlic) -Sarin gas (nerve agent) *Mechanism:* Inhibit acetylcholinesterase (by phosphorylation), has the effect of excess ACh on muscarinic receptors. At *muscarinic* (parasympathetic) receptors causes SLUDGE. At *nicotinic* (somatic) receptors causes fasciculations (twitching) and paralysis (dangerous for diaphragm). *Treatment:* Pr*a*lidoxime (paralysis) and *A*tropine (all others) -*Atropine blocks muscarinic receptors*: dries secretions, decreases bronchoconstriction, and increases heart rate → does not work on nicotinic receptors (skeletal muscles still paralyzed) -*Pralidoxime reverses Ach-ase phosphorylation* (treat ASAP before covalent bond formation; *treats paralysis*)

Sympathomimetics (adrenergics) vs. Anti-Cholinergic (antimuscarinic)

*Similarities:* ↑HR ↑temperature (NE: no vasodilation; ACh: no sweat) ↑pupil size *Differences:* -NE: ↑BP (vasoconstriction); cannot happen in muscarinic blockages (with anti-cholinergics) -ACh: ↑dry mouth; cannot happen with NE stimulation (with sympathetics) Since BP is affected, it cannot be due to anything cholinergic (not parasympathetic)

Autonomic Outflow

*Sympathetic*: T1-L2 *Parasympathetic*: S2-S4 + CN III, VII, IX, X

Dominant Nervous System in Each Organ System Reminder: Parasympathetic post-ganglionic neurons (which have nicotinic receptors) are cholinergic and release ACh onto muscarinic receptors

*Sympathetic:* V-SLAK -Vascular smooth muscle: *no parasympathetic innervation exists* (no muscarinic receptors) -Skin: sweat glands active -Liver: gluconeogenesis -Adipose Tissue: lipolysis -Kidney: renin secretion *Parasympathetic:* -Heart -Bronchioles: faster to get O2 into alveoli -GI tract: stomach grumbles when it wants -Eye: pupil and ciliary muscle contracted

Rejection is an immunological response mediated by...

*T cells.* *Evidence:* 1. A second skin graft from the same donor to the same recipient shows accelerated rejection (memory). 2. Primed T cell transfer accelerates rejection from a sensitized donor to a naive recipient (specificity).

Adaptive Cellular Immunotherapy

*The transfer of cultured immune cells that have anti-tumor reactivity into a tumor-bearing host.* *These T cells do not need to see MHC on tumors.* They have extracellular (CAR) domains that recognize tumor antigens and intracellular domains (ITAMs/CD28) that signal activation of killing mechanisms in the T-cell's nucleus.

Hereditary Genes Associated with Cancer BRCA1/2: DNA repair genes involved in: -Cell cycle checkpoint control -Ubiquitination -Transcriptional regulation

*Tumor Suppressor Genes* 1. APC (can't bind b-catenin) → FAP 2. PALB2 (LOF mutation) → breast cancer (high risk) *DNA Repair Genes* 1. BRCA1 (17q21)/BRCA2 (13q12) → 40-80% risk 2. Lynch Syndrome (Hereditary NonPolyposis Colorectal Cancer (*HNPCC*)) 3. Fanconi Anemia *Oncogenes* 1. RET gene -GOF: Multiple Endocrine Neoplasia Type 2, Medullary Thyroid Cancer (Chernobyl = papillary; RET-PCT1 gene fusion allowed for nutrient production and faster growth) -LOF: Hirschprung's Disease (no ganglia; congenital ganglionic megacolon)

Tumor-Related Mechanisms of Escape

*Tumors evolve to evade recognition in the host and can even resist immune effector mechanisms.* Immune responses must kill all tumor cells to be effective Failure of tumor to provide a suitable antigenic target or an effective immune response: -Lack of tumor antigen -Lack of MHC-I -Deficient antigen processing -Antigen modulation -Resistance of tumor to tumoricidal pathways -Lack of co-stimulatory molecules -Production of inhibitory cytokines -Shedding of tumor antigens

Volume of Distribution equation Equation: Vd=D/C Give more drug (D), it reaches more areas (Vd).

*Vd=D/C0* Vd= Volume of distribution D=Amount of drug in body (administered) C=Concentration of drug (theoretical C0 at t=0) *To determine C0 and t(1/2):* 1. Plot the y-intercept (drug concentration) on a log scale. 2. The elimination phase will be a straight line 3. The y-intercept of the line will be the theoretical C0. 4. Using C0, one can then calculate Vd based on the amount of drug administered. 5. Vd can then be used to calculate the dose aimed at achieving desired concentration (see below).

How is cAMP degraded?

*cAMP phosphodiesterase* hydrolyzes cAMP, yielding AMP

CDKIs of Interest -Prevent Cyclin D/CDK4 from phosphorylating Rb -Both end up halting the G1-S transition checkpoint

*p21 (G1/G2/M)(KIP):* induced through the p53 pathway in response to DNA damage *p16 (G1)(INK):* mutated in many human tumors; important in cell growth control

Drug's Half-Life Equation

*t(1/2) = 0.693 (Vd/CLt)* = ln 2/Ke Vd = volume of distribution CLt = total body clearance Ke = first-order elimination rate constant *t(1/2) is longer if Vd is greater or CLt is lower. If the drug enters more compartments or is cleared more slowly, it will degrade less quickly and remain in the body for a longer duration.*

Factors that determine drug dosing Reminder Variability: -Bioavailability of oral meds can range from 5-100% -Protein binding can range from 0-95%

-Bioavailability (if IV route is not being used) -Urinary excretion -Binding to plasma proteins -Clearance through non-kidney routes -Volume of Distribution (Vd) -Half-life of the drug in the plasma -(Time to) Peak concentration

Errors in the cell cycle associated with cancer

1. Gene amplification due to gene rearrangement results in the over expression of Cyclins D/E → breast cancer, leukemia, testicular carcinomas 2. Mutational inactivation of Rb → retinoblastoma, small cell lung carcinoma, osteosarcoma 3. Sequestration of Rb by HPV E7 protein → cervical cancer 4. Deletion or mutation of CDKI genes → many cancers (including prostate)

How does a GPCR work?

1. Hormone binds receptor 2. G-protein exchanges GDP for GTP 3. This causes Gα to dissociate from βγ and activate (or inhibit) Adenylyl Cyclase 4. Adenylyl Cyclase generates cAMP for second messenger signal transduction 5. Bound GTP is hydrolyzed back to GDP (by Gα) The downstream effects of cAMP: 1. cAMP activates PKA 2. PKA phosphorylates cellular proteins, causing a cellular response to the initial hormone binding 3. cAMP is degraded, reversing the activation of PKA

Less Common Hereditary Breast Cancers

-Cowden Syndrome (PTEN gene) -Li-Fraumeni (TP53 gene) -Peutz-Jeghers (STK11 gene)

Classes of Drug Targets

1. Ligand-Gated Ion Channels (cholinergic nicotinic receptors) -Change membrane polarity and intracellular ion concentration 2. GPCRs (α and β adrenoreceptors) -Change protein phosphorylation 3. Enzyme-linked receptors (insulin receptor) -Change protein phosphorylation 4. Intracellular targets (enzymes, nuclear DNA, structural proteins) -Modulate metabolism and gene expression

4 Types of Breast Cancer ER = estrogen receptor PR = progesterone receptor

1. Luminal A (ER+, PR+, Her-2/neu -) -Low grade 2. Luminal B (ER+, low levels of hormone receptors) -High grade 3. Her-2/neu + (HER2 enriched) -Amplification and high expression of ERBB2 4. Basal type (ER-, PR-, and HER2-) -Triple negative tumors (more common with BRCA1 mutation)

Genetic Barriers to Transplantation

1. MHC-I and MHC-II have a high level of genetic variability (polymorphic) and HLA alleles can be inherited and expressed in various combinations. 2. As such, every individual likely expresses some MHC proteins that appear foreign to another individual's immune system. 3. Minor histocompatibility antigens (associate with MHC on cell surface) also serve as targets of rejection (even when MHC is identical between the donor and recipient) *Recognition of non-self MHC by alloreactive T cells is what leads to transplant rejection.*

Family and Personal History Flags for BRCA Mutations

1. Multiple cases of early onset breast cancer in the family 2. Ovarian cancer (with family hx of breast/ovarian cancer) 3. Breast and ovarian cancer in the same woman 4. Bilateral breast cancer 5. Male breast cancer 6. Ashkenazi Jewish heritage (1:40 compared to 1:400 in Europeans, Africans, and Asians) 7. Basal phenotype (BRCA1): ER-,PR-,HER2- 8. High grade prostate cancer with family hx of ovarian, breast, prostate, or pancreatic cancer 9. Pancreatic cancer with family hx of ovarian, breast, or prostate cancer

Tumor Immunology

-Immune response against tumor can eradicate and prevent growth of some tumors -Treatment: *Enhance immunity against tumors*

Tumor Antigens

1. Products of mutated genes (neo-antigens) 2. Products of oncogenes or mutated tumor suppressor genes -HER2/neu amplification in breast carcinoma → over-expressed on surface, leading to T cell detection 3. Aberrantly expressed proteins (de-repressed expression) -Fetal testes antigen expressed outside of fetal development due to demethylation of gene

Urinary Excretion is affected by:

1. Protein binding (determines the proportion that can be filtered) 2. Charge and efficiency of reabsorption 3. Renal function (creatinine clearance) 4. Urine pH (basic urine holds weak acids) 5. Metabolism (conjugation, conversion to hydrophilic forms)

Options for cells to exit cell cycle

1. Quiescence (G0) 2. Senescence (by terminal differentiation) 3. Apoptosis (if DNA is not accurately replicated) Examples: 1. Cells can enter G0 if they become confluent (shut down signaling pathways for ribosomes etc.) 2. If telomeres get short, cells enter senescence (permanent growth arrest). 3. Adipocytes can become terminally differentiated and not enter the cell cycle (senescence). 4. Injury upon reperfusion → apoptosis.

Ribosomal development during G1

1. Ribosomal proteins are encoded by RNAs that have 5' oligopyrimidine tracts (5'TOP mRNAs) that are synthesized by RNApol-I. 2. RNApol-I transcription is under control of a number of proto-oncogenes (including *c-Myc*). 3. Activation of 5'TOP transcription is under the control of *mTOR* (mechanistic target of rapamycin; a nutrient-sensing kinase). 4. mTOR provides a mechanism whereby a cell can detect nutrient abundance and proceed (or not) to grow and divide.

Testing Strategy for Hereditary Breast/Ovarian Cancer

1. Test the most informative individual 2. If a family member has a known familial mutation, test for that first. 3. Ashkenazi Jews: test for 2 BRCA1 and 1 BRCA2 founder mutations first 4. In other patients, full sequencing/deletion and duplication testing of BRCA1/2 5. Multi-Gene panel (that includes BRCA)

CKI content is regulated at the level of

1. Transcription 2. Protein degradation

Cyclin content is regulated at the level of

1. Transcription 2. Translation 3. Protein degradation (occurs via ubiquitination to allow for cycling)

Bioequivalence Requirements

2 drugs are considered equivalent if: 1. They contain the same active ingredients 2. They are identical in strength and concentration 3. They are in the same dosage form (e.g., capsules vs. tablets) 4. They have the same route of administration 5. They have the same bioavailability

Biosimilar Drug

A biologic that is similar, but not identical, to another approved drug.

*Non-Competitive (Allosteric) Inhibition* -The same dose of a drug has a reduced effect

A drug/inhibitor binds to an allosteric site, changing the shape of the normal agonist binding site, preventing binding. -No change in EC50 (potency) -Lowers Vmax (maximal relative effect of drug) -Curve shifts down (same dose has reduced effect)

Potentiation

A potentiator binds to one part of the drug receptor and amplifies the effect of the agonist (drug).

EGF-R (Epidermal Growth Factor Receptor) EGFR is a HER family member EGFR = HER1

A receptor tyrosine kinase (RTK). Can be thought of as an allosteric enzyme, with hormone (e.g., growth factor) functioning as the allosteric activator of auto-phosphorylation activity. Mechanism: 1. Upon binding EGF, EGF-R dimerizes 2. Trans-phosphorylation (self-phosphorylation) of tyrosine kinase domains activates kinase (relieves inhibition by opening up ATP-binding site) 3. Activation of EFG-R to phosphorylate substrates (at tyrosine residues) 4. Threonine phosphorylation (via other protein kinases) shuts it off.

Post-ganglionic *sympathetic* neurons are ___________ or __________ and bind to respectively _____________ and _____________ receptors

Adrenergic (secrete NE) (bind adrenoreceptors) or Cholinergic (secrete ACh) (bind muscarinic receptors) (seen in thermoregulatory sweat glands)

Adrenoreceptor Mechanism -Located on all sympathetic effectors -Respond to NE released right onto the receptor from post-ganglionic sympathetic neurons -Respond to NE/E form Chromaffin cells in adrenal medulla

Adrenoreceptors are bound to G-Proteins. When E/NE binds, alpha subunit activates second messenger system.

Testing in BRCA1/2 Carriers

Age 25-35: annual or semi-annual clinical breast examination -Increased surveillance at age 25 (via MRI) -Annual mammography/breast MRI at age 30

Evolutionary Theory of Aging

Aging confers evolutionary advantages (getting rid of the old to make room for the new)

Repeated Dosing

An alternative to constant infusion. *Option 1:* Dose = 1 unit of drug divided n times per day *Option 2:* Dose = 1/2 unit of drug divided 2n times per day More frequent doses will reduce fluctuations (amplitude) in plasma concentration. Time to steady state will not be affected. Note: with antibiotics, we give the drug less often to maximize peaks and minimize troughs. We want the maximized peaks for max efficiency of the drug.

Drug:Receptor Interactions -The "receptor" is the *drug target*

Analogous to Michaelis-Menten: Maximal Effect ≈ Vmax EC50 ≈ Km; [drug] at 1/2 Max Effect

Rapamycin -Anti-fungal antibiotic -Anti-transplant rejection drug -Targets mTOR → blocks IL-2 production (immunosuppressive) -Prevents cancer/aging → current studies underway

Anti-fungal agent that has *immunosuppressive properties*. *Targets mTOR*, a kinase involved in a network that detects nutrients and regulates cell growth, motility, survival, protein synthesis, autophagy, and transcription. In this process, rapamycin blocks IL-2 production (immunosuppressant). Pre-treating a liver with rapamycin before a "mitogenic" protocol prevents HCC development in 95% of cases. Rapamycin has anti-aging effects in mice, fruit flies, and worms by an unknown mechanism.

BRCA Genes in Male Breast Cancer

BRCA1: 17q21 -Increased risk of breast cancer -Risk is less than BRCA2 mutation -Pancreatic cancer BRCA2: Fanconi Anemia Gene (13q12-13) -5-10% risk by age 70 (normally 1:1000; significant) -Prostate, pancreatic, bladder, bile duct, melanoma cancers

Potency -A drug on the left will be more potent as it requires a lower concentration for 1/2 Max Effect (lower EC50)

Based on the drug concentration at which you reach 1/2 maximal effect (EC50). A drug is more potent if its EC50 is lower for the same maximal effect.

Rb tumor suppressor and Cyclins/CDKs -Rb is a *tumor suppressor* gene because it binds E2F and inhibits progression through the cell cycle

Before the G1 restriction point, Rb is hypophosphorylated and sequesters E2F (inactive). Cyclin D/CDK-4 complex (activated by mitogenic stimulation) phosphorylates Rb to release E2F. Unbound E2F binds DNA and induces transcription of genes necessary (e.g., Cyclin E) for passage through the G1-S restriction point and continuation of the cell cycle.

Nicotinic receptors mechanism

Binding of ACh to nicotinic receptor triggers opening of Na+/K+ channels causing hypopolarization (depolarization)

Cyclin B

CDK-1 in M

Cyclin A

CDK-2 in S and G2 CDK-1 in G2

Cyclin E

CDK-2 in late G1 and S

Cyclin D

CDK-4 (adult) and CDK-6 (fetal) in G1

Cyclin Dependent Kinases -Most are *serine/threonine (ST) kinases* -Protein levels are fairly constant in cell cycle -Phosphorylate proteins during checkpoints

CDKs are the kinases *responsible for phosphorylating proteins during cell cycle checkpoints*. Cyclin / CDK Pairs / When Present: 1. *D / 4 and 6 / G1* 2. *E / 2 / G1→S* 3. A / 2 / S→G2 4. A / 1 / G2 5. B / 1 / M Reminder: CDK1 = CDC2

Total Clearance Rate Equation

CLt = 0.693*(Vd/t(1/2)) Reminder: 1. t(1/2) = 0.693*(Vd/CLt) [reciprocal equation] 2. Vd = D/C0 (administered dose/concentration of dose)

Primary Estrogen Receptor Mutation

Can result in a VERY tall man at a young age who had normal puberty. Estrogen was not able to limit growth at the epiphyseal plate.

Warburg Effect OXPHOS (normal) → aerobic glycolysis (tumor) -Quicker ATP -Increased nucleotides (G6P→HMP shunt) -Metabolic intermediates for cellular components *PKM2 favors aerobic glycolysis* Reminder: TCA fully oxidizes glucose to generate CO2, ATP, and produce FADH2 and NADH for OXPHOS

Cancer cells exhibit glucose conversion to lactate even when O2 supply is adequate (reduced OXPHOS). Aerobic glycolysis provides rapidly dividing tumor cells with metabolic intermediates they need for synthesis of cellular components. OXPHOS does not. Aerobic glycolysis also provides faster ATP. *Increased aerobic glycolysis also increases the Pentose-P pathway (HMP shunt via G6P) that increases nucleotide production, which benefits growing cells.* PKM2 (dimeric) isoform of PK (pyuvate kinase, PEP→pyruvate) is upregulated in cancer cells. The normal isoform is tetrameric M1 (PKM1). *Tumor PKM2 favors aerobic glycolysis.*

MAP Kinase Pathways (3-kinase pathways) Characterized by: 1. Small G-protein signal intiation (e.g., Ras) 2. Cascade of 3 protein kinases (triple kinase cascade) 3. Terminal kinase that can be phosphorylated twice at nearby threonine and tyrosine residues

Cascades of protein kinases for signal transduction usually have three key kinases. 1. Proximal Kinase (first in pathway) 2. Intermediate Kinase 3. "MAP" Kinase (leads to biological effect) e.g., Raf→MEK→ERK 1,2→→Growth stimulation

Biotransformation Example

Chloramphenicol: -Pro-drug: chloramphenicol succinate -Converted to active drug: chloramphenicol -Further converted to inactive product: chloramphenicol glucuronide Codeine (active drug) → Morphine (active product). Acetominophen (active drug) → NAPQI (toxic product)

Cholera Toxin (CTX) and GPCRs Mechanism: NAD+ + Gs-α → ADP-ribose-Gs-α + H+ + nicotinamide Symtoms: Secretory diarrhea

Cholera Toxin catalyzes ADP-ribosylation of Gs-α at Arginine 201 (R201). This *inhibits GTPase activity*. Cannot hydrolyze GTP to GDP. Effects: 1. *Gs-α is constantly active.* 2. Ribosylation promotes release of Gs-α from the other subunits, leading to *increased adenylyl cyclase stimulation*. 3. Increased cAMP production in the brush border epithelial cells of the large intestine 4. Hypersecretion of Na+ and Cl- (electrolyte loss), which carries H2O with it. 5. *Non-systemic effects* because CTX cannot make it past the gut mucosa.

All preganglionic neurons are ________

Cholinergic (secrete Ach)

Drug Elimination (Clearance) (volume/time)(ml/min)

Clearance = (Vmax x C) / (Ke + C) -Ke occurs at 1/2 Vmax Clearance follows first order kinetics until clearance metabolism is saturated. Total body clearance of a drug is the *volume of plasma from which all drug appears to be eliminated in a given time (volume/time).* NOT the amount of drug removed. CL(tot) = CL(renal) + CL(hepatic) + CL(other)

Fanconi Anemia (FA) -Autosomal Recessive -Hypersensitivity to DNA damage -*BRCA2 = key in Fanconi complex for DNA repair*

Clinical Findings: -Hematological malignancies (BM failure→low RBC, low WBC, low platelets) -Skeletal abnormalities -Developmental abnormalities Fanconi Anemia can happen in children whose parents are both BRCA2 carriers. Children have mutations on both alleles of Chromosome 13.

McCune Albright Syndrome Cause: Gs-α mutation leads to constitutive activation and inappropriate production of excess cAMP. Occurs during embryogenesis (somatic).

Clinical Manifestations: 1. Hyper-functioning endocrine disease -Gonadotropin-independent precocious puberty -Hyperthyroidism -Growth hormone excess -Neonatal Cushing syndrome (↑cortisol; fat) 2. Fibrous dysplasia 3. Café au lait macules

Pseudohypoparathyroidism Cause: Gs-α mutation (Met→Val, truncated protein): blocks initiation

Clinical Manifestations: 1. Hypocalcemic (seizures) 2. High PTH (to increase serum Ca2+), but resistant to it 3. Short stature 4. Obesity 5. Short metacarpals and phalanges 6. Migratory subcutaneous ossification 7. *Resistance to cAMP-mediated hormones (e.g., PTH, TSH, LH)*

Chi-Squared

Compare two *proportions* (ratios) to see if they're different (expected vs. observed)

Relationship between Infusion Rate and Steady State Concentration

Css = Ro / (Ke*Vd) Css = steady state concentration (mg/vol) Ro = infusion rate (mg/min) Ke = elimination constant (1/min) Vd = volume of distribution (vol) For first order kinetics, Ke and Vd are unchanging, so *Css ∝ Ro (directly proportional)*. Infusion rates determine concentration, not time to steady state. Changing the infusion rate does NOT change the time it takes to reach steady state. *You can double the infusion rate and double the plasma concentration, but it will take just as long to reach steady state as would the initial infusion rate.*

How to create a cumulative frequency distribution curve -"Quantal concentration-effect curve" (sigmoid in shape)

Curves are made by using the frequency distribution of patients responding to what concentration they felt an effect (frequency distribution) and stacking those bars on top of each other (cumulative frequency distribution)

Cyclin-CDK complexes

Cyclin, CDK, CDKI. All three components are required for the active cyclin-CDK complex. CKIs are required at sufficient levels to assist in cyclin-CDK assembly, but at a low enough level to permit CDK activity.

*Law of Mass Action* -The higher the Kd, the weaker the affinity of the drug for the receptor. -When drug concentration is equal to Kd, half of the receptors are occupied

D + R → DR, where D=drug; R=receptor [D]/(Kd +[D]) = [DR]/[Rt] = bound receptors/total receptors [D] = concentration of drug [DR] = concentration of drug:receptor complex [Rt] = total receptor concentration Kd = dissociation constant *When D=Kd, half the receptors are occupied.*

How do we age?

Damage Theory: oxidative stress and environmental factors induce DNA and protein damage -- however antioxidants have not been shown to enhance lifespan Rate of Living: finite number of heartbeats

2017 Screening Methods for Hereditary Cancer

Day 1: diagnosis Day 7: STAT breast panel results Within 1 month: *Positive for Mutation:* -Bilateral mastectomy (90% risk reduction if BRCA present) -No radiation ± adjuvant therapy (post surgery) -Family testing *Negative for Mutation:* -Expanded panel (~35 genes): half of all pathogenic variants in breast/ovarian cancer are in genes other than BRCA 1/2 -Lumpectomy -Adjuvant therapy as needed (can include radiation)

Insulin Receptor Structure

Dimer: α: extracellular β: extracellular, TM, intracellular α: cysteine-rich domain binds insulin β intracellular: *tyrosine kinase domain* with many auto-phosphorylation sites

GTP addition to Hormone-Receptor complex causes ...

Dissociation of hormone:receptor complex

*Competitive Inhibition* -Need a higher concentration of the drug for the same relative effect

Drug Interactions: -No change in maximal effect -Increases EC50 (lower potency) -Curve shifts to the right (higher EC50; same Max Effect) Michaelis-Menten: -No change in Vmax -Increased Km (need more enzyme for 1/2 Vmax)

Binding of Drugs to Plasma Proteins

Drugs absorbed from the gut → enter the vascular space → interact with proteins (mainly albumin, which *binds weak acids and hydrophobic drugs best*). Only free drugs (not-bound) can traverse the endothelial barrier and enter the interstitial space, where they are available to cells and cell surface receptors. *Class I Drugs*: Do NOT saturate the binding capacity of albumin in a typical dose *Class II Drugs*: Exceed the binding capacity of albumin in a typical dose Note: Administration of a Class II drug to someone receiving a Class I drug can result in higher free concentration of the Class I drug (Vd increases as the drug is free to leave the vascular space).

Catecholamines

Epinephrine (E) and Norepinephrine (NE) are the primary neurotransmitters/neurohormones in the autonomic PNS. Dopamine functions in the CNS. High levels of circulating catecholamines (mainly E from the adrenal medulla) are associated with stress. *Pheochromocytoma*: tumor of the adrenal medulla that originates in Chromaffin cells → ↑circulating E/NE

Major hormonal determinant of *senescence in resting zone* of epiphyseal plate

Estrogen. Estrogen limits the number of times that cells in the resting zone can divide.

Pharmacodynamic Variability

Example: Lisinopril (ACE inhibitor that reduces BP) affects are attenuated by NSAIDS, but magnified by other BP reducing drugs.

Renal Clearance of a Drug

Excretion Rate = Renal Clearance x Plasma Concentration Renal Clearance = UV/P *Excretion Rate = UV* (only looks at urine, mg/min) U = urine flow rate (ml/min) V = urine concentration (mg/ml) P = plasma concentration (mg/ml)

Dendritic Cell Vaccines

Facilitate the delivery of tumor antigens. *Vaccination Process:* 1. DCs are purified from patients 2. DCs are incubated with tumor antigens (pulsed) 3. Tumor cells are injected back into patients *Mechanism:* 1. Tumor antigen presentation occurs to patient's tumor-specific T cells (CTLs) 2. CTLs are activated and kill tumor cells

Kinetics of Continuous IV Administration

Features: 1. Rate of drug entry into the body is constant 2. Elimination is first order -The fraction of the drug cleared per unit time is constant -The amount cleared per unit time is related to the plasma concentration 3. At the start of the infusion, concentration rises until a steady state is reached.

Passive Diffusion of a Drug Across a Plasma Membrane

Flick's Law: movement of the drug is affected by the surface area of the membrane, the lipid solubility of the drug, membrane thickness, and the concentration gradient across the membrane. Diffusion = ((DAK)/h)*(C1-C2) D = diffusion coefficient of the drug A = surface area of the membrane K = lipid-water partition coefficient h = membrane thickness (thick = slower) C1-C2 = concentration different from one side of the membrane to the other

G(olf) and Olfaction

G-protein for olfactory cells Activated by chemoreceptors in the cribriform plate. Mechanism: 1. Odorants bind chemoreceptors 2. G(olf) is activated, leading to activation of olfaction-specific adenylyl cyclase 3. In olfactory neurons, cAMP leads to opening of Na+ channels, resulting in signal propagation down an axon (membrane depolarization).

Answers to Grappuso Lecture Questions on Signaling

G-proteins MUST be able to bind GTP. Transducin is a G-protein. Most of the metabolic effects of *cAMP-mediated* hormones involve activation of a protein kinase. The proto-oncogene, c-src, is specific for phosphorylation of proteins on tyrosine. JAKs phosphorylate STATs in the JAK/STAT signaling pathway. p21 (KIP1) binds to and regulates the kinase activity of CDK2.

Cell Cycle Overview

G1: cell contents double (8+ hours, variable) G1-S checkpoint: check for DNA damage S: DNA replication (10 hours) G2: preparation for mitosis (5 hours) G2-M checkpoint: check for damaged or unduplicated DNA; make repairs M: cell contents divide (1 hour) G0: state of quiescence (cell cycle arrest) from which cells can re-enter G0

Creutzfeld-Jakob Disease -Caused by prions -Led to creation of *Protropin*: recombinant human GH -$10,000-$30,000/year of treatment -Currently used to treat "idiopathic short stature" (>2.25 SD below the mean) → mean 1.5" gain

Growth hormone was originally isolated from cadavers (which could be contaminated with prions). GH was used to treat pituitary dwarfism in the 1960s and 1970s → some patients developed CJD. CJD: the progressive death of the brain's nerve cells, which is associated with the build-up of abnormal prion protein molecules forming amyloids.

Passive Diffusion Depends Largely on Drug Charge

Henderson-Hasselbalch Equation: pH = pKa + log ([A-]/[HA]) Reminder: pH>pKa → deprotonation The low pH within the stomach lumen results in a high proportion of the drug being in the protonated (non-polar) form, which effectively traverses biological membranes more readily than the ionized (polar) form. At the intestinal lumen pH (6.4), less of the drug is in the protonated (non-polar) form, so transport into the plasma is reduced.

Amphetamine Poisoning (sympathomimetics; adrenergics) -HIGH everything (but appetite) -Affects BP (not muscarinic)

High HR, high BP, hyperthermia, mydriasis (pupil dilates). Appetite loss. *Mechanism:* *Amphetamines stimulate NE (sympathetic) and dopamine (CNS) release.* Contracts blood vessels and increases AV/SA node signaling (↑heart rate). -NE stimulates *b1 → ↑heart rate (SA/AV nodes; ↑contractility)* (*b*etas make the heart *b*eat) -NE stimulates *a1 → ↑BP (vasoconstrict vascular smooth muscle)* *Treatment:* benzodiazepines (e.g., Valium)

Trastuzumab (tras*2*zumab) -Blocks growth factor signaling via HER family -Used to treat EGF-related breast cancer

Humanized monoclonal antibody against HER-2. 1. Blocks shedding of HER-2 EC domain 2. Prevents dimerization with HER-2 3. Promotes ADCC (immune cell killing) and endocytosis (for degradation)

Bioavailability Gold Standard

IV Administration. Relative levels are measured as the integrated plasma drug concentration. If the bioavailability +/- SD ~ 100, then the drug has the same effect orally as through IV administration.

Conserved domains for protein-protein interactions (in response to Tyrosine phosphorylation) SH2 and SH3 show up in many different domains (well-conserved)

Important Conserved Domains: 1. SH2: interact with tyrosine-P proteins 2. SH3: interact with cytoskeletal proteins Others: 3. PTBs (phosphotyrosine binding) domains 4. PH (Plekstin Homology) domains

Spare Receptors

In the case of drugs that interact with true receptors, maximal effect can often be achieved when receptor binding is far below maximum. EC50 < Kd, where: EC50 = 50% occupied receptors Kd = 1/2 Emax (max biologic effect) e.g., Cardiac β-adrenergic receptor shows near-max effect (Emax) when 10% of receptors are occupied e.g., Skeletal muscle insulin receptor shows near-max effect (Emax) when 50% of receptors are occupied

Adenylyl Cyclase Activation

Indirectly activated by: 1. GTP 2. cAMP-dependent hormones 3. Cholera Toxin (CTX) Directly activated by: -Gs-α → forms an active complex

Pseudo-irreversible Inhibition

Inhibitor binds so tightly to the site where the agonist normally binds that it's effectively irreversible. *Drugs that do this have high potency (can be used at low concentrations).*

Drug Development

Initial Steps: 1. Start with desired effect (e.g., lower cholesterol) 2. Identify a target (e.g., HMG-CoA reductase) 3. Determine if the target is "drugable" Questions to ask for "drugability": 1. Is it subject to inhibition or activation by small molecules? 2. Is the target in an accessible place (e.g., on the cell surface)? 3. Is the development of the drug economically viable? Once a potential drug is identified, preclinical research determines: 1. Specificity and effectiveness 2. Absorption, distribution, metabolism, excretion 3. Toxicity (requires 2 species) 4. Carcinogenicity? Genotoxicity? Reproductive toxicity? If ok, then the company files an *IND (investigational new drug) application* with the FDA before starting clinical trials. IND Application describes: 1. Rationale for drug development (and efficacy) 2. Preliminary data on pharmacology, toxicity 3. Plan for clinical trials

Insulin Stimulation of Skeletal Muscle Glucose Uptake Impaired skeletal muscle glucose transport in response to insulin is the primary reason for hyperglycemia in *Type 2 diabetes (not due to a mutation in GLUT-4)*. IRS = insulin receptor substrate

Insulin Effects: 1. ↑Vmax for glucose transport 2. Km remains the same *Due to the increase in active glucose transporters rather than their affinity for glucose* Mechanism: 1. Insulin binds insulin receptor (IR) 2. IRS-1 is a docking protein with SH domains that get phosphorylated (acts as a binding site for other signaling proteins, e.g., Grb2) 3. Leads to increase in active glucose transporters via PI3K (i.e., *GLUT-4* is translocated to cell surface, mainly in skeletal muscle; unknown mechanism) Note: *Brain, pancreatic β cell, and liver transporters are NOT insulin-sensitive.* Brain cell glucose levels must be independent of whether or not you've just eaten (express Glut-2).

Calculation of Loading Dose (LD)

LD = (Vd)(desired Css) Css = desired steady state concentration Vd = volume of distribution

Risk Factors: Breast Biopsy Findings

Lobular Carcinoma in situ (LCIS) -Abnormal cells form in the lobules or milk glands of the breast -*LCIS is NOT cancer* -Diagnosis with LCIS *increases risk of developing breast cancer* Ductal Carcinoma in situ (DCIS) -Abnormal cells inside a milk duct in the breast -*DCIS is the earliest stage of breast cancer (Stage 0)* -DCIS is non-invasive (pre-malignant lesion)

Beta 2 Receptors -Vasodilation of skeletal muscle vasculature (more blood) -Bronchodilation (↑O2 into the blood) -Only kicks in when there is a BIG fight or flight response (due to E)

Location *VASCULAR smooth muscle* that feeds: -Skeletal muscle -Smooth muscle of GI tract and bladder -Smooth muscle of bronchioles Activation causes: -*Relaxation of smooth muscle* -Vasodilation of skeletal muscle vasculature (more blood to skeletal muscle) -Relaxation of GI smooth muscle (stops peristalsis) -Bronchodilation (↑O2 into the blood) Response to Catecholamines: -Very sensitive to E -Not innervated by post-ganglionic sympathetic neurons (NE), so *ONLY activated by E released from the adrenal medulla*.

Beta 1 Receptors -Increase metabolic functions (fight or flight) -Heat beats faster and more forcefully -Mobilize fatty acids for energy -Kidneys increase renin secretion for ↑BP

Location and Activation Effect: -SA and AV nodes (↑heart rate, tachycardia) -Ventricular myocardium (↑contractility, more forceful contractions) -Adipose tissue (↑lipolysis of FA for energy) -Kidneys (↑renin ↑BP) -Salivary glands (↑salivation) Response to Catecholamines: -More sensitive to E than NE -Due to [NE]>>[E], b1 receptors respond to both E and NE -Activated by post-ganglionic neurons (NE) and the adrenal medulla (E)

Alpha 1 Receptors -↑BP, peripheral resistance -Contraction of vascular smooth muscle

Location: -*In VASCULAR smooth muscle (arteries) of skin, skeletal muscle, and GI.* -In the smooth muscle that forms the sphincters of the GI tract and urinary bladder, and the dilator papillae muscle of the eye *Activation causes contraction of smooth muscle.* -Increase BP -Fight or Flight: decrease blood flow to skin/GI/brain → pale skin -Vascular contraction -Closing of GI sphincters -Dilated pupils Response to Catecholamines: -Equally sensitive to E and NE -[NE]>>[E] at the receptor plate -NE: dumped on a1 by post-ganglionic sympathetic neurons -E: circulating (from Chromaffin cells) *-Therefore, a1 receptors are only activated by post-ganglionic sympathetic neurons.*

Alpha 2 Receptors -Relaxation of GI smooth muscle -Stops digestion (peristalsis)

Location: *Smooth muscle walls of digestive tract.* Activation causes *relaxation of GI smooth muscle* (pauses peristalsis, inhibiting digestion). Response to Catecholamines: -More sensitive to E than NE -Due to [NE]>>[E], a2 receptors respond to both E and NE -Activated by post-ganglionic neurons (NE) and the adrenal medulla (E)

Beta Blocker Poisoning -LOW everything (HR, BP, glucose) -Affects BP (not muscarinic)

Low HR, low BP, Low glucose. Bronchospasm. *Mechanism:* Beta blockers block beta receptors. *Treatment:* -"-olol" drugs (used to treat hypertension) -Glucagon: binds glucagon receptors on heart and triggers cAMP production to open Ca2+ channels (alternative to NE on beta receptors). Ca2+ acts to cross link actin/myosin to increase heart rate (muscle contraction).

Drug Duration of Action

MEC: mean effective concentration The duration of action is defined as the how long a concentration in the therapeutic window is maintained (between the mean effective concentration for desired and adverse responses).

Biologic

Manufactured in a living system (microorganism, plant cells, animal cells). Most are produced by recombinant DNA technology. Tend to be expensive (not replaced by generics). e.g., Insulin

RTK Mechanism 3: Formation of a *multi-subunit signaling particles* that activate small G-Proteins. e.g., Formation of signaling complexes that activate kinase cascades

Mechanistic Pathway: 1. Activation of receptor tyrosine kinase (RTK) 2. Phosphorylation of Shc adaptor 3. Shc binds Grb2 (activating it) 4. Shc/Grb2 binds SOS (activating it) 5. Activated SOS (nucleotide exchange factor) induces GTP for GDP exchange on Ras 6. Activated Ras recruits Raf (a serine/threonine kinase) to the cell membrane 7. Raf activates MEK 8. MEK activates MAPK (mitogen activated protein kinase) *MAPK effects:* 1. Further kinase activation 2. Phosphorylation of TFs 3. *Entry into and progression through the cell cycle*

Competitive Binding Assays Scatchard Plot: -X-intercept: receptor number -Y-intercept: [DR]/[Rt]

Mix all 3 together. Centrifuge. 1. Ligand (drug or hormone) 2. Same ligand radio-labeled 3. Receptor Unlabeled ligand competes with labeled ligand, reducing the amount of labeled ligand that is recovered in the pellet resulting from centrifugation. Competitive binding data can be used to derive values for the affinity of the receptor for the particular ligand and the amount of receptor present in the tube. If you know the amount of ligand added to each tube, and you measure the percent bound using the radio-labeled ligand, then you can calculate the amount of bound ligand and the amount of free ligand. *Scatchard Plot (see image):* -*Slope= -1/Kd* = affinity -*Lower Kd = steeper slope = higher affinity* for receptor leads to higher y-intercept (percentage of bound receptors) -X-intercept: free ligand is maximal, so bound ligand is maximal, which can be used to determine theoretical receptor number (all bound ligand has a receptor).

ANOVA

More than two samples → compare their means

Muscarinic receptors mechanism

Muscarinic receptors are bound to G-proteins, which are activated when ACh binds to the muscarinic receptor (parasympathetic, or sympathetic thermoregulatory sweat glands). When ACh binds, alpha subunit may directly open an ion channel in the cell membrane, but more often the *alpha subunit activates a secondary messenger system (cAMP/cGMP)* that activates an intracellular process that changes cell physiology.

cAMP target

PKA (cAMP-dependent protein kinase). PKA is a serine/threonine kinase.

Cholinergic Drugs -Slow down the heart -Stimulate the parasympathetic system -Muscarinic receptors

Parasympathomimetics = Anti-Adrenergic. Mimic parasympathetic innervation either by blocking sympathetic pathway or exciting parasympathetic pathway. *Direct Acting:* bind cholinoreceptors and activate them (like ACh) -*Methacholine is a muscarinic agonist* that excites muscarinic receptors of the parasympathetic system → causes *bronchial smooth muscle contraction* -Bronchial Test for Asthma: a stronger than average response to methacholine may be a sign of asthma or COPD *Indirect Acting:* increase ACh release, inhibit ACh breakdown (acetylcholinesterase inhibitors), or inhibit the sympathetic nervous system. -A drug that has no effect on the parasympathetic system, but blocks receptors of the sympathetic system is a cholinergic (mimics parasympathetic stimulation) -Prazosin (a1 blocker): prevents NE from binding a1, which prevents contraction of the vascular smooth muscle in vessels that supply the skin → vasodilation -Beta blockers (b1 blocker): bind to and block b1 receptors on the heart, reducing the likelihood of cardiac arrhythmia → low BP and dry mouth (less salivary activity)

Pertussis Toxin (PTX) and GPCRs Mechanism: NAD+ + Gi-α → ADP-ribose-Gi-α + H+ + nicotinamide Symptoms: -Coughing -Lymphocytosis (due to demargination) -Hypoglycemia

Pertussis Toxin catalyzes the ADP-ribosylation of Gi-α, which *inhibits nucleotide exchange*. Effects: 1. Gi-α remains in the GDP-bound form (cannot bind GTP to activate inhibition). 2. *Gi-α is unable to inhibit adenylyl cyclase.* 3. Increased cAMP production due to lack of inhibition. 4. *Systemic effects* because PTX can circulate and reach all tissues.

RTK Mechanism 2: Conformational change of an enzyme's regulatory subunit leads to localization and activation of catalytic subunit. e.g., Formation of signaling complexes that activate signaling enzymes. Similar to cAMP binding to PKA, allowing catalytic domains to act by binding regulatory domains.

Phosphoinositide 3-Kinase (PI3K) has 2 subunits: 1. p85: regulatory subunit that binds to docking proteins that are phosphorylated on tyrosine 2. p110: catalytic subunit p110 co-localizes to p85, changes shape, and is activated. PI3K phosphorylates PIs on the 3rd position, activating downstream signaling kinases.

RTK Mechanism 1: Direct activation of signaling enzyme by tyrosine phosphorylation

Phosphorylated PLCγ catalyzes hydrolysis of PIP2 to IP3 and DAG. -DAG activates Protein Kinase C -IP3 activates intracellular Ca2+ channels (Ca2+ influx)

Time to Reach Steady State (Css)

Plasma concentration doubles with every t(1/2). *About 90% of the steady state concentration (Css) is reached at 3.3 x t(1/2).* *The Css can be reached more rapidly by administering a loading dose.* -Loading Dose = (Vd)(desired Css)

Over-expression of oncogene MUC1 is associated with...

Poor response to *tamoxifen*, an estrogen receptor inhibitor.

Potency vs. Efficacy

Potency: x-value at 1/2 Max Effect Efficacy: y-value at Max Effect

In addition to direct neural input by post-ganglionic neurons, sympathetic signaling can occur by ____________

Pre-ganglionic cholinergic stimulation of Chromaffin cells in adrenal medulla. Chromafin cells release E and NE into bloodstream (as neurohormones) to bind adrenoreceptors

Parasympathetic Pathways

Pre-ganglionic parasympathetic cell bodies are located in cranial nerve nuclei of CN III, VII, IX, and X and at spinal levels S2-S4. All pre-ganglionic parasympathetic neurons are cholinergic and secrete ACh onto *nicotinic receptors*. All post-ganglionic parasympathetic neurons are cholinergic and release ACh onto *muscarinic receptors* on their target organs (smooth and cardiac muscle, glands).

Ras (proto-oncogene) Ras activates 2 pathways: (1) Raf → MEK → MAPK → cell proliferation (2) PI3-K → AKT → mTOR → cell survival

RAS is classic example of a small G-Protein. 1. Regulated by GTP exchange. 2. Activation occurs in response to growth factors. 3. Requires a Nucleotide Exchange Factor (SOS) to activate (GDP→GTP). 4. Needs GAP (GTPase Activating Protein) to turn off signal, hydrolyzing GTP→GDP. *Mutations in Ras are common to human cancers, often due to loss of GTPase activity.* Ras remains active and leads to uncontrolled growth.

Renal Clearance of a Drug Equation

RC = UV/P RC = (urine flow rate)(urine concentration)/(plasma concentration). Creatinine clearance can be estimated using the patient's age, weight, serum creatinine, and sex. Fraction of normal renal function (the patient's creatinine clearance as a proportion of the usual creatinine clearance of 100 ml/min): CL(cr) / (100)

Receptor Desensitization aka tachyphylaxis, refractoriness, adaptation, downregulation

Receptor agonists will often show reduced effect with repeated drug administration. This is especially true for agonists that interact with GPCRs. *Mechanism of GPCR Downregulation:* 1. Ligand (agonist) binds GPCR 2. GPCR kinase (GPK) adds Pi internally to GPCR, recruiting β-arrestin (β-ARR), which recruits clathrin and the AP-2 complex. 3. Endocytosis occurs via clathrin, removing the GPCR from the cell membrane with dynamin-dependent fission The period of time between desensitization and return to normal sensitivity is called the refractory period. When an antagonist is withdrawn, one can see supersensitivity.

Cyclins -Protein levels fluctuate in cell cycle -DEAAB; 42211; start at G1, G1→S, S→G2, G2, M

Regulate the activity of Cyclin Dependent Kinases during cell cycle. Binding of Cyclin to CDK activates CDK to phosphorylate proteins involved in cell cycle. Cyclin / CDK Pairs and Time Present: 1. D / 4 (adult) and 6 (fetal) / G1 2. E / 2 / G1→S 3. A / 2 / S→G2 4. A / 1 / G2 5. B / 1 / M

Grappuso Pharmacology Review Questions

Relationships between Vd and Total Body Water: 1. Drug is charged and large (remains in plasma compartment) -Vd = plasma volume (4L; 30% of 1/3 total) 2. Drug is lipophilic and readily crosses cell membranes to distribute to total body water -Vd = total body water (=weight x 0.6) 3. Drug does not readily cross cell membranes, but can diffuse through aqueous channels within membranes and between cells in the interstitial space. -Vd = 2/3 total body water (?? why not 1/3) 4. Drug is distributed to an extravascular site -Vd >> total body water

Rous sarcoma virus

Retrovirus that inserts its genomic material into host cells (not killing them). Results of Transformation: growth advantages -Loss in contact inhibition -Cells proliferate indefinitely (immortalized) -Cells don't need growth factor (serum) -Loss of anchorage-dependence -Glycolysis and glucose transport increase, supporting increased growth

Size of a cell is best correlated with....

Ribosome Number (related to synthesis). *A cell must double its size (and number of ribosomes) to pass from G1→S.* For a cell to double its complement of ribosomes, it must synthesize a sufficient amount of rRNA and ribosomal proteins.

STAT5b Mutation

STAT5b is required for signaling by growth hormone and a number of cytokines. Clinical Manifestations: -Short stature -Impaired immune function (no IL-2 production) -No response to growth hormone therapy STAT could not be activated as a TF for transcription of nuclear genes.

Rhodopsin-Transducin System -Light reception

Same as a GCPR with different names. Proteins involved 1. *Rhodopsin*: light-sensitive equivalent of hormone receptor. 2. *Transducin* is a G-protein. 3. *Phosphodiesterase* (PDE) is the effector protein (cGMP→GMP). Mechanism: 1. Photon capture by rhodopsin causes isomerization. 2. Isomerized rhodopsin interacts with transducin (G protein), leading to GDP→GTP exchange. 3. Transducin-GTP dissociates from rhodopsin and reacts with PDE; cGMP→GMP. 4. Reduced cGMP levels close Na+ channels, causing hyperpolarization. 5. The hyperpolarization is an electrical signal to the visual cortex. 6. The signal is terminated when transducin hydrolyzes GTP→GDP

Insulin Signaling

See image. IRS (insulin receptor substrate) is phosphorylated and binds many signaling molecules (e.g., Grb2-SOS and PI3-K), serving as a binding site for signaling proteins. Insulin results in activation of many protein phosphatases, which dephorphorylate many enzymes in metabolic pathways (rate-limiting step).

G1 Restriction Point *Mechanism*: 1. External growth factors bind 2. MYC/RAS→MAP signaling→Cyclin D (starts cell cycle) 3. Cyclin D/CDK4 phosphorylate RB, which releases E2F 4. E2F (TF) induces Cyclin E transcription (also Cyclin A, DNApol, and other genes needed for G1→S) 5. Cyclin E joins CDK2 to allow access through the restriction point (G1-S).

Separates 2 functionally different parts of G1. 1. G1 requires continuous mitogenic signals and a high rate of protein synthesis. Interruption of these signals or attenuation of protein synthesis leads to cell cycle exit and entry into quiescence G0. 2. Once the cell passes the "restriction point," it becomes committed to DNA synthesis and cell division. Note: *p21* inhibits CyclinD/CDK4 complex when DNA damage or cell stress are detected (p53 pathway). *p53* is degraded by MDM2 for feedback regulation. *p14ARF* binds MDM2 to stimulate the tumor suppression pathway.

PKA (Protein Kinase A)

Serine/threonine (ST) kinase. *Heterotetramer*: 2R-2C R: regulatory subunit binds cAMP C: catalytic subunit catalyzes the transfer of Pi from ATP to proteins Mechanism of Action: 1. cAMP binds 2R-2C 2. 2R-2cAMP dissociates from C proteins 3. C has phosphorylation effects Since 2R protects cAMP from hydrolysis, PKA activity is regulated by cAMP levels.

What are the biggest risk factors for breast cancer?

Sex: *Women are 100x more likely to get breast cancer than men.* Family History: 1 first degree relative = 2x risk 2 first degree relatives = 5x risk Knudson's Two-Hit Hypothesis: -5-10% of women with breast cancer have a hereditary form of the disease -One germline copy is mutant; a second somatic mutation is required for disease expression and tumor development -Highly penetrant genes (45-85% lifetime risk)

Drug Effect

Strongly related to drug binding to its receptor. Effect ≈ [DR]/[Rt] (filled receptors/total receptors) A low Kd for binding indicates high affinity of the drug for its receptor, but DOES NOT mean that the receptor is *specific* for that one drug only. E/Emax = [D]/(Kd+[D]) = [DR]/[Rt] Implications: 1. The higher the [D], the greater the effect 2. As [D] approaches infinity, E approaches Emax (at max dose, max effect is reached) 3. The lower the Kd, the greater the drug effect at any given [D]

Pharmacogenetics

Study of the role of inheritance in variation in drug response (across a population). -Requires the genomes of many people to find SNPs that correlate with drug effects.

Reminder for Sympathetic vs. Parasympathetic

Sympathetic: NE/E Parasympathetic: ACh

Anti-Cholinergic Drugs

Sympathomimetics = Adrenergic. Mimic sympathetic innervation either by blocking parasympathetic pathway or exciting sympthetic pathway. *Direct Acting:* excite adrenergic receptors, mimicking the effect of sympathetic stimulation. -Epinephrine -Dobutamine (b1 agonist): excites b1 receptors on the heart and *increases cardiac output* → used with NE during heart failure -Albuterol (b2 agonist): used in rescue inhalers for *bronchodilation* (relaxation of smooth muscle) *Indirect Acting:* block muscarinic receptors, blocking the effects of the parasympathetic system, and mimicking the effects of sympathetic stimulation -Spiriva (anti-muscarinic inhaled powder): prevents bronchospasm associated with COPD (blocks constriction)

Care Plus Model of Care -Targeting safety net patients who were failing usual care. -Patients used the emergency room quite a lot, had many chronic illnesses (medical, mental health), were in poverty, and had diminished cognitive function.

Tenets: -Multidisciplinary team -Population based -Value-based care -Immersed in social determinants (screen and treat) -Get out into the community -Attend to mental health and substance abuse (adverse childhood events increased risk of depression, substance abuse, and suicidal ideation by 4-12x) -Focus on care transitions

PARP Inhibitor (Lynparza)

The PARP family of enzymes plays a critical role in the maintenance of DNA integrity as part of the *base excision pathway* of DNA repair. -PARP 1 is over-expressed in many cancers -PAPR 1/2 inhibitors have demonstrated important clinical activity in BRCA 1/2 gene carriers (*"synthetic lethality"*) -Simultaneous loss of the capacity to repair DNA damage may have anti-tumor activity In other words, PARP helps tumors grow (repairs DNA). By inhibiting this process, the tumor is more susceptible to chemotherapy and cell death.

Sympathetic Pathways

The cell bodies of pre-ganglionic neurons are in there lateral horn of the grey matter of the spinal cord at levels T1-L2. *Rules:* 1. All pre-ganglionic neurons are cholinergic (ACh). 2. All post-ganglionic neurons have nicotinic receptors that bind ACh. 3. Most post-ganglionic neurons are adrenergic (secrete NE onto target receptors). *Exceptions:* 1. Post-ganglionic neurons that innervate *thermoregulatory sweat glands* are cholinergic (NE), and their receptors are *muscarinic*. 2. Greater Splanchnic Nerve (GNS) -Cell bodies in T5-T9 (lateral horn) -Cholinergic neurons (GNS) release Ach onto nicotinic receptors of Chromaffin cells of the *adrenal medulla* (NCC origin, cannot form post-ganglionic axons → secrete into the bloodstream) -Secrete neurohormones into the bloodstream: 80% E, 20% NE

Single Compartment Model

The concentration of the drug is determined by rate constants. -Assumes a single, rapid IV dose. -Assumes rapid distribution of the drug. -Assumes the concentration is measured in a single compartment. *Drug at Absorption Site* ↓ Ka = absorption → (*Distributed*) ↓ Ke = excretion Km = metabolism *Equation: Vd=D/C* Vd= Volume of distribution D=Amount of drug in body (administered) C=Concentration of drug in the compartment measured In the image, only the *elimination* phase is shown. The *distribution* phase shows the spike and initial decline in drug concentration.

Renal Extraction Ratio

The decline in drug concentration in the plasma from the arterial side of the kidney (C1) to the venous side (C2). *Extraction Ratio: C2/C1* Affected by filtration (glomerulus), active secretion (proximal tubule), and passive reabsorption of the non-polar drug (distal tubule and collecting ducts). Passive reabsorption in the distal nephron is affected by the ionization state of the drug (dependent on urine pH). *Raising the urine pH (e.g., bicarbonate) can "trap" a drug that is a weak acid in the urine.*

Bioavailability

The fraction of an administered drug that reaches the systemic circulation in a chemically unchanged form. Affected by: 1. *First pass* hepatic metabolism (for oral drugs) 2. *Solubility* and other chemical characteristics (might need to be protein bound) 3. *Stability* of the drug (e.g., in the acidic stomach) 4. *Drug formulation* (particle size, coatings, etc.) Bioavailability is MAXIMAL when a drug is administered intravenously (IV). Bioavailability = [(AUC-oral)/(AUC-IV)] * 100%

Efficacy -A drug with a higher end y-value is more efficacious

The physiologic effect of a drug. The higher the Maximal Effect, the more efficacious.

Therapeutic Window

The plasma concentration range for which more than 50% of subjects have a therapeutic response, but fewer than 5% have adverse effects.

Drug Distribution

The process by which a drug leaves the circulation (plasma compartment) and enters the fluid around and in cells. Affected by: 1. Blood flow to a specific tissue 2. Capillary permeability (e.g., BBB) 3. Binding of drugs to plasma proteins *Total Body Water*: 42L (60% of body weight) *Intracellular Volume*: 28L (2/3) *Extracellular Volume*: 14L (1/3) >Plasma: 4L >Interstitial Water: 10L

Pharmacodynamics

The study of the biochemical and physiological effects of drugs and their mechanisms of action (the effects of drugs on the body). Drugs act through existing physiologic mechanisms (i.e., they do not create effects de novo). "Drug:Target" interactions are mainly dependent on *electrostatic and hydrogen bonds.* Covalent interactions would be irreversible.

Therapeutic Index

Therapeutic Index = LD50/ED50 (*higher is better*) Note: LD50 (animal studies) = TD50 (human pharma) Determines how selective a drug is in producing desired effects relative to toxicity. *We want a large therapeutic window (high lethal dose, low effective dose).*

T-tests

Two samples/groups → compare the mean results between the two samples/groups -Used when samples have normal distributions

Src → tyrosine kinase v-Src: oncogene (viral) c-Src: proto-oncogene (human homologue)

Tyrosine kinase that auto-phosphorylates. *v-Src autophosphorylates at Y416: activation* -No inhibition site for regulation *c-Src autophosphorylates at 2 sites* 1. First: Activation 2. *Second: Inhibition* (Y527: regulation)

Pharmacogenomics

Using knowledge of an individual's DNA sequence to modify therapy (personalized medicine, in an individual).

Drug Metabolism (Biotransformation) -The elimination of a drug by chemical modification (can be spontaneous or enzyme catalyzed) -Most biotransformation occurs in the *liver* -Oral administration has the greatest potential for biotransformation (first pass phenomenon) -Biotransformation is more broad than drug metabolism and can include gut flora drug alterations

Vmax = max rate of metabolism Km = concentration at which half the max rate of metabolism is achieved First-Order Kinetics: At (lower) *concentrations below and near the Km*, the rate of metabolism is dependent on the *concentration of the drug to the first power* (slope of curve becomes less and less steep, but still rises). Zero-Order Kinetics: At *(high) concentrations* that greatly exceed Km, the rate of metabolism will be *independent of the drug concentration*. A constant amount will be excreted per unit time (curve levels out at max rate of metabolism).

Pure Aging vs. Disease

What happens if you survive, no matter how well you live your life (e.g., diets, habits, exercise). Inevitable and irreversible. *Pure Aging Changes in Organs* -Lungs: reduced elastic recoil and alveolar exchange area (survival probability with pneumonia) -Immunity: decreased antibody production and clonal expansion -Body composition: increase fat, decline in lean mass -Metabolism: insulin resistance (diabetes risk) -Renal: decreased C(cr) rate -Heart: LV stiffness; decreased max HR -Bones: decreased density, increased fragility -Skin: decreased D3 production -Urogenital: decreased bladder control -Brain: wider sulci (valley); narrower gyri (mountain); enlarged subdural space

Overview of Adrenoreceptors

a1: contraction of smooth muscle (↑BP), tight sphincter a2: relaxation of smooth muscle (in the gut); no peristalsis b1: increase metabolic function and heart rate; saliva b2: relation of smooth muscle (vasodilation/bronchodilation); no peristalsis