MBT254 Final Exam

Five stages of manufacture

1. Genetic development 2. Fermentation: upstream process (USP) 3. Purification: downstream process (DSP) 4. Formulation, filling 5. Quality control

Bacterial Inhibition Assay (1963)

Agar plate Add B-2 thienylalanine (Inhibits bacterial growth) Spread with bacteria (e.g. Bacillus subtilis) Add disc containing blood spot If phenylalanine present, bacteria will grow near disk Detects down to 180-240 μmol/L (Healthy < 120 μmol/L)

Knock-Out

Basic research in biotech uses knock- out experiments, which are very helpful for learning about the function of a gene. A knock-out is created when an active gene is replaced with DNA that has no functional information. Without the gene present, it may be possible to determine how the gene affects the organism (its function)

F. picrosperma to therapeutic drug

Cannot be commercially synthesized Fruit are currently harvested from wild populations Commercial plantations recently been established

Heterogametic males (XY) Feminization

Female (androgen treatment) → neo-male X female → 100% female

Pharmacogenomic Drugs

Herceptin Gleevec Erbitux Tumoricide

Bisphenol A (BPA)

In Industrialized regions levels of 7-8 μg/ml could be detected in water. In parts of Japan water contained 17 μg/ml and precipitation 100 μg/ml. Half life 1 to 4 days. High affinity to the estrogen receptor. 1 L X 17 μg/ml = 17 mg

Analyte

What you are trying to detect

Tandem Mass Spectroscopy (2006)

Analyse 20 metabolites in ~2 minutes Comprehensive assessment from a single blood-spot specimen Simultaneously detect Phenylalanine & tyrosine (and determine the ratio) Reduces false positives Determine the ratio Detects femtomole to picomole quantities of each metabolite.

Sexual differentiation in crustaceans

Androgenic gland hormone: key regulator Freshwater prawn: case study for AG removal and RNAi Environmental friendly sex reversal Gene silencing through dsRNA

Aquatic Applications

Aquaculture is a common aquatic application of biotech. Aquaculture is the process of raising finfish or shellfish in controlled conditions for food sources. Products include: transgenic salmon (increased growth rates), disease-resistant oysters, vaccines against viruses that infect aquatic species Overall, aquatic organisms are thought to be rich & valuable sources for new genes, proteins, & metabolic processes.

2. Fermentation: bioreactors

Batch / batch fed Single use Continuous

Sensing molecules

Binds to what you want to detect

CRISPR REVOLUTION: Genome editing "made easy"

CRISPR/Cas9 Pubmed search: - 2012: 3 publications - 2013: 100 publications - 2014: 367 publications - 2015: 827 publications - 2016: 1213 publications Runner-up for 2013 Breakthrough of the Year (Science): CRISPR - Genetic microsurgery for the masses

Diversification of seaweed species and products

Carbohydrates: Very high levels (30-60% dry weight) due to structural polysaccharides - Low digestibility Lipids: energy from lipids similar to vegetables, but small percentage (1-15% dry weight) - Up to 50% fatty acids are PUFAs Protein: High protein content, especially green seaweeds (10-35% dry weight) - Some species are similar to soybean Trace Elements/Minerals: High mineral content (ash content = 10-40% of DM), including Iron, Iodine - Iron: more in seaweed than meat and spinach (especially nori) Vitamins: can provide enough Vitamin A, B2 and B12 (and majority of Vitamin C) Antioxidants: pigments, phenolics and secondary compounds

1b. Genetic development: transfection

Cationic reagents - Polymers or lipids

Transducer/Signal output

Converts the binding into something we can detect

Stem Cells

Different chemicals can coax them to develop into different cell types. Newest, most promising area Most controversial

Biopharmaceutical:

Drugs extracted/derived from natural sources For example: - Whole blood products - Organs and tissue transplants - Human breast milk - Fetal microbiota - Sperm/egg cells

Tracy

Engineered to produce Human protein alpha 1-antitrypsin in her milk (Potential treatment for cystic fibrosis)

Marine Biotechnology Summary

Enormous biological diversity of marine organisms There is chemodiversity of the NPs isolated between and within different genera Diversity of biological activities displayed by marine NPs Continual advances in technological methods, including instrumentation and screen protocols Marine sponges currently provide the most NPs Biotechnology can be used to develop heterologous synthesis of NPs

Omics

Epigenomics Genomics Metalloproteome Transcriptomics miRNAomics Metabolome Cistromics Proteomics Microbiomics

Drug Safety

Essentially, in Australia, the regulator asks: - Does the drug work? - Is it safe? - Is it manufactured using the highest standards?

In Vivo Studies

Experimentation using a whole, living organism. Gives information about: metabolic profile, toxicology, drug interaction

Phase III

Extended Clinical Trials. Most expensive & time consuming. 250-1000 Patients. Controlled Double Blind Technique. Concerned With: Safety, Efficacy, Comparison with other drugs, Package insert

Enzyme Assays Important Considerations

Extremely rapid (minutes) Requires the analyte to be either an enzyme or a substrate Useful for abundant molecules Leaky/cross-reactive (affects specificity) Most enzymes cross-react with structurally similar substrates Detects only 1 type of analyte per reaction (no multiplexing) Sensitivity averages in the μmol - mmol range (i.e. 10^17 - 10^20 molecules

Biologic medical products

Higher molecular weight Biological production

Sexual differentiation in vertebrates

Hormonal regulation Sex hormones Sex reversal (steroid administration) Environmental endocrine disruptors Steroidogenesis

Biochemical Classes of Drug Targets

G-protein coupled receptors: 45% Enzymes: 28% Hormones and factors: 11% Ion channels: 5% Nuclear receptors: 2%

Use of Pharmacogenomics

"One-size-fits-all drugs" only work for about 60 percent of the population at best The other 40 percent of the population increase their risks of adverse drug reaction because their genes do not do what is intended of them.

What prior knowledge is needed?

Genetic background (WZ; known through AG removal) What gender is preferred (male, ZZ) Mechanism of sex differentiation (AG) Timing of sex characters appearance (hand segregation) Timing of sex reversal (known through AG removal) Sex specific DNA markers (to tell between a sex reversed individual and a false-identified one)

PKU

Genetic disease Mutation in the enzyme phenylalanine hydroxylase Body unable to metabolize the amino acid phenylalanine Causes brain damage, seizures, and intellectual disability.

CRISPR/Cas9 concerns

Human germ-line editing: - Scientists called for a moratorium on the use of the technique for human genome editing - Non-viable embryos destroyed after three days - Deliberately made viable human embryos with a genetic aberration - Fixed the aberration to show it could be done, before destroying the cells

Lead Discovery

Identification of small molecule modulators of protein function The process of transforming these into high-content lead series.

Insulin History

In 1950s/60s, Hemophiliacs were treated with fresh frozen plasma Mid-1960s: extracted factor VIII could be stored in frozen form. Late-1960s: freeze-dried Factor VIII and IX extracted from pooled plasma Thousands of donations combined as starting material Discovered to contain HIV/Hepatitis - most patients infected 1984: cloned factor VIII gene 1987 recombinant factor VIII clinical trials 1982: cloned factor IX gene 1997: Factor IX recombinant product became available

Dolly the Sheep

In 1996, Dolly the sheep became the first cloned animal created by the somatic cell nuclear transfer process. Born: July 5, 1996 Announced: February 22, 1997 Died: February 14, 2003 Dolly was cloned from a cell taken from a six-year-old ewe She became the center of much controversy that still exists today

Tigilanol tiglate drug development: human oncology

In early stage development as a human oncology therapeutic - Phase I/II clinical trial complete - First-in-human study with a primary focus on assessment of safety parameters - "the drug was well tolerated" - "signs of efficacy were reported in 8 different tumour types with full tumour destruction"

What is intellectual property?

Includes rights related to: - Literary, artistic and scientific works - Performances - Inventions and innovations - Trade marks - Geographic indications of origin

Anti-cancer mechanism of action

Induction of cellular necrosis and vascular damage - Rapid removal of tumour Activation of innate immune response - Removal of residual tumour cells?

Pathway to IND/NDA

International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH): 'Guidance documents' from FDA Good Laboratory Practice: set of principles intended to assure the quality and integrity of non-clinical laboratory studies Good Manufacturing Practice: set of principles intended to assure the quality and integrity of manufacturing therapeutic drugs Good Clinical Practice: set of principles intended to assure the quality and integrity of clinical studies

Chemical Synthesis

Involve production of lead compound in suitable quantity and quality to allow large scale animal and eventual, extensive human clinical trials. Optimization of chemical route for bulk industrial production. Suitable drug formulation. (In glass) studies using component of organism i.e. test tube experiments

Cell-Disease Models

Isogenic human disease models: A family of cells that are selected or engineered to accurately model the genetics of a specific patient population, in vitro Stem cell disease models: Adult or embryonic stem cells carrying or induced to carry defective genes can be investigated in vitro to understand latent molecular mechanisms and disease characteristics

Example for the use of Bioinformatics

Laboratory procedures Patent sample (blood or saliva) Extract DNA from cells Sequence DNA In silico Bioinformatics analysis Compare patient DNA sequence to reference sequence Search database to determine if patient mutation is associated with disease

Asparagopsis

Light microscopy Transmission electron microscopy - Metabolites are localised inside a refractile inclusion in the cell Gland cell is a specialised cell for halogenated metabolite production What we need to know in the marine context: Ecology Genomics Aquaculture What we are finding out for agriculture: Post-harvest processing and logistics Meat and milk quality Different animals Palatability - feed additive

Imaging: Positron Emission Tomography (PET)

Liquid radioactive material injected into body Pick up where it accumulates using PET scanner E.g. Fluorodeoxyglucose (gives off gamma rays) accumulates in tumours as these need sugar to grow

Ingenol mebutate: Pharmacodynamics

Loss of adherent cells from culture after 2 days of treatment - SKMel28 human melanoma - B16 mouse melanoma Atypical dose-response

AMH role in vertebrate embryogenesis

Low AMH: mullerian ducts develop High AMH: mullerian ducts apoptosis

Small molecules drugs

Low molecular weight (<900 Daltons) Simple manufacturing process (produced by chemical synthesis)

Homogametic males (ZZ) Masculization

Male (estrogen treatment) → neo-male X female → 100% male

Heterogametic males (XY) Masculization

Male (estrogen treatment) → neo-male X female → 66%/75% male → YY male X female → 100% male

The Androgenic Gland

Male specific, unique to crustaceans Regulates masculinity development and maintenance AG removal results in sex reversal

Ingenol mebutate → Therapeutic drug?

Marketing authorisation Since 1938, every new drug has been the subject of an approved NDA before U.S. commercialisation

Medical applications

Medical applications of biotech include preventative, diagnostic, and treatment. The Human Genome Project is very useful within this field. Gene therapy and stem cell technologies are two up-and-coming fields within the medical area of biotech. Stem cell technologies include immature cells that have the potential to develop and specialize into a variety of other cell types.

Animal Patents

Method for reducing total gas production and/or methane production in a ruminant animal Growth performance improvements in pasture and feedlot systems Asparagopsis targets the archaea portion of the microbes in the rumen microbiome Almost entirely related to bromoform (tri- bromomethane) No other seaweed accumulates bromoform in its tissue

Surrogate Technology

Microinjection of donor-derived germline stem cells into the abdominal cavity of rainbow trout hatchlings. - Intraperitoneally transplanted germline stem cells migrate to the genital ridges of the recipient, are incorporated, and initiate oogenesis or spermatogenesis Produce one species using another Improve breeding efficiency Use cryopreserved germ cells to produce the breed Induces fertility

Mirena® (levonorgestrel-releasing intrauterine system) contraceptive

Mirena is a small, T-shaped intrauterine device (IUD). Placed into the uterus by healthcare provider Release progestin hormone (levonorgestrel) Thickens cervical mucus Inhibits sperm from fertilizing egg Thins uterine lining The cylinder of the device is coated with a membrane that regulates the release of the drug. Prevents pregnancies (no more daily contraceptives)

Molecular Pharming

Molecular pharming is the use of genetically modified plants (or animals) as a source of pharmaceutical products. These are usually recombinant proteins with a therapeutic value. This is an emerging but very challenging field that requires: manipulation (at the genetic engineering level) of protein glycosylation (addition of polysaccharide chain), subcellular protein targeting in plant cells

Biotechnology Laboratory

Mostly recombinant proteins Cell culture - the technique of growing cells in a lab under controlled conditions; similar to in vitro "in vitro" - working in a controlled environment outside of a living organism Bioreactors (large culturing "containers" where DNA of interest can be mass produced)

What have Pfizer trademarked?

Name Composite (logo) Colour and shape (in some countries)

Case Study: Fontainea picrosperma

Native, dioecious, rainforest understory tree First described in 1985 Found only in the southern Atherton Tablelands of Far North Qld Extracts of blushwood fruit contain therapeutically interesting NPs

Immune response: primary tumour

Neutrophils are required for tumour cure Antibodies are required for tumour cure

Viagra Patents

New use of old substance Novelty - s 18(1)(b)(i) - Patentable inventions, when compared with the prior art base as it existed before the priority date of that claim is novel Limited protection - Compound (sildenafil citrate) to be used to treat erectile dysfunction - You can treat erectile function in other ways

Aquaculture Bottlenecks

No or unpredictable ovulation/spawning Seasonal spawning Impaired sperm production Sterility Timing of puberty (early, late) Growth/reproduction interactions

HiSeq2500 - Rapid mode

Number of reads: 100-150M/lane Read length: 2 x 150 bp Yield per lane (PF data): Up to 45 Gb Instrument time: ~2 days Pricing per Gb: $53 (PE150)

MiSeq

Number of reads: 12-15M (v2), 20-25M (v3) Read length: 2 x 300 bp (v3) Yield per lane (PF data): Up to 15 Gb Instrument time: ~2 days Pricing per Gb: $108 (PE300)

HiSeq2500 - High output

Number of reads: 150-180M/lane Read length: 2 x 100 bp Yield per lane (PF data): Up to 35 Gb Instrument time: ~12-14 days Pricing per Gb: $59 (PE100)

PacBio RSII

Number of reads: 50-80K/SMRT cell Read length: ~10-20 kb Yield per lane (PF data): Up to 0.4 Gb Instrument time: ~2 hours Pricing per Gb: $697

CRISPR Corrects Sickle cell-causing Gene in Human cells

October 13, 2016 Design reverse of glutamate-to-valine substitution in haemoglobin using CRISPR Electroporate RNA/DNAs into hematopoietic stem progenitor cells (HSPCs) Check for off-target mutations (<0.0001%) Xenograft into mouse 2-6% of the corrected cells retained the edits after 16 weeks once administered to the mice

Australian Regulations

Once registered, the medicines may be marketed and distributed, but: - are subject to GMP inspections by the Manufacturing Quality Branch (mutual recognition agreements for audits done by the FDA, Japanese PMDA, Health Canada, Swissmedic, the EDQM and some member countries); - For biotechnological medicines, samples of first five batches, all adverse report samples & regular surveys must be submitted for analysis; - present Product Information (PI) and Consumer Medicine Information (CMI) with any medicine. - must report all adverse events; and - must update any changes (Category 3) to the TGA.

Other Biotechnology Products

Other biotech products include proteins in: home pregnancy tests (monoclonal antibodies), frost-resistant strawberry plants Although many are focused on medical and agricultural applications, some are for our own fashion interests (specialty apparel)

Therapeutic Goods Administration

Part of the Department of Health Responsible for: regulating therapeutic goods including prescription medicines, vaccines, sunscreens, vitamins and minerals, medical devices, blood and blood products Almost any product for which therapeutic claims are made must be entered in the Australian Register of Therapeutic Goods (ARTG) before it can be supplied in Australia Quality, safety and efficacy

Experimental use exemption

Patents are aimed at encouraging innovation and invention Problems may arise where there is uncertainty about the extent to which patent rights restrict freedom to research ('freedom to operate') The exemption applies to tests, trials and procedures that a researcher or follow-on innovator undertakes as part of discovering new information or testing a principle or supposition the exemption does not apply where the main purpose is to commercialise a patented invention, or to manufacture it for sale or use for commercial purposes

Euphorbia peplus

Petty spurge milkweed - Native to europe Early 1800's sap used topically to treat dermatological conditions (warts, corns, waxy growths, skin cancers) Also used orally - Purgative, cancer of the stomach, liver and uterus Effective home remedy to treat skin cancers

Pharmacogenomics

Pharma = Drug or Medicine Genomics = The study of genes Studying response of genetic make up of an individual to a drug or pharmaceutical products Some barriers faced are: complexity of finding gene variation that affect drug response, limited drug alternatives, disincentives for drug companies to make multiple pharmacogenomic products, educating healthcare providers

Phases of Clinical Trials

Phase I Screening for safety Pharmacodynamics & pharmacokinetics Phase II Dose/regimen optimisation Determine optimal dose and regimen Phase I/II Screening for safety (in 'serious' diseases) Primarily a safety study, but with efficacy too Phase III Pivotal studies for marketing application

Animal Functional Feed

Polishing/finishing diets: Taste (flavour changing) Similar applications for farm animals Ruminant livestock belch the methane produced by their gut microbes Livestock methane has a global warming potential 28-times that of carbon dioxide Belching is responsible for more than 6% of global GHG emissions In Australia, 15% of total emissions come from agriculture, two-thirds is from ruminants

Genetic Engineering

Protein engineering Immunochemistry In vitro cell cultivation Bioprocess technology

Drug Discovery Methods

Random Screening Molecular Manipulation Molecular Designing Drug Metabolites Serendipity

Lateral flow strips considerations

Rapid (5-10) minutes Great for at home testing Detects anything an antibody will bind to Often doesn't need sample extraction Sensitivity similar (or worse) than ELISA Only one analyte per strip

Combinatorial Chemistry

Rapid synthesis of or computer simulation of large no. of different but structurally related molecules - Search new leads - Optimization of target affinity & selectivity. - ADME (absorption, distribution, metabolism, and excretion) properties - Reduce toxicity and eliminate side effects

Plasma Membrane Damage

Rapid uptake of propidium iodide and release of 51Cr1 - LK2 mouse UV-induced SCC - B16 mouse melanoma Rapid disruption of plasma membrane - Suggestive of primary necrosis

Phase 0

Recent designation, also known as human microdosing studies. First in human trials, conducted to study exploratory investigational new drug. Designed to speed up the development of promising drugs. Concerned with: preliminary data on the drug's pharmacodynamics and pharmacokinetics, efficacy of pre-clinical studies.

Dereplication databases

SciFinder: a research discovery database containing all natural products MarinLit COMET AntiBase

High-Throughput Screening

Screening of drug target against selection of chemicals. Identification of highly target specific compounds.

Ligand-Based Strategy

Search for similar compounds Database → known actives → structures found

Real Time PCR (Molecular Assays)

Sensitivity: 1 copy of genetic material/mL Specificity: Very specific Speed: 3 hours Cost: Expensive

Tandem mass spectroscopy (e.g. for PKU metabolites) (Direct analyte measurement)

Sensitivity: Femtomolar (10^8 molecules/mL) Specificity: Detects specific analyte Speed: 2 mins Cost: Expensive

Glucose oxidase assay (Enzyme Assays)

Sensitivity: Micromolar (10^14 molecules/mL) Specificity: Specific to D glucose (rare) Speed: Mins Cost: Cheap

Nudibranchs

Smallshell Most well studied group for NPS Some metabolites show antimicrobial &/or antitumor activity Dietary origin An anti‐HIV protein, bursatellanin‐P, was isolated from the purple secretion of an opisthobranch

Patents, drug research, & development

Some people argue that a monopoly (e.g. patent) hinders scientific research, and the development of and access to medicines E.g. millions of people in the developing world do not have access to the medicines they need to treat disease or alleviate suffering

Microorganism Group

Sponges → culturing marine isolates → bioactive compounds → Pseudovibrio sp. W19 (Example) Sponges → culturing marine isolates → bioactive compounds → Sporeformers Bacillus sp. (Example) Clones analysed by activity profile End-sequenced to determine phylogenetic origin Selected clones fully sequenced

The process of gene therapy is of two types

Stem cell gene therapy: In this gene therapy is applied on a fully developed organism and the effects of gene therapy lasts only to the operated organism Germ line gene therapy: In this process gene therapy is done on a fertilized egg or an early embryo and the altered genome is followed in next generations.

Target Selection

Target selection in drug discovery is defined as the decision to focus on finding an agent with a particular biological action that is anticipated to have therapeutic utility — is influenced by a complex balance of scientific, medical and strategic considerations. Target identification: aim to identify molecular targets that are involved in disease progression. Target validation: to prove that manipulating the molecular target can provide therapeutic benefit for patients.

Drug Affinity

The ability of a drug to bind to its biological target (receptor, enzyme, transport system, etc.)

Phase 3: 1st assessment

The data are assessed & evaluation reports (for manufacturing and QC [Module 3]; toxicology [Module 4] and clinical [Module 5]) are completed. Any missing or required data are highlighted. If adequate, recommendations for a decision are made.

Phase 4: 2nd assessment

The data sent in response to the s31 letter are assessed for completeness. - If not the submission is assessed on the basis of available data which usually results in a recommendation of rejection. If the data are adequate they are assessed in round 2 evaluation reports and recommendations to accept or reject are made to the clinical delegate. - Reports sent to sponsor who may review them.

Genetic Testing

The examination of a patient's DNA molecule to determine his/her DNA sequence for mutated genes The genome of an individual is scanned for this purpose by a scientist Uses: forensic/identity testing, determining sex, conformational diagnosis of symptomatic individuals, newborn screening, prenatal diagnostic screening Better drugs can be obtained by the knowledge of genetics Genetic testing can be used to detect the mutations regarding genetic disorders like cystic fibrosis, sickle cell anaemia, Huntington diseases, etc. Tests are also being developed to detect various cancers

Environmental Applications

The major environmental use is for bioremediation. Bioremediation is the use of biotech to process or degrade a variety of natural and manmade products, especially those contributing to pollution Therefore, cleaning up environmental hazards produced by industrial progress is a major application of this type of biotechnology. There is a strong tie to microbial biotech (since many microbes are helpful for this area).

Building the Brand

The trade marks have allowed Pfizer to protect what's trademarked from use and/or misuse by competitors while building trust and brand loyalty among repeat customers.

Tigilanol tiglate

Tigilanol tiglate discovered as the anti-cancer lead from F. picrosperma Complex phorboid terpenoid Natural Product Discovered by the Australian company, EcoBiotics

Stents (Cook medical, Brisbane)

Titanium mesh Hand sewn in Brisbane Customised to fit each and every heart Regulatory approval of the process, not the product.

AK Phase 3: Study design

Treatment Location Non-Head (trunk & extremities) or Head (face & scalp) Patients with 4-8 AK lesions in a 25 cm2 treatment area Number of Patients ~500 total patients Design Randomized, double-blind, vehicle-controlled conducted at multiple sites 2 days of treatment (non-head) / 3 days treatment (head) Concentration = 0.05% (non-head) / 0.015% (head) Endpoints Primary: Complete clearance rate Secondary: Partial clearance rate (clearance of majority of AK lesions within the area) Secondary: Median reduction in lesion count

Viagra Biologics

Unlike traditional drugs made from chemicals, biologics are made from living organisms, engineering by scientists Biologics can cost up to $100,000 annually Biologic drugs produced from living organisms or contain components of living organisms Medicines made up from living cells A highly complex process handled & administered under monitored conditions

Imaging: Computed Tomography (CT)

Use X-rays to take very fine slices (tomographic images) through the body Reconstruct a 3D image Develop anatomic pictures based on ability to absorb X-ray beam. Useful for determining head injuries, lung damage, heart damage etc.

Immunoassays

Use antibodies as the sensing molecules

Some common ways are

Using fat droplets in nose sprays Using cold viruses that are modified to carry alleles ,go into the cell and affect them The direct injection of DNA (might include electroporation or biolistic method)

Are human drugs patentable?

Yes The Patents Act 1990 (Cth) sets out the requirements of a standard patent (s 18(1)) including: - Patentable subject matter ('manner of manufacture') - Novel - Inventive Step - Useful - Not secretly used But s18(2) of the Patents Act excludes 'Human beings, & the biological processes for their generation' from patentability

Anti müllerian hormone

a glycoprotein secreted by fetal Sertoli cells active hormone is 140 kDa homodimeric glycoprotein linked by disulfide bonds The human gene encoding AMH is located on chromosome 19 (in Tilapia is on the Y chromosome) AMH expression is controlled by a male specific protein located on the Y chromosome AMH regulates levels of sex hormones

Theories of IP

stimulate interest (and funding) in research find solutions to problems promote the broader good reward/incentive - drug development is risky & costly

FDA Orphan Drug Approvals

Big pharma: 43% Small biopharma: 19% Established biopharma: 17% Small & medium biopharma: 19%

Canine mast cell tumours

14 yo boxer Cutaneous mast cell tumour on dorsal back Tumour volume: 5.3 cm3 Single injection with 2.6 mL tigilanol tiglate (1 mg/mL) gel

Basis for regulation

Manufacturers have to comply with four levels of governmental control: - The Law: Food, Drug and Cosmetics Act & Public Health Service Act (USA), Directive 2001/83/EC (EU) & Therapeutic Goods Act (Aus) - very general but it is where any penalties are embedded. - Regulations: appendices to the Act changed by Parliament at the request of regulatory body. Specific issues e.g. labelling or fees. - Guidelines: documents produced and maintained by regulatory body. Most specific directions given at this level. - Codes of GMP: Most regulatory bodies have Codes of Good Manufacturing Practice that comply with the Pharmaceutical Inspection Co-operation Scheme (PIC/S)

1b. Genetic development: cell banking

Transfection: Transfect host cells with recombinant plasmids encoding protein of interest Selection of Pool of Transfected Cells: Select cells that are stable & producing protein of interest Clonal Screening & Selection: Screen & select clones for high expression of protein of interest Cell Line Characterisation: Validate & characterise each clone for stability & productivity Expansion & Downstream Evaluation: Expand clones & perform downstream bioprocesses; cell banking is also performed Operation of two-tiered cell banking system - Genetic engineering techniques are used at laboratory scale to create the cells that make up the Master Cell Bank (MCB) - For simplification, the diagram shows 5 vials of MCB stock - In practice, however, there are likely hundreds of vials within the MCB - A single MCB vial is used to prepare a Working Cell Bank (WCB) containing many vials - Over time, new WCBs can be created as others are depleted - Like the MCB, WCBs are stored in an inactive state at -150°C in liquid nitrogen - Typically, each batch of biopharmaceutical produced requires a WCB vial (PR1) to be taken from storage, reactivated & used to initiate the production run

Biotechnology Defintions

use of living organisms, or their products, to create new ways to improve human health and the environment (Department of Industry, Innovation and Science, Australia) the application of science and technology to living organisms as well as parts, products and models thereof, to alter living or non-living materials for the production of knowledge, goods and services (OECD definition)

Research Question and Objectives

"What are Australian consumers' current perceptions of and attitudes toward seaweed as a food product and what would encourage inclusion of seaweed in their diet?" RO1: To determine the current consumption patterns of seaweed foods in Australia RO2: To explore Australian consumers' current perceptions of and attitudes toward seaweed as a food product RO3: To explore demographic and psychological characteristics and influences on seaweed consumption RO4: To identify strategies for encouraging consumption of seaweed in Australia and potential for growing the Australian seaweed industry

Mass Spectroscopy

1. Place sample in vacuum chamber 2. Bombard with electrons to turn into ions (ionization) 3. Accelerate ions through a powerful electric field - highly charged ions accelerate the most 4. Bend the ions using a magnetic field - lighter and more positively charged ions will bend more 5. Detect the number of ions arriving for each mass/charge

Radio Immunoassays

1960: Radio immunoassay (Berson and Yalow) Competitive assay: Known and labelled insulin competes for binding with unknown amount of insulin in sample Problem: Radiolabelling is hazardous. Conjugate an enzyme (e.g. HRP) to an antibody 1971: Perlmann and Engvall (Sweden), Schuurs and Van Weemen (Netherlands), Avrameas and Guilbert (Pasteur Institute, France)

Need for regulation

1970s - Industrial Biotest Laboratories (40% of all US toxicological testing) 3 officers guilty of fraud - 1y or 6min prison (1983) 1996 onwards - nvCJD - from BSE-infected cows Mar → 3 suspected infections from blood in Europe 2003 - Pan Laboratories (70% of Australian non-prescription meds) no GMP & fraud. Company fined & closed. 2008 - Adulteration of heparin - OSCS added in China → 81 deaths, 785 injuries in USA more internationally.

Genetic Engineering Timeline

>20 years ago - No precise control over genetic engineering. - Zinc finger nucleases (Chandrasegaran, PNAS, 1996) TALENS (Transcription activator-like effector nucleases; Voytas, Nucleic Acids Res. 2011) CRISPR: Clustered regularly interspaced short palindromic repeats • Bacterial mechanism to fight bacteriophages Doudna and Charpentier (Science, June 28 2012) - Demonstrated a dual RNA structure can target dsDNA - Engineered dual RNA structure as a single RNA chimera (guide RNA = gRNA)

Cnidarians

A diverse group defined by the presence of stinging cells, called cnidocytes NPs include toxins and venoms, and others that are highly potent antitumor compounds Many species of jellies are bioluminescent. - The largest scyphozoans have tentacles 100+ meters long with a body 2 meters in diameter The sea wasp (Chironex fleckeri) is a member of the Cubozoa class - Its poison, which can subdue fish & other large prey, is more potent than cobra venom Sea anemones & other members of the Anthozoa class only exist as polyps

Dereplication

A drug discovery program aims to search for: novel bioactive NPs, have some for of potent biological activity but isolation of known or undesirable NPs with no bioactivity of interest is inevitable $US 50,000 and 3 months of work to isolate and identify an active NP from its source The process of identifying known compounds responsible for the activity of an extract prior to bioassay-guided isolation is referred to as dereplication Many advanced methodologies and protocols that distinguish novel NPs from known NPs Need bioassay-guided fractionation ,in-house screening, access to NMR and mass spectrometers With the advent of new hyphenated spectroscopy technologies such as HPLC-MS, HPLC- NMR and HPLC-NMR-MS, further means of rapid compound identification and dereplication are now possible

Selectivity

A drug should bind to specific receptor site on the cell

Patent Fees

A full list of fees is available in Schedule 7 of the Patent Regulations 1991 (Cth) - Application - Renewal - Examination - Acceptance Plus, the legal fees

What is a patent?

A patent is a legally enforceable right for a device, substance, method or process A patent gives exclusive commercial rights an invention (i.e. a monopoly) Different types of patents: - A standard patent lasts for up to 20 years. - An innovation patent only lasts for up to eight years. - Pharmaceutical patents can last up to 25 years (Standard patent with an extension of term (EoT))

Vaccines & Drugs

A pharmaceutical drug is a drug used to diagnose, cure, treat, or prevent disease. Important properties include: Molecular structure & production Route of administration (oral (enteral), blood stream (parenteral), intranasal, topical, inhalation, rectal) Biological system affected (circulatory, digestive, respiratory, reproductive, urinary, skeletal, endocrine (hormones), immune, muscular, nervous, integumentary (skin, hair, fat, nails)) Therapeutic effect/mechanism Therapeutic effect/mechanism Stability/Half-life/dosage/break-down products

Implants

Man-made devices (c.f. transplant) Requires special materials (titanium, silicone) - e.g. heart valve/stents; orthopaedic implants; breast implants May contain electronics - E.g. pacemakers and cochlear implants May be bioactive - drug delivery devices - E.g. contraceptives

Echinoderms

About 7,000 species including starfish, sea urchins, and sea cucumbers The bioactive compounds are mainly saponins soap-like foaming when shaken (also found in plants) Properties include dietary supplements, food ingredients, vaccine adjuvants

Ingenol mebutate

Activity guided fractionation led to selection of ingenol mebutate as the anti-cancer lead from E. peplus - Extraction of sap → Test for anti-cancer activity - Fractionate extract → Test for anti-cancer activity - Fractionate semi pure fractions → Test for anti-cancer activity

Enzyme Assays

Add an enzyme that metabolises glucose (e.g. glucose oxidase) Measure the products produced 1962 Clark and Lyons: enzyme electrode for glucose

Microbiology/Animal biology group

Educated knowledge or luck Traditional medicine - at least 266 marine animals Taxonomy

Cone Snails

Numerous small peptides - conotoxins 50,000 identified, 10‐30 amino acids Used in therapeutics Modulate the activity of ion channels Examples - α‐conotoxin inhibits nicotinic acetylcholine receptors at nerves and muscles. - δ‐conotoxin inhibits the inactivation of voltage‐dependent sodium channels. - κ‐conotoxin inhibits potassium channels

Pathway to NDA

Safety and efficacy in target indication and patient population - Clinical trials Access to unapproved therapeutic goods - Clinical Trial Notification scheme (CTN) - Often connected with IND with FDA - Clinical Trial Exemption Scheme (CTX) Investigational New Drug Application (IND) with FDA1

Ingenol mebutate: API & DP stability

API shown to be stable in amber borosilicate glass vials: At least 12 months at various temperatures and humidity's DP shown to be stable in amber borosilicate glass vials: At least 3 months at various temperatures and humidity's

Actinic keratosis

Actinic keratosis (AK) - Chronic UV exposure - Precursor for squamous cell carcinoma - 67% of cases had prior AK diagnosis - 45% of Australians over 40 have AK

Natural product research workflow

Aim: to harness the power of marine organisms to improve the human condition through the discovery of better drugs, alternative fuels and industrial products. Microbiology/Animal Biology group: identify target species and locations Chemistry group: isolate and purify NPs, characterize Marine microorganisms group: isolate and culture microorganisms, bioassay Cell biology group: screen for bioactives, determine the mechanism of action Biotechnology group: identification of biosynthesis pathways, use bioinformatics to find NPs

Marine Algae

Algae are relatively simple eukaryotic photoautotrophs that lack the tissue of plants Unicellular and filamentous algae are found in the ocean Provide oxygen, food for animals, prolific production of unique NPs

Genetic sex markers: DNA level

All cells share the same genomic DNA sequence in their nucleus Every cell type express different sets of RNA and Protein at any given state / developmental stage, environment, etc. Female specific sex markers identified through Amplified Fragment Length Polymorphism (AFLP)

Efficiency and Off-target Mutations

Efficiency favourable compared to TALEN or ZFNs Human cells - >70% in zebrafish and plants, - 2-5% in pluripotent stem cells - 78% in one-cell mouse embryos Off-target mutations are a problem - Need to do whole-genome sequencing to confirm

Summary of biosynthesis

All secondary metabolites, no matter how complex, are biosynthesized via discrete chemically-reasonable steps. The biosynthetic transformations are classified as: hydrolysis, esterification, oxidation (hydroxylation, epoxidation or oxygenation of alkene, dehydrogenation, halogenation), reduction (hydrogenation, deoxygenation), carbon-carbon bond formation (aromatic radical coupling, Claisen condensation, aldol condensation), cationic rearrangement (1,2-migration, Wagner-Meerwein), rearrangement under control of orbital symmetry, Sn2 displacement, E2 elimination, carboxylation / decarboxylation Each step is presumed to be mediated by a specific enzyme. All chemical transformations are accounted for by the system of six enzyme classes: oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase The enzymes are located in specific parts of the cell, and in some cases may be immobilized on a membrane. The enzymes are coded for in the plant's genome whose expression can be controlled at the level of the gene.

Glucose oxidase enzyme assay

Analyte: Glucose Sensing molecules: Glucose oxidase enzyme Transducer/Signal output: Colorimetric: HRP making a colour change Electronic: Electron moving to an electrode

PKU test bacterial inhibition diagnostics terminology

Analyte: Phenylalanine Sensing molecules: Bacteria that require phenylalanine to grow Transducer/Signal output: Visual monitoring of bacterial growth

Animal Feed

Animal feed trials using mixtures with grass Protein (nutrition) and other basic nutrients Protein versus functional components Animal feed trials using mixtures of seaweed with grass or in formulated feeds

From trials to commercialisation

Animal studies - Ethics approval required: ARE & HREC Clinical trials - Exploratory - Phase I - Phase II - Phase III - Phase IV Approval - Data about the drug & trials assessed by the Therapeutic Goods Administration (TGA) Subsidy - Pharmaceutical Benefits Scheme (PBS) Commercialisation - Patent enforcement - Trademarks - Broader marketing strategies - Ongoing regulation

Animal Applications

Animals can be used as bioreactors Many human therapeutic proteins are needed in massive quantities (>100s of kgs), so scientists create female transgenic animals to express therapeutic proteins in milk. Goats, cattle, sheep, & chickens are sources of antibodies (protective proteins that recognize & destroy foreign material) Transgenic refers to containing genes from another source

Phase 3-4: Milestone 4 - s31 response

Any requests for clarification or more data are consolidated into a single section 31 letter which is sent the to the sponsor. The sponsor must reply within the timeframe they nominated (either 1 or 2 months).

Imaging: Magnetic resonance imaging (MRI)

Atomic nuclei absorb and emit radio frequency energy when placed in a magnetic field E.g. Hydrogen ions from water and fat - can help map the location of water and fat in the body. E.g. blood flow in the heart and throughout the body Sometimes uses a gadolinium contrasting dye (e.g. in the liver)

Pharmaceutical Patents: Extension of Term

Australian law provides for extensions of patent term (EoT) of up to five years for certain patents relating to pharmaceutical substances where: - goods containing those substances are included on the Australian Register of Therapeutic Goods (ARTG); and - the time or 'delay' between filing a patent application and listing of the goods on the ARTG is more than five years EoTs 'impose considerable costs on consumers, government, & ultimately taxpayers through the PBS': Productivity Commission (2016)

Transplants

Autograft (take from same body) Transplant (take from different body) - E.g. heart, kidney, liver, lungs, pancreas, intestine, thymus. - Bones, tendons, cornea, skin, heart valves, nerves, veins, - Bone marrow, blood transfusion - Hand (Issues with transplant rejection (requires immunosuppressant drugs)) Xenograft (take from different species) - E.g. porcine heart valve transplants

Microbial Applications

Bacteria & yeast are the most frequently used microbes Better enzymes and organisms for making foods, simplifying manufacture and production processes, and making decontamination processes for industrial waste product removal more efficient. Microbes used to clone and produce batch amounts of important proteins

Antibiotics (Penicillin)

Before penicillin: Infections were as feared as cancer. You could die in days, or have limbs amputated to save your life. Sir Alexander Fleming (1928): discovered a fungus (Penicillium notatum) destroyed staphylococcus bacteria in culture plate Breaks down the cell wall production in bacteria Howard Florey (1939) & team purified and demonstrated they could save the lives of infected mice. Moved production to the USA - Mary Hunt (mouldy Mary) found mould growing in cantaloupe that produced higher levels of penicillin - Mass produced by growing in liquid byproduct of the corn-milling process (requires production under stress, otherwise the antibiotic is not produced) - Developed efficient extraction & purification chemistry By June 1945, over 646 billion units per year were being produced Problem: rapid renal clearance (3-4 hours) & short lived action - Solution: co-deliver with uricosuric agent probenecid (increases blood plasma concentrations of antibiotic) Nowadays: semi- synthetic/synthetic production Additional modifications change the mechanism of action and specificity of the compounds (e.g. Ampicillin, which has a broader spectrum)

Sterility Induction

Benefits: prevent fish from becoming sexually mature - desirable since maturation reduces flesh quality and makes fish more susceptible to diseases, reduce environmental impact in preventing farmed fish from being introduced into wild populations by escapees. Current sterilisation method of choice for aquaculture is to induce triploidy. High pressure or high temperature early in development. Drawbacks: reduced growth rates, skeletal deformities Alternative approaches: CRISPR, RNAi Key issue: delivery

Insulin

Biopharming can use a variety of source organisms to produce drugs Bacteria (e.g. E. coli): engineered to produce Human Insulin - Type 1 Diabetes used to be a death sentence - The body produces little or no insulin (required to help convert glucose into energy) - Without Insulin, blood glucose levels go too high (hyperglycaemia), which causes damage to eyes, kidneys, nerves (& heart disease, stroke) - Originally used extracts from cows or pigs, but these caused harmful side effects and were hard to grow in large quantities and purify - Recombinant Insulin product has less side-effects than purified version, and is easier to produce Mammalian cells (e.g. Chinese Hamster Ovary, CHO cells): engineered to produce coagulation factors

Viagra Biosimilars

Biosimilars are drugs that have proven to be same & effective as originator biologic drugs but at a lower cost Biosimilars are estimated to cost 20-30% less than the originator biologic Biopharmaceutical drugs designed to have active properties similar to the one that has been previously licenced A type of biologics, similar to another biologic drug, which is already approved by the FDA Have no clinically meaningful difference in terms of safety & effectiveness with the reference product

Diabetes

Body unable to metabolise glucose Type 1: immune system destroys pancreas cells, which produce insulin Type 2: body loses capacity to utilise insulin (still being produced), or the body stops making it.

Ebola

Breadth of techniques: Electron microscopy, cell culture, neutralisation, fluorescence Earlier techniques: difficult to mass-produce, lengthy (days), require highly skilled scientists to perform and analyse results, dangerous (having to grow the virus), restricted to PC4 facilities Modern/current techniques, elisa, polymerase chain reaction, easier to mass-produce, faster (hours), not as much skill required to perform assays, and automated results analyse

Quality Control Factors

Buildings and Facilities Equipment Equipment Maintenance, Cleaning, and Calibration GMP Validation Cleaning Validation Production & In-process Controls Materials Management Packaging and Labeling Laboratory Controls Personnel Complaints Recalls Change Control Computer Systems

Pathway to IND

Manufacture of Active Pharmaceutical Ingredient (API) Manufacture of Drug Product (DP) API & DP stability Pre-clinical - Includes toxicology, pharmacokinetics, pharmacology & mechanism of action

NPs from sponges

More than 280 new NPs since 2014 Sponge peptides show a variety of biological activities Example 1: Jasplamide (from genus Jaspis) is antimicrobial towards Candida albicans and antimicrobial against Heliotis virescens Example 2: Haligramides show toxicity against lung and colon cancer cell lines

ELISA Considerations:

Takes ~ 3-4 hours Antibodies can non-specifically bind to related or even unrelated antigens Sensitivity averages in the fmol - pmol range i.e. 10^8 - 10^11 molecules One well = 1 reaction (detects only 1 analyte at a time)

Natural Products

Chemical compound produced by a living organism Often structurally complex and exquisitely optimised to modify specific biological activities Morphine was isolated from opium in 18171 - first demonstration that the activity of a medicinal plant could be attributed to a single chemical constituent

Animal natural alternatives

Climate-friendly farming and a promise of carbon-neutral farming by 2030 Asparagopsis knocks out enteric methanogenesis in cattle when added at <2% of the organic matter Feed additive = small doses

Waste Water Treatment for Aquaculture

Combination of sand filter (biological) and seaweed Additional licence has been granted for Pacific Reef - "zero net discharge" of nitrogen Requires treatment area of <20% facility and a treatment cost of $30/kg nitrogen New licence for 260 ha facility at Guthalungra is now very close - development approval since 2001

Crop Production (Fertiliser)

Compost production Ratio of C:N in the material (mixing leaves [carbon] & seaweed [nitrogen]) Target CN range: 22:1 - 30:1

In Silico Screening

Computer simulated screening of chemicals Helps in finding structures that are most likely to bind to drug target Filter enormous Chemical space More economic than HTS

Monosex in Aquaculture

Culture all-male or all-female populations Why practice monosex? superior growth of males/females (species-specific), gender-specific tailored conditions, better breeding programs (no unplanned reproduction), higher product uniformity, environmentally safer - leakage risk is minimized, avoid theft of genetically improved lines How to produce monosex? Manual segregation, sex reversal: direct or indirect What prior knowledge is needed? genetic background (XY/WZ), what is the preferred gender, molecular mechanism of sex differentiation, timing of sex characters appearance, timing of sex reversal, sex specific DNA markers (to tell between a sex reversed individual and a false-identified one)

Monitoring: Electroencephalography (EEG)

Cumulative signals from 1000s of neurons release change in voltage across the skull. Can be measured by putting a piece of conductive metal against the skull (plus voltmeter) Changes in patterns indicate neural diseases (e.g. epilepsy, sleep disorders)

The Future of Monosex in Aquaculture

Current: mostly steroid administration. Available alternatives: RNAi / recombinant proteins. Setback: delivery and cost.

Biopharming:

Deliberate re-engineering of a biological system to produce/grow a drug Genes for bioproducts are inserted ("recombined") into a host organism The host produces the "recombinant" bioproduct, which can then be extracted and purified.

Recommendations for the Seaweed Industry

Develop convenient and sophisticated seaweed products (healthy, tasty and convenient seaweed snacks) Accentuate and emphasise health and nutritional benefits. Harvesting, production and processing and marketing claims and appeals need to reflect the desire for more sustainable and safer food (safety and quality procedures and regulations (CEVA 2014)) Provide opportunities for trial and development of innovative seaweed products (facilitate trial and experimentation by ensuring seaweed products are on menus, cooking shows, cooking websites and recipe books) Manage sensory characteristics - appearance, smell and texture (Disguise seaweed as a minor ingredient) Capitalise on the association of "you are what you eat" (potential for seaweed to be 'chic' or trendy food choice) Manage perceptions of affordability (Value for money) Gain wider distribution in mainstream food outlets (supermarkets)

Commercial Challenge

Discovery, isolation and development of Natural Products as pharmaceutical drugs is exceptionally challenging - Isolation of active constituents - Inherent variability of source material - Incompatibility of crude extracts with screening techniques - Co-elution of compounds that interfere with bio-assays - Availability of source material for drug manufacture - Uncertainty of ownership of biomaterials posed by the 1992 Rio Convention on Biological Diversity - Inability to gain Composition of Matter patent claims

Ex-Vivo Studies

Experimentation on tissue in an artificial environment outside the organism with the minimum alteration of natural conditions. Counters ethical issues. Examples: measurement of tissue properties, realistic models for surgery

Homogametic males (ZZ) Feminization

Female (androgen treatment) → neo-male X female (75%) → WW female X male → 100% female

Dolly

First cloned mammal from an adult somatic cell Adult cell DNA + unfertilized egg with nucleus removed Place into surrogate Has three mothers

Bisphenol A (BPA) History

First synthesized in the 30's as a synthetic estrogen More potent products (like DES) were found. Rediscovered as a cross-linker for the petrochemical industry. Present in fungicides and general pesticides. Present in industrial products.

Ingenol mebutate: DP manufacture

Formulated to produce a product that is 'fit-for-purpose' Easy to manufacture: stable, easy to use, pleasant to use Formulated in: isopropyl alcohol, hydroxyethyl cellulose, citric acid monohydrate, sodium citrate, benzyl alcohol

Biotechnology Group

Genome | Proteome | Metabolome | Transcriptome Gene identification and cloning to establish heterologous NP biosynthesis Enzyme engineering → cell engineering → reaction engineering Isolate sponge DNA → construct library → transform into E. coli → screen for activity Determination of the biosynthetic pathway enables us to understand the dynamic flow of the compounds Opens the door for biotechnology: Cloning of the biosynthetic genes and biotechnological production Substrate 1 → (enzyme 1) Substrate 2 → (enzyme 2) Substrate 3 → (enzyme 3) End product

Metabolic Reconstruction

Genomic sequence from EuPathDB Template metabolic pathways from MPMP & KEGG Sequence homology search by bidirectional BLASTP Genomes & predicted proteotomes of related species from EuPathDB / KEGG Expression / proteomics evidence from EuPathDB Biochemical / physiological data from scientific literature Protein motif identification by InterPro Predicted proteome from EuPathDB

Application of GnRHa Sustained Release Technology to Induce Spermiation, Ovulation and Spawning

Gilthead seabream Red seabream European seabass Striped bass White bass American shad Gray mullet Grouper Turbot Plaice Flounder Catfish Yellowtail Bluefin tuna Red porgy (pagrus) Dentex Sturgeon Snook Pompano Atlantic salmon Coho salmon Sockeye salmon Chinook salmon Rainbow trout Brown trout Amberjack High hat Pufferfish

What is regulation?

Government regulation Self-regulation (Industry rules & codes) Co-regulation

Monitoring: Blood glucose measurements

Helps monitor the amount of insulin required for Diabetes Type 1 patients Enzyme: Glucose oxidase Sample: containing Glucose Signalling molecule: Gold-plated electrode to accept donated electron (electronics)

Functional Foods

How to test functional foods? - High carbohydrate - high fat rat model - Wistar rats fed HCHF diets for 8 weeks - Seaweed replacement at 5% w/w for 8 weeks - HUO (5% Ulvaohnoi) High carbohydrate, high fat outcomes - Reduced body fat mass with Ulva - No change in triglycerides and cholesterol in plasma High carbohydrate, high fat outcomes - Improved insulin sensitivity - Improved glucose utilization - Reduced plasma ALT activity - Reduced plasma AST activity Salt in our diets: We consume more than twice the daily recommended intake of salt Majority of this salt (90%) is in packaged food, bakery goods and canned foods Food ingredient producers looking to lower salt in their products because of the strong links between salt, blood pressure and increased risk for heart attacks and strokes. Sustainable aquaculture production → fresh seaweed → dried seaweed → existing seaweed processing → carrageenan gel (existing product) → packaged food & beverages New commercial opportunity for co-products → extraction technology → solar salt production → new salt production → packaged food & beverages High carbohydrate - high fat rat model - Wistar rats fed HCHF diets for 8 weeks - Prevention protocol Replacement at 5% w/w for 8 weeks - H (HCHF control) - C (control - corn starch diet) - HRS (5% Kappaphycus whole seaweed) - HSS (2.2% seaweed salt) Substantial change in blood pressure No change in blood glucose relative to control

Can seaweed help feed the world?

Human food Food (basic nutrients, taste) - expanding existing markets Functional food (nutrition and health) - marketing Nutraceuticals (derived health product) - R&D Animal feed Feed (basic nutrients) - poor source of protein Functional feed (specific nutrition or effect) - research leads to additives Feed additive (derived health product) - R&D & marketing Crop production Fertiliser (nutrients: nitrogen, phosphorus, potassium [NPK]) - $ value Soil conditioning (minerals and carbon, soil health) - $ value Fortification (derived health claims) - R&D & marketing Biorefinery turns a single raw seaweed material into multiple products

Fundemental Principles

Human, animal, plant physiology Molecular & cell biology Immunology Microbiology Biochemistry Genetics Chemical engineering

Commercialisation of a therapeutic drug: summary

Identification of lead compound Patent protection Manufacture/quality of API Manufacture/quality of DP Non-clinical safety Clinical safety Clinical efficacy New Drug Application Sales & Marketing

Australian Drug Regulations

In Australia, all new drug applications are classified as Category 1 applications. - Clinical trials must be notified or exempted (CTN/CTX). - Data evaluated by the TGA: - Clinical by the Medicines Authorisation Branch; - Toxicological by the Pharmaceutical & Chemical Evaluation Section of Scientific Evaluation Branch (SEB); - Manufacturing & QC by Biological Sciences Section, SEB assisted by the Laboratories Branch. The data must comply with - the TG Act (1988), - Associated TG Regulations - Australian Regulatory Guidelines for Prescription Medicines. - Australian Code of GMP. If further data are required they are requested under Section 31 of the Act. Final decision made by the clinical delegate, with advice from primary evaluators, Australian Committee for Medicines and the Pharmaceutical Subcommittee.

Tigilanol tiglate drug development: veterinary oncology

In late stage development as a veterinary therapeutic for canine mast cell tumours - Canine MCT is a common neoplastic disease ~16-21% of all cutaneous canine neoplasms Phase III clinical trial complete Regulatory submissions to EU and US Q4 2018 Recently signed a marketing and distribution agreement with Virbac, one of the worlds largest dedicated animal health companies

Ingenol mebutate: API manufacture

Ingenol mebutate is a complex NP and cannot be synthesised commercially API isolated from dried E. peplus using chromatography - E. peplus grown in SE Qld - Ingenol mebutate purified at a custom-built facility on the Gold Coast

Ecology

Involved in a host of ecological functions1, for example - protection of plants from herbivores and pathogens (e.g. alkaloids) - triggers of social responses within species (e.g. pheromones) - influence seed germination, plant growth, plant survival, and reproduction of competing organisms (e.g. allelochemicals)

Physiology

Involved in many essential physiological processes, for example - cellular respiration (e.g. haemoglobin) - photosynthesis (e.g. chlorophyll) - cell membrane structure (e.g. cholesterol) - regulation of growth and development (e.g. oestrogen, testosterone & thyroxine)

Sustainability

It is difficult to cultivate most marine invertebrates on a large-scale. For example, ET-743 (trabectedin) is a promising anti-cancer agent yet 1 metric ton (wet weight) of the tunicate Ecteinascidia turbinate has to be harvested and extracted in order to obtain ~1 g ET-734 Therefore, economically viable chemical synthesis techniques are essential for the large-scale development of these products. Chemical synthesis - depends on complexity, or can produce an analog Controlled harvesting - mostly not an option Aquaculture In vitro production Microbial fermentation Coral reefs declining

Monitoring: Pulse oximetry

Measures oxygen saturation Haemoglobin in our blood carries oxygen away from the heart/lungs (red) When depleted it returns to get reloaded (blue) LED lights (one red 600nm, one infrared 940nm) shines through a translucent part of the body. The amount of light absorbed is measured. The ratio of red to infrared indicates the amount of haemoglobin with and without oxygen.

Real Time PCR

Mechanism: Similar to PCR except an additional probe region enables conversion to a fluorescent signal

Nutraceuticals

Need to extract and concentrate the functional components (fibre or salt) Ulvan is 9-15% DW; Fucoidan from brown seaweed is 3-16% DW, US$500/kg

GLP: Good laboratory practice

Non-clinical health and environmental safety studies - Covers work done in a laboratory, in an animal house, in greenhouses, and in the field. Australia has adopted the OECD guidelines & ISO 17025 Compliance at the level of: - Personnel responsibilities - Quality assurance programs - Facilities: general, test systems, handling test and reference items, archives and waste - Equipment, materials and reagents - Test systems (chemical/biological) - Reference items: Receipt, handling, sampling, storage, characterisation - Standard operating procedures - Study plan: content, conduct, reporting - Storage and retention of materials

Tigilanol tiglate in vivo anticancer activity

Over 300 companion animals treated (e.g. dogs, cats & horses) Single intra-lesional injection with EBC-46 >100 cases have been formally monitored Cure resulted in more than 30% of cases - >50% reduction in tumour size in a further 40% of cases

Natural Products in Marine Biotechnology

Over 62% of small molecule agents approved for use as drugs can be traced back to NPs Examples: aspirin (willow/birch), morphine (poppy), penicillin (fungus), EBC‐46 (plant) Requires skills in the areas of organic chemistry, medicinal chemistry,ethnobotany, traditional medicine and ethnopharmacology Other biological areas include chemical biology, chemical ecology, chemogenomics, and systems biology

Seaweed industry development

Pathway to an Australian seaweed industry: - Work closely with existing aquaculture and agriculture companies - Develop local seaweed markets with global relevance - Link environmental benefits to existing business needs Locations: Moreton Bay, Bribie Island Research Centre - Seawater intake - Ultra-purified water; flow through system - Temperature controlled rooms and outdoor facilities

Marine Algae Applications

Phycoremediation Food & nutraceuticals Animal & fish feed Biofertilisers Cosmeceuticals & pharmaceuticals Food colourants, natural dyes, & fluorophores Biofuel Papermaking Bioplastics

Primary necrosis

Pronounced swelling of mitochondria - B16 mouse melanoma - Ca flux? Rapid disruption of plasma membrane and organelles - Suggestive of primary necrosis

Typical Patenting Process & Cost

Provisional (12 months): $5-$10 thousand Complete (18 months): $10-$15 thousand National phase (2-24 months): $2-$200 thousand Renewal (ongoing): $1-$10 thousand Challenge (years): $1 million & greater

Spawning Induction

Published the first induced spawning in fish, 1930 Won the Nobel Prize in Medicine, 1947 for the role of the pituitary in carbohydrate metabolism and in diabetes the central role of the pituitary gland Application of GnRHa Spermiation Technology to Induce Ovulation and Spawning Sustained Release , Works in ~30 fish pieces Requires R&D to understand where the issue is. Understanding the endocrine axis: GnRH → LH/FSH → Steroids GnRH analog implant can induce oocyte maturation which leads to ovulation and spawning

PCR Considerations

Takes ~3-4 hours Can be very specific (primer sequence dependent) Can be extremely sensitive (1-10 molecules!) Highly susceptible to contamination Susceptible to PCR inhibitors Requires nucleic acids to be extracted & purified from the sample first Performed in centralised facilities by skilled personnel Limit to the number of fluorophores (detect up to ~4 analytes in a single reaction)

Orphan Drugs Advantages

Tax incentives. Enhanced patent protection and marketing rights. Clinical research financial subsidization. Rise in research and development.

Use of CRISPR in treating cancer

Remove T cells from patents with cancer 1. Insert gene to detect cancer and instruct T cells to target them 2. Remove natural T- cell protein that interferes with this process 3. Remove the gene for a protein that prevents the cancer cells from disabling them

Environmental outcomes to economic development

Seaweed extracts nitrogen as it grows and this is valuable to polluters Wastewater Treatment: Industry standards: $20 to $45/kg of nitrogen Diffuse sources: $50 to >$250/kg of nitrogen ($150/kg recommended) Aiming for win-wins: environment, agriculture and economy

Seaweed or crickets

Seaweed is a superfood UK chef Jamie Oliver describes it as "the most nutritious vegetable in the world". Seaweed traditional diet of many Asian countries - Malaysia, China, Iceland, Canada, Japan, Korea, Singapore and some areas of Scandinavia traditionally eat seaweed (Brownlee et al. 2012; Fleurence et al. 2012; Prager 2017). Less commonly consumed in Western countries - not part of the traditional diet Globally the seaweed industry is growing - more than 20 million tonnes of seaweed grown/harvested annually - about half from China (Paul, Tseng & Borowitzka 2013). - global crop is US$6 billion - about half for human consumption (Flannery 2017). - 12,000 species - 500 currently used for human consumption (Prager 2017). - Australia has an extensive coastline and over 6000 species of seaweed - seaweed increasingly featured on menus, TV cooking shows and in cooking books - What are western consumers' perceptions of seaweed as a food product? - unfamiliar (novel) food product - consumers may find it intrinsically unappealing

ELISA (Immunoassays)

Sensitivity: Femtomolar to picomolar (10^11-10^8 molecules/mL) Specificity: Antibodies can cross-react Speed: 4 hours Cost: Cheap

PKU bacterial inhibition assay (Bacterial Assays)

Sensitivity: Micromolar (10^14 molecules/mL) Specificity: Other diseases may cause growth Speed: Days Cost: Cheap

Lateral Flow (Immunoassays)

Sensitivity: Picomolar (10^8 molecules/mL) Specificity: Antibodies can cross-react Speed: 5 mins Cost: Cheap

Culturing for specific diseases (Bacterial Assays) Bacteria: agar Viruses: cell culture

Sensitivity: Single infectious unit (~1 molecule/mL) Specificity: Other things may grow Speed: Days Cost: Cheap

Ingenol mebutate: Preclinical toxicology, PK & pharmacology

Series of rat, rabbit and minipig studies aimed toward the development of a short course topical application - Contracted to various Contract Research Organisations - Covance Laboratories, Charles River Laboratories, Battelle, Huntingdon Life Sciences

Chemistry group

Several sample preparation, pre‐purification and clean‐up steps are used prior to isolation and/or analysis of natural products Initial extraction with low‐polarity solvents yields the more lipophilic components, while ethanolic solvents obtain a larger spectrum of non‐polar and polar material If a more polar solvent is used for the first extraction step subsequent solvent partition allows a finer division into different polarity fractions High performance liquid chromatography - a form of liquid chromatography used to separate the components of a mixture - The separation occurs because each component in the mixture interacts differently with the stationary phase - Molecules that interact strongly with the stationary phase (yellow component) will move slowly through the column, while the molecules that interact less strongly (blue component) will move rapidly through the column Equipment: GC-MS, HPLC, Mass spectrometer, Nuclear magnetic resonance

Sex determination and sex differentiation

Sex determination is a genotype dependent mechanism (according to the sex chromosomes) whereby an individual acquires the gonad type characteristic of one gender of the species (with the exceptions of TSD and sexual plasticity) Sexual differentiation is the process of changes in cells of indifferent gonads that lead to formation of embryonic testes or fetal ovaries as well as processes related to the acquisition of phenotypic sex Heterogamy XY/XX; WZ/ZZ; X/Y; W/Z

Direct masculinization, any genetic system

Sexually undifferentiated fish (androgen treatment) → all-male stock Direct masculinization: Nile tilapia - faster growth. Efficient, routine throughout the world. Indirect masculinization: Nile tilapia (YY males). Blue tilapia (ZZ females).

Direct feminization, any genetic system

Sexually undifferentiated fish (estrogen treatment) → all-female stock Direct feminization: Rainbow trout - late sexual maturity, faster growth, superior flesh quality. Incomplete sex reversal, hermaphrodites with ovotestes. Indirect feminisation: Grass carp - faster growth. Implants (not eating artificial food).

Natural Products Benefit to Humans

Significant source of important commercial products: food flavourings, fragrances, pigments, therapeutic drugs Products from plant Natural Products estimated to be worth in excess of $US150 billion worldwide per year

Marine Sponges

Simple animals: no nervous system, no internal organs Sessile filter feeders: 1000 L/kg/hr Chemical defence system: important source of new bioactive NPs ~7,000 species: ~1,400 species described in Australian waters (of ~5,000), Queensland museum has 400 species, becoming more dominant A drug treasure First marine NPs from marine sponge, C-nucleosides, Spongouridine, Spongothymidine Possess antiviral activity Sponge samples for antimicrobial screens

Where are the natural products?

Since less than 10% of the world's biodiversity has been evaluated for potential biological activity, many more useful natural lead compounds await discovery with the challenge being how to access this natural chemical diversity The dominant source of knowledge of NP uses from medicinal plants is a result of man/woman experimenting by trial and error for hundreds of centuries through palatability trials or untimely deaths, searching for available foods for the treatment of diseases

Phase 1: Pre-submission

Six weeks before the proposed submission date, the sponsor lodges an online pre-submission planning form (PPF), including a summary of the data and some critical reports. The TGA assesses whether this information is sufficient. - If so a planning letter detailing milestone dates is sent to the sponsor. - If not, the PPF is rejected, though a new PPF may be submitted a month later.

Bisphenol A (BPA) in vivo effects

Skin exposure damages kidney, liver, spleen and lungs. Earlier sexual maturity In young female rodents changes in the ovaries and uterus were detected

Chemical Synthesis Advantages

Studies can be completed in short period of time. Reduces risk in later trials Permits an enormous level of simplification of the system • investigator can focus on a small number of components

Subsidies (Pharmaceutical Benefits Scheme)

Subsidised prescription medication pursuant to the National Health Act 1953 (Cth) 80% subsidy for approved drugs 100% subsidy for hospital patients Additional concessions for eligible people (concession card)

Cochlear Implant (Australian invention, 1977/78)

Surgically implanted device Provides sound to profoundly deaf people Microphone outside the skin bypasses normal hearing process Transmits signal to electrodes placed in the cochlear, which stimulate the cochlear nerve.

Aquaculture Biotechnology Solutions

Surrogate technology Cryopreservation Sterility induction Spawning induction Monosex

F. picrosperma in vivo anticancer activity

Survival of C57BL/6J mice with B16-F0 tumours. Mice with two tumors were treated with a single injection of - vehicle alone (20% propylene glycol in water; light grey) - 50 nmol (30 mg) PMA (mid grey) - 50 nmol (30 mg) EBC-46 (black) Difference between survival following treatment - EBC-46 was significant (p = 0.0004) - n = 12.

5. Quality Control

Testing of active drug substance & drug product - Identity - Content - Activity/potency - Quantitation of impurities - Process-related (solvents, leachates, HCP and DNA) - Product-related (oxidation, deamidation, proteolysis, aggregation & dissociation) - Glycosylation (monosaccharides, sialic acid, glycan map) - Sterility - Endotoxins Validation - All tests are done by standard operating procedure (SOP) to specification with a reference standard. - Tests are validated for specificity,accuracy, precision & robustness. - Each chromatography step is validated for: specificity; capacity; purification and yield; and viral & TSE clearance. - Cleaning, re-use & of columns, storage & testing of any intermediates must all be justified and specified.

Cell Biology Group

Tests ability of compounds to alter specific signaling pathways All assays are cell based Target‐based screening assays Molecules that regulate cell proliferation and survival Molecules that regulate inflammation Molecules that affect mast cell degranulation and migration Used assays should be rapid and simple, not very expensive, specific Using cell biological assays to identify the active samples

Phase 2: Submission

The application is submitted in electronic CTD format (with fees) at least two weeks before the proposed submission date. The TGA assesses whether the data are complete and what was promised in the PPF. - If so, a notice of acceptance is sent to the sponsor and evaluation begins on the agreed date. - If not, the submission is rejected (with loss of 25% of fees). - A new PPF may be submitted.

Phase 5: expert advice

The clinical delegate produces a summary for the Advisory Committee for Medicines (ACM) to which the sponsor may respond. These external experts discuss the reports at the meeting each month and make a recommendation in the ACM resolutions to the clinical delegate. If necessary, issues of pharmaceutical concern are referred to the Pharmaceutical Subcommittee who also advise the delegate.

Phase 6: decision

The clinical delegate reviews the evaluation reports and recommendations of the TGA evaluators and the resolutions of the expert advisory committees. On the basis of these he/she makes a decision to accept or reject the medicine for registration in Australia. - He/she prepares and sends a letter informing the sponsor of the decision.

1a. Gene discovery or production

The gene is isolated or made by: Probing genomic or cDNA libraries with degenerate primers (from amino acid sequence) or precise probes from human genome. Analogues of known or novel proteins were made by: site-directed mutagenesis or direct synthesis. The gene is tested for viral safety. Depending on the plasmid used, expression factors are added or modified.

Secondary metabolites (Natural Products)

The mechanism by which an organism biosynthesizes compounds called ‛secondary metabolites' (natural products) is often found to be unique to an organism, or is an expression of the individuality of a species and is referred to as "secondary metabolism" Generally not essential for the growth, development or reproduction of an organism Are produced either as a result of the organism: adapting to its surrounding environment, or as a possible defense mechanism against predators to assist in the survival of the organism Derived from the fundamental processes of photosynthesis, glycolysis and the Krebs cycle to afford biosynthetic intermediates - Examples: Acetyl coenzyme A (acetyl‐CoA), shikimic acid, mevalonic acid, 1‐deoxyxylulose‐ 5‐phosphate 4 structural classes: Alkaloids, Phenylpropanoids, Polyketides, Terpenoids

Phase 7: post-decision

The sponsor may appeal the decision. - As soon as the decision is ratified, the product details are registered onto the Australian Register of Therapeutic Goods (ARTG). A summary of the evaluation of the sponsor's data and the factors leading to the decision (called the AusPAR) is prepared and, with the agreement of the sponsor, is published on the TGA website.

GCP: Good clinical practice

This is an internationally accepted standard for the designing, conducting, recording and reporting of clinical trials. The TGA has adopted the EU/ICH guideline and ISO 14155 Compliance at the level of: - Institutional Review Board / Independent Ethics Committee - Investigator - Qualifications; Care of Trial Subjects; Compliance with Protocol; Informed Consent of Trial Subjects; & Records and Reports. - Sponsor - Quality Management, Assurance and Control; Trial Design, Management, Data Handling, and Record Keeping; Compensation to Subjects and Investigators; Manufacturing, Packaging, Labelling, Coding, Supply and Handling Investigational Product(s); & Adverse Drug Reaction Reporting. - Monitoring - Auditing - Clinical Trial Protocol and Protocol Amendment(s)

E. peplus sap: Human clinical trial

Topical treatment using ~200 μl E. peplus sap Once a day for three consecutive days 44 patients, 61 NMSC lesions ~80% lesions cured

Assay Development

Used for measuring the activity of a drug. Discriminate between compounds. Evaluate: expressed protein targets, enzyme / substrate interactions.

Genetic Engineering Process

Uses the cellular double-strand break repair mechanism to reattach broken DNA strands (two different mechanisms)

Regulation of sex differentiation (induce sex reversal)

Usually through administration of synthetic estrogens/androgens: not environment friendly Temperature manipulation: does not apply to most species

ELISA

enzyme-linked immunosorbent assay

Therapeutic Drugs

~1560 novel drugs were approved between 1981 & 2014 - Of those, ~1210 were small molecules - Of those, ~50% of those are Natural Products - NPs (N), derivatives of NPs (ND), synthetic analogues of NPs (S* & S*/NM)

Isogenic human disease models

A family of cells that are selected or engineered to accurately model the genetics of a specific patient population, in vitro

Biotechnology

A form of technology that uses living organisms, usually genes, to modify products, to make or modify plants and animals, or to develop other microorganisms for specific purposes.

CYP450 applications in Biotechnology

Activity, stability Bioremediation Medicine Biotechnology CSM Rational Activity, peroxide Expression, stability Directed evolution (random mutagenesis → screening selection)

Stem cell disease models

Adult or embryonic stem cells carrying or induced to carry defective genes can be investigated in vitro to understand latent molecular mechanisms and disease characteristics

Agricultural Applications

Agricultural Biotechnology is estimated to be $6 billion market (2005), including applications such as: - Pest-resistant plants - Higher protein & vitamin content in foods - Drugs developed and grown as plant products - Drought-resistant, cold-tolerant, and higher-yielding crops

Orphan Drugs

An orphan drug is a pharmaceutical agent that has been developed specifically to treat a rare medical condition, the condition itself being referred to as an orphan disease. National Organisation for Rare Disorders European Organisation for Rare Diseases 7,000 Orphan Diseases (each affecting < 2 million persons; total 300 million affected) Unprofitable drug development Orphan Drug Status given to <1,000 Drugs 1985 Amendment: Marketing Exclusivity

Top 10 Selling Biotechnology Drugs

Betaseron (Multiple sclerosis) Ceredase (Gaucher's disease) Engerix B (Hepatitis B vaccine) Epiver (Anti-HIV) Epogen (Red blood cell enhancement) Genotropin (Growth failure) Humulin (Diabetes) Intron (Cancer & viral infections) Neupogen (Neutropenia reduction) Procrit (Platelet enhancement)

Bioinformaitics Target Selection

Bioinformatics is a branch of molecular biology that involves extensive analysis of biological data using computers, for the purpose of enhancing biological research. It plays a key role in various stages of the drug discovery process including target identification computer screening of chemical compounds pharmacogenomics Computer based (in silico) approaches. Omics data crunching used to transform the raw sequence into meaningful information (eg. genes and their encoded proteins) and to compare whole genomes (disease vs. not). $600 US per genome

Bioinformatics

Bioinformatics is a branch of molecular biology that involves extensive analysis of biological data using computers, for the purpose of enhancing biological research. It plays a key role in various stages of the drug discovery process including target identification, computer screening of chemical compounds, pharmacogenomics Computer based (in silico) approaches. Omics data crunching used to transform the raw sequence into meaningful information (eg. genes and their encoded proteins) and to compare whole genomes (disease vs. not). $600 US per human genome Once target is identified - computer simulations can be used to identify potential drugs: Structure prediction → core binding pocket 3D alignment → identify drug templates → docking simulations and molecule refinement

Chemical Synthesis Examples

Cells derived from multicellular organisms Subcellular components (Ribosomes, mitochondria) Cellular / subcellular extracts (wheat germ, reticulocyte extract) Purified molecules (DNA, RNA, proteins)

Phase 1

Clinical Pharmacologic Evaluation First stage of testing in human subjects. 20-50 Healthy Volunteers Concerned with: human toxicity, tolerated dosage range, pharmacology & pharmacodynamics Types of Phase-I Trials: SAD (Single Ascending Dose), MAD (Multiple Ascending Dose), food effect (on drug ADME properties)

Library Development

Collection of stored chemicals along with associated database. Assists in High Throughput Screening Helps in screening of drug target (hit) Based on organic chemistry

Molecular Docking

Computational determination of binding affinity between molecules (protein structure and ligand). Given a protein and a ligand find out the binding free energy of the complex formed by docking them.

In Silico Screening Medicinal Chemistry

Computer simulated screening of chemicals Helps in finding structures that are most likely to bind to drug target. Filter enormous chemical space More economic than HTS

Phase II

Controlled Clinical Evaluation. 50-300 Patients Controlled Single Blind Technique Concerned With: Safety, Efficacy, Drug Toxicity, Drug Interaction

Forensic Applications

DNA fingerprinting is the classic example of a forensic application. It is used most commonly for law enforcement and crime scene investigation (CSI). It was first used in 1987 to convict a rapist in England. Other applications of DNA fingerprinting include: identifying human remains, paternity tests, endangered species (reduces poaching), epidemiology (spread of disease)

Drug Development Cost Break-Up (%)

Discovery/Basic Research - Synthesis & Extraction: 10.0 - Biological Screening & Testing: 14.2 Preclinical Testing - Toxicology & Safety Testing: 4.5 - Pharmaceutical & Dosage Formulation: 7.3 Clinical Trials - Phase I, II, & III: 29.1 - Phase IV: 11.7 - Manufacturing & QC: 8.3 - IND & NDA: 4.1 - Bioavailability: 1.8 - Others: 9.0 Total: 100.0

Drug Affinity & Selectivity

Drug affinity is the ability of a drug to bind to its biological target (receptor, enzyme, transport system, etc.) Selectivity: A drug should bind to specific receptor site on the cell Salicylic acid (aspirin) produced from willow (Salix alba) Binds COX and relieves pain. Higher affinity to COX-1 Since then >10 new drugs developed with varying specificity

Whole genome sequencing now possible for IVF

For the first time, a team of international scientists have successfully used a specific technology to edit human DNA, to prevent disease. Their study, published in Nature, showed that, through the use of CRISPR-Cas9 gene editing, a gene mutation causing hypertrophic cardiomyopathy (HCM) could be removed from an embryo. By then implanting the modified embryo into a uterus using IVF, the inheritance of this disease can be avoided. Prior to this, there was no prevention or cure for HCM, which can cause sudden death and affects 0.2 percent of people.

Future Biotechnology Treatments

Gene therapy (replace "defective" gene with "normal") Tissue engineering (designing & growing tissues for use in regenerative medicines). 1st Genetically Modified Organism (GMO) to produce human protein was E. coli introduced with DNA to produce somatostatin (hGH - human growth hormone - 1977)

Omics cascade: From genome to phenome

Genome: what can happen Transcriptome: what appears to be happening Proteome & Metalloproteome: what makes it happen Metabolome & Ionome: what has happened & is happening Phenotype

The top four most utilized omics (Google Scholar articles)

Genomics (genes): 654 000 Transcriptomics (mRNA): 69 300 Proteomics (proteins): 93 900 Metabolomics (metabolites): 31 800

Business Applications

Healthcare & pharmaceuticals Food innovations & food processing Fermentation technology Diagnostics Animal agriculture Plant, agriculture, & crop improvement Energy & environmental management

Funding Distribution

Healthcare: 69% (20.28% of market) Plant agriculture: 13% Animal agriculture: 8% Chemical & food: 5% Other: 4% Energy & environment: 1%

Structure-Activity Relationship (SAR) Studies

Helps identify pharmacophore The pharmacophore is the precise section of the molecule that is responsible for biological activity Enables to prepare more active compound Allow elimination of excessive functionality Molecule modified in many different ways Defines the active part Refines the activity 50% of world Morphine and Codeine produced from poppy fields grown in Tasmania

Importance of Pharmacogenomics

Helps in the development of tailor made medicines Ensures more appropriate methods of determining drug dosages Improve process of drug discovery and approval Obtaining of better and safer vaccination Decrease in the overall cost of Health Care Advanced Screening for Disease

Cellular & Genetic Targets

Identification of the function of a potential therapeutic drug target and its role in the disease process. For small-molecule drugs, this step in the process involves identification of the target receptors or enzymes whereas for some biologic approaches the focus is at the gene or transcription level. Drugs usually act on either cellular or genetic chemicals in the body, known as targets, which are believed to be associated with disease. Scientists use a variety of techniques to identify and isolate individual targets to learn more about their functions and how they influence disease. Compounds are then identified that have various interactions with the drug targets that might be helpful in treatment of a specific disease.

Oil Spill

In the 1970s, the first U.S. GMO patent was granted to a scientist for a strain of bacteria capable of degrading components in crude oil. In 1989, the Exxon Valdez oil spill in Alaska used Pseudomonas species (oil-degrading bacteria) to clean up the spill It was 3x faster & without increased environmental effects

Medicinal Chemistry

Intersection of synthetic organic chemistry and pharmacology. Focuses on small organic molecules (and not on biologics and inorganic compounds) Used in: drug discovery (hits), lead optimization (hit to lead), process chemistry and development

Proteomics

It is the study of the proteome, the complete set of proteins produced by a species, using the technologies of large - scale protein separation and identification. It is becoming increasingly evident that the complexity of biological systems lies at the level of the proteins, and that genomics alone will not suffice to understand these systems. It is also at the protein level that disease processes become manifest, and at which most (91%) drugs act. Therefore, the analysis of proteins (including protein-protein, protein- nucleic acid, and protein ligand interactions) will be utmost importance to target discovery. Proteomics is the systematic high-throughput separation and characterization of proteins within biological systems. Target identification with proteomics is performed by comparing the protein expression levels in normal and diseased tissues. 2D PAGE is used to separate the proteins, which are subsequently identified and fully characterized with LC- MS/MS. Further complexity lies at the post-translational modification level: dynamic and integrated pathways. Upon binding to its receptor on fat cells, insulin phosphorylates more than 5,000 proteins within 10 minutes

Proteomics Target Selection

It is the study of the proteome, the complete set of proteins produced by a species, using the technologies of large - scale protein separation and identification. It is becoming increasingly evident that the complexity of biological systems lies at the level of the proteins, and that genomics alone will not suffice to understand these systems. It is also at the protein level that disease processes become manifest, and at which most (91%) drugs act. Therefore, the analysis of proteins (including protein-protein, protein- nucleic acid, and protein ligand interactions) will be utmost importance to target discovery. Proteomics is the systematic high-throughput separation and characterization of proteins within biological systems. Target identification with proteomics is performed by comparing the protein expression levels in normal and diseased tissues. 2D PAGE is used to separate the proteins, which are subsequently identified and fully characterized with LC- MS/MS. Further complexity lies at the post-translational modification level: dynamic and integrated pathways. Upon binding to its receptor on fat cells, insulin phosphorylates more than 5,000 proteins within 10 minutes

Functional Imaging

Method of detecting or measuring changes in metabolism, blood flow, regional chemical composition, and absorption. Tracers or probes used. Modalities Used: MRI, CT-Scan

Top Companies by R&D Expense ($b)

Novartis: 7.9 Merck & Co: 8.1 Roche: 7.8 GlaxoSmithKline: 5.7 Sanofi: 5.8 Pfizer: 9.1 Johnson & Johnson: 4.5 Eli Lilly: 4.7 AstraZeneca: 4.2 Takeda: 3.4 Bayer: 2.3 Bristol-Myers Squibb: 3.3 Boehringer Ingelheim: 3.1 Amgen: 2.8 Novo Nordisk: 1.7

General Bioinformatics

Omics provides platform to discover disease causative at a rapid pace Bioinformatics enables shortening the time from hit to lead Machine learning techniques might provide computer-based pipelines from disease to lead Comparison of Sequences: Identify targets Homology modelling: active site prediction Systems Biology: Identify targets Databases: Manage information In silico screening (Ligand based, receptor based): Iterative steps of Molecular docking. Pharmacogenomic databases: assist safety related issues

tPA

One of the first genetically engineered (GE) products sold was tissue plasminogen activator (tPA) tPA is a blood clot dissolving enzyme used immediately after a heart attack or stroke to clear blocked vessels

Lead Candidate Refinement

Optimizing chemical hits for clinical trial is commonly referred to as lead optimization The refinement in structure is necessary in order to improve: potency, oral availability, selectivity, pharmacokinetic properties, safety (ADME properties)

Ecdysteroid in plants as pesticide?

Phytoecdysteroids - Plant-derived ecdysteroids synthesized for defense against phytophagous (plant eating) insects. When insects eat the plants with these chemicals they may prematurely molt, lose weight, or suffer other metabolic damage and die. Over 250 ecdysteroids identified so far, > 1,000 possible structures might occur in nature.

Phase IV

Post Marketing Surveillance. Designed to detect any rare &/or long-term adverse effects Adverse Drug Reaction Monitoring. Pharmacovigilance.

History of Drug Discovery

Pre 1919: herbal drugs, serendipitous discoveries 1920s-1930s: vitamins, vaccines 1940s: antibiotic era, R&D boost due to WW2 1950s: new technology, discovery of DNA 1960s: breakthrough in etiology 1970s: rise of biotechnology, use of IT 1980s: commercialisation of drug discovery, combinatorial chemistry 1990s: robotics , automation 2000s: pharmacogenomics, machine learning

Top CROs by Revenue

Quintiles: $2.5 Billion Pharmaceutical Product Development: $1.8 Billion Covance: $1.4 Billion Charles River Laboratories: $1.2 Billion Parexel: $930 Million Icon: $887 Million Kendle: $590 Million Pharmanet: $470 Million PRA International: $410 Million 4G Pharmacovigilance: $391 Million

Genomics Target Selection

RAD-Seq: quick way to compare ~1% of the genome between individuals Advantage: easier to identify SNPs

Synthesis & Isolation

Separation of mixture Separation of impurities In vitro chemical synthesis Biosynthetic intermediate

Clinical Trials

Set of procedures in medical research and drug development to study the safety and efficacy of new drug. Essential to get marketing approval from regulatory authorities. May require up to seven years

Genes for Jeans

Stonewashed jeans use genetically engineered enzymes (amylase & cellulase) to create a faded look Originally, pumice stones were used (jeans washed with the stones) This method damaged the machines

Animal models of disease states

Test conditions involving induced disease or injury similar to human conditions. Must be equivalent in mechanism of cause. Can predict human toxicity in 71% of the cases. Eg. SCID mice - HIV, NOD mice - Diabetes, Danio rerio - Gene function

Plant Advantage

The Ag-Biotech field boasts about the plant advantage over microbial biotech. Plant advantage refers to the fact that the cost of producing plant material with recombinant proteins is often significantly lower than bacteria Also, the Ag biotech may combine with medical biotech in order to produce drugs with molecular pharming

Gene Therapy

The process in which a faulty gene is removed or replaced with its healthy copy to restore the normal function of that gene Replacing a mutated gene that causes disease with a healthy copy of the gene Inactivating or "knocking out" a mutated gene that is functioning improperly Introducing the new gene that help fight a disease

Drug Production

The process in which pharmaceutical products are produced through application of biotechnological techniques Medicines are produced for: diagnosis, cure treatments, disease prevention Recently, plants are being genetically modified to produce pharmaceutical products instead of their natural compounds For Example: A drug Elelyso for treating Gaucher is being produced by genetically engineering carrots Insulin: Human insulin is being produced using genetic engineering technique known as humulin and it is used for the treatment of diabetes.

Genomics

The study of genes and their function. Genomics aims to understand the structure of the genome, including the mapping genes and sequencing the DNA. Seeks to exploit the findings from the sequencing of the human and other genomes to find new drug targets. Human Genome consists of a sequence of around 3 billion nucleotides (the A C G T bases) which in turn probably encode 35,000 - 50,000 genes. Estimated number of genes implicated in disease (both due to defects in single genes and combinations of genes): ~ 1,000 Based on 5 or 10 linked proteins per gene, number of potential drug targets ~5,000 to 10,000. Single Nucleotide Polymorphism (SNP) libraries: are used to compare the genomes from both healthy and sick people and to identify where their genomes vary. RAD-Seq: quick way to compare ~1% of the genome between individuals

Modern Biotechnology

There are more than 80 biotech drugs, vaccines, and diagnostics with more than 400 biotech medicines in development targeting over 200 diseases Nearly 50% of new drugs target cancer 1982 - Genentech developed Humulin (human insulin) to treat diabetes - first FDA approved biotech drug.

Behavioural Studies

Tools to investigate behavioral results of drugs. Used to observe depression and mental disorders. Example: despair based-forced swimming/tail suspension, reward based, anxiety Based

Ligand Torsions: Receptor Based Methods

Uses the 3D structure of the target receptor to search for the potential candidate compounds that can modulate the target function. These involve molecular docking of each compound in the chemical database into the binding site of the target and predicting the electrostatic fit between them. The compounds are ranked using an appropriate scoring function such that the scores correlate with the binding affinity. Receptor based method has been successfully applied in many targets

Market Scenario

~$800 M spent to bring a new drug to market. $127 Billion spent on Pharma R&D in 2010 Share of CROs in research operations is 27% World CRO market is 16.3 B