PHMD 274B Pharmaceutics II Lecture 1 Introduction PDF

PHMD 274B Pharmaceutics II Introduction September 26, 2024 Kevin Wang, Ph.D.  Learning objectives o Differentiate between drugs, biologics, and biopharmaceuticals o Compare chemical entities to biologics o Describe the process of biopharmaceutical development/manufacturing o...

PHMD 274B Pharmaceutics II Introduction September 26, 2024 Kevin Wang, Ph.D.  Learning objectives o Differentiate between drugs, biologics, and biopharmaceuticals o Compare chemical entities to biologics o Describe the process of biopharmaceutical development/manufacturing o List the challenges for biologics o Explain recombinant DNA technology and its advantages o Interpret QA and QC 2 Medicine: Past and Future Loading… MIT News 3 Some terms o Drugs and Pharmaceuticals o Biopharmaceuticals, Biologics (biological products), and TIDES o Pharmaceutics and Biopharmaceutics (recall) 4 Drug (legal use only) o A substance recognized by an official pharmacopoeia or formulary. o A substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease. o Loading… A substance (other than food) intended to affect the structure or any function of the body. o A substance intended for use as a component of a medicine but not a device or a component, part or accessory of a device. o Biological products are included within this definition and are generally covered by the same laws and regulations, but differences exist regarding their manufacturing processes (chemical process versus biological process.) https://www.fda.gov/drugs/drug-approvals-and-databases/drugsfda-glossary-terms#D 5 Pharmaceuticals o Medicinal products (both active agents and formulated products), including therapeutics, prophylactics, and in vivo diagnostics o There is no clear distinction between drugs and pharmaceuticals, as these two terms often overlap. o In the medical field, "drugs" and "pharmaceuticals" are often used interchangeably to refer to therapeutic substances. 6 Biologics (Biological products ) o Include a wide range of products such as vaccines, blood and blood components, allergenics, somatic cells, gene therapy, tissues, and recombinant therapeutic proteins. o Biologics can be composed of sugars, proteins, or nucleic acids or complex combinations of these substances, or may be living entities such as cells and tissues. o Biologics are isolated from a variety of natural sources — human, animal, or microorganism — and may be produced by biotechnology methods and other cutting-edge technologies. o Gene-based and cellular biologics, for example, often are at the forefront of biomedical research, and may be used to treat a variety of medical conditions for which no other treatments are available. Biotechnology: The manufacture of products by or from living organisms, usually involving bioprocessing. https://www.fda.gov/drugs/drug-approvals-and-databases/drugsfda-glossary-terms#B 7  Biopharmaceuticals o Biopharmaceuticals: noun A pharmaceutical inherently biological in nature and manufactured using biotechnology. Biopharmaceutical: adjective Relating to biopharmaceuticals, for example, products, technologies, companies, or industries. o Also, biologics and biopharmaceuticals often overlap. 8 TIDES o Therapeutic Peptides (P) and Oligonucleotides (IDES) Peptides: Short chains of amino acids (2-50); Mimic proteins found in the body Oligonucleotides: Short sequences of nucleic acids (DNA/RNA); Regulate gene expression or alter RNA o Bridge the gap between chemical drugs and biologics o Commonly considered biologics, but some may overlap with chemical drugs Al Shaer et al., Pharmaceuticals (Basel). 2024, 7(2):243 de la Torre et al., Molecules. 2024,29(3):585 9 Aptamers o Short, single-stranded oligonucleotides (DNA or RNA) that can bind to specific targets, such as proteins, small molecules, or cells. o Similar to oligonucleotides, but more focusing on the ability to bind to particular targets Nature Biotechnology 26, 2008 10 New drugs approval by FDA New drugs can be simply classified into Chemical entities and Biologics Loading… de la Torre et al., Molecules. 2024,29(3):585 11 Comparison Chemical Entities Biologics Large molecular masses (sometimes not even single Size Small molecules (relatively few atoms) macromolecules) Structure Well-defined, simple Complex Chemical name, Molecular formula, and specific biological source, sequences, and specific biological Identity structure activity. Active ingredient and well-defined excipients Multiple components including stabilizers and Formulation (more options: tablets, capsules, injections) preservatives (usually injections) Manufacture Chemical synthesis or natural sources Derived from living organisms using biotechnology Stability Stable Typically temperature sensitive Emphasize on biological activity and the Quality control Focus on the quantity of the active component(s) characterization of complex structures Intellectual Unique biological processes, specific protein Chemical structures and synthetic methods Property sequences, and methods of production Regulation FDA CDER FDA CBER 12 Production o Production of a pharmaceutical or biopharmaceutical involves many different, complex, and lengthy steps: Synthesis (chemical or cell culture) Purification Formulation Final dosage form preparation 13 Step 1: Synthesis o Order the raw materials needed to make the product. o Test all raw materials to be sure they meet quality standards. o For biomanufacturing, equipment and materials need to be sterilized to avoid bacterial and other contamination of the cell cultures. o The product then is created. For chemical drugs, chemical processes are usually involved (chemical synthesis). For biologics, cell culture or fermentation is often involved. The product is referred to as the “active ingredient.” 14 Recombinant DNA Technology Bacterium with functional gene of interest Journal of Animal Science and Biotechnology 10, 28 (2019) Adapted 15  Example: Insulin o 1921: first discovered by Frederick Banting and Charles Best at the University of Toronto o 1936: produced commercially using traditional method (extract from animal tissues) o 1978: first genetically engineered, synthetic "human" insulin Biopharmaceuticals: Biotechemistry and Biotechnology, Walsh, 2nd Ed, Wiley, 2003 16 What are the advantages of recombinant DNA technology over traditional methods? o Overcomes the problem of source availability. o Overcomes problems of product safety. o Provides an alternative to direct extraction from inappropriate/dangerous source material. o Facilitates the generation of engineered therapeutic proteins displaying some clinical advantages over the native protein product. 17 o For the biopharmaceuticals, the original cell culture is started in small bottles (around the size of large soda bottles) (laboratory scale). o As the cells grow and multiply, they are introduced into a small bioreactor (pilot scale). o Eventually they are grown in large bioreactors, which can be several stories tall! (commercial scale) Laboratory scale Pilot scale Commercial scale 18 https://www.engr.colostate.edu/CBE101/_images/bioreactor_ethanol.png Step 2: Purification o After the active ingredient is synthesized, it must be purified. o Purification involves removing the chemicals used in the process. o For biopharmaceuticals, purification involves separating the cells from the cellular nutrients and byproducts (the “soup”) they grew in. o The end result of purification is called the bulk product. o The bulk product may be sold as is, processed further at the same plant or shipped to another plant for further processing. 19 Step 3: Formulation o Several other operations are required to get the bulk product into its final form. o Formulation involves chemical mixing operations to blend the active ingredient with other substances, such as fillers, needed in the final form. o The final form may be a solid (tablet or capsule), liquid, gels/creams or aerosols. o Biopharmaceuticals usually are sold as sterile liquids or sterile powders. 20 Step 4: Final dosage form preparation o The formulated preparation is made into its final form. o The final form is dispensed into containers. o The containers are labeled and packaged. 21 Quality matters (QA and QC) o The standards of quality are high because the stakes are high. o Poor quality products can harm or even kill consumers. o Companies must conform to the stringent Good Manufacturing Practice (GMP) regulations established by the FDA. 22 Quality assurance (QA) o QA ensures product quality by setting up and checking the systems of standard operating procedures (SOPs) and documentation. o SOPs guide every task by defining each procedure in detail so it can be performed exactly the same way every time. o Companies are required to prepare and follow SOPs by the FDA. o Any deviation from the SOP must be documented and approved by the QA department. o Critical deviations that could affect product quality are investigated further. 23 Quality assurance (QA) o Documentation proves that a company has done what it said. o A company is required to have a traceable, written record of all processes and checks. o “If it isn’t written down, it doesn’t exist. If it isn’t written down, it never happened.” 24 Quality control (QC) o Involves testing and inspection of products to identify defects or deviations from specifications. o Includes the testing of raw materials, in-process materials, and finished products to verify their quality, purity, potency, and consistency. 25 Validation o Validation proves that a manufacturing process will consistently produce the product to predefined specifications. o The operation of every part of the plant that affects quality must be validated. 26 Example: Interferon-Based Biopharmaceuticals (production, purification, and formulation) Vaccines 2021, 9(4), 328 27 Cell-free protein synthesis (FYI) 28 Downstream Processing (FYI) Loading… Purification of tPA produced by E. coli 29  Protein expression systems (FYI) Post-translational modifications include: o Polypeptide folding into a globular protein with the help of chaperone proteins to arrive at the lowest energy state o Modifications of the amino acids present, such as removal of the first methionine residue o Disulfide bridge formation or reduction o Protein modifications that facilitate binding functions: Glycosylation Prenylation of proteins for membrane localization Acetylation of histones to modify DNA–histone interactions o Addition of functional groups that regulate protein activity: Phosphorylation Nitrosylation GTP binding 30 Vaccines 2021, 9(4), 328 Protein expression systems (FYI) Microorganisms 2018, 6(2), 38 31  Summary o Definitions of drugs, biologics, and biopharmaceuticals o Chemical entities and biologics o Biopharmaceutical manufacturing: Synthesis (chemical or biotechnology); Purification; Formulation; Final dosage form preparation o Recombinant DNA technology and its advantages o Challenges for biologics: complex (structure, development, manufacturing processes, etc.), high production costs, stability and storage issues, potential immunogenicity, difficulties in QA/QC, etc. o QA and QC: QA ensures proper processes to maintain product quality; QC involves the testing and inspecting of products to meet specific standards o Validation: to prove that a manufacturing process can consistently produce the product to predefined specifications. 32 PHMD274B Class activity 1. Which of the following best describes biologics? a) A small molecule synthesized through chemical processes, such as traditional pharmaceuticals. b) A therapeutic product derived from living organisms, such as vaccines, proteins, and allergenics. c) A completely synthetic chemical compound used in various applications d) Any product derived from biological origins, including animals and plants 2. What is the primary difference between biologics and small chemical drugs? a) Biologics are prepared using living cells, while chemical drugs are synthesized through chemical processes. b) Biologics are smaller molecules, while chemical drugs are larger molecules. c) Biologics are taken orally, while chemical drugs are injected. d) Chemical drugs are mainly derived from proteins, while biologics are made from small molecules. 3. What is the role of recombinant DNA technology in the production of biologics? a) To combine small molecules to create synthetic drugs b) To modify the genetic material of organisms to produce therapeutic proteins  c) To produce vaccines using viral particles d) To chemically synthesize large protein molecules 4. What are the challenges in the development and manufacturing of biologics? a) Complex structures and intricate development processes b) Stability during storage and transportation c) High water solubility d) Difficulty in scaling up production e) Lower potency compared to chemical entities 5. Which of the following is true about the manufacturing of biologics? a) The process is simpler and faster than traditional chemical drug manufacturing. b) Biologics are manufactured using living cells, which makes the process more complex and variable. c) Once the biological drug is formulated, it can be easily replicated with little quality control. d) Biologics can be produced in large batches with less batch-to-batch variation. 6. Why are QA and QC especially critical in biological drug manufacturing? a) Because biologics are chemically synthesized and more complex b) Because biologics are highly stable and unlikely to degrade over time c) Because biologics are produced using living  organisms, leading to potential variability in the product d) Because biologics can have a wide range of effects and potential side effects due to their complex nature 7. What is the purpose of validation in the manufacturing of biologics? a) To verify that the biologics are highly effective and safe for their intended use b) To ensure that the production process consistently produces a product that meets predetermined quality standards c) To demonstrate that the biologic can be stored under specified conditions to maintain its stability and integrity throughout its shelf life d) To simplify the manufacturing process and eliminate the need for quality control 8. How should etanercept (Enbrel) be stored to maintain its stability? a) room temperature b) In a freezer c) Refrigerated between 2°C to 8°C (36°F to 46°F) d) In a warm, dry place 2 Drug development Oct 1, 2024 Kevin Wang, Ph.D. Learning objectives o Explain the key phases of drug discovery and development o Identify and select appropriate clinical study designs o Explain the objectives of Phase I-IV clinical trials 2 Outline o Drug discovery and development o Preclinical study o Clinical studies 3 Revenue and R&D expenses of top Pharmaceutical Companies Company Revenue ($b) R&D expenses ($b) 1 Johnson & Johnson 85.2 15.09 2 Roche 66.4 15.56 3 Merck & Co 60.1 30.5 4 Pfizer 58.5 10.58 5 Abbvie 54.3 7.68 6 Sanofi 46.9 7.18 7 AstraZeneca 45.8 10.94 8 Novaritis 45.4 11.37 9 Bristol-Myers Squibb 45.0 9.21 10 GSK 38.4 6.2 11 Eli Lilly 34.1 9.31 12 Novo Nordisk 33.7 4.71 17 Bayer 51.7* €5.84 * Pharmaceutical business revenue 26.1 $b 4 Data based on https://www.pharmaceutical-technology.com; www.google.com Discovery and Development Targets/Receptors; Drug Discovery Small/Macro molecule drugs; …. Preclinical (animal) studies: Drug Development PK/PD; Study procedures (Compliance with Toxicology; following GLPs regulatory Delivery systems requirements is (formulation) necessary) Study procedures Clinical Trials: following GCPs; Phase I, II, III Manufacturing procedures following GMPs Marketing application (FDA, EMA, etc) 5 How are drugs discovered and developed? Investigational New Drug application Preclinical Post Marketing Development Clinical Trial Surveillance New Drug Application Formulation Animal test Phase I Phase 2 Phase 3 Phase 4 1-2 Years 6-7 Years 10 K 250 5 2 1 Compounds ~2M ~15 M ~30 M Up to 1 B 6 Drug Discovery o Identify the target(s) that cause or lead to diseases. o Screen chemicals or biological molecules that can interact with the target. 7 Drug Target examples o Proteins o G protein-coupled receptors o Enzymes (especially protein kinases, proteases, esterases, and phosphatases) o Ion channels o Nuclear hormone receptors o Structural proteins such as tubulin o Membrane transport proteins o Nucleic acids 8 Lead compound A compound (small chemical entities or biologics) that has pharmacological or biological activity o Natural Compound Collections o Rational drug design o Combinatorial chemistry https://futorolabs.com/natural-compound/ Satyanarayanajois, et al., Drug Design and Discovery, Human Press http://www.daylight.com/meetings/mug00/Weber/ac/sld016.htm 9 Drug development o Preclinical study o Clinical studies o Marketing application 10 Preclinical study o Toxicology Toxicity Carcinogenicity Genotoxicity Reproductive toxicology o PK/PD Pharmacokinetics Pharmacodynamics (pharmacology) 11 Clinical studies HHS and NIH: Research study in which one or more human subjects are prospectively assigned to one or more interventions (which may include placebo or other control) to evaluate the effects of the interventions on biomedical or behavioral health-related outcomes. ICH E6: Any investigation in human subjects intended to discover or verify the clinical, pharmacological and/or other pharmacodynamic effects of an investigational product(s), and/or to identify any adverse reactions to an investigational product(s), and/or to study absorption, distribution, metabolism, and excretion of an investigational product(s) with the object of ascertaining its safety and/or efficacy. The terms clinical trial and clinical study are synonymous. o Assessment of efficacy (superiority, non-inferiority, or equivalence), safety, or benefit/risk o To find out right drug, right dose, and right time for right patients 12 Product Manufacture for clinical studies o Drug designed for clinical trial has to be manufactured in compliance with current Good Manufacturing Practice. o A quality control system has to be set up to ensure drug manufactured in accordance with approved procedures. 13 Clinical Trial Design o Randomization o Controls Placebo Non-treatment Active control Dose response External control 14 Randomization o Randomization is the process of assigning clinical trial participants to treatment groups. o R.A Fisher first introduced the idea of randomization in 1926. o Randomization gives each participant a known (usually equal) chance of being assigned to any of the groups. o Successful randomization requires that group assignment cannot be predicted in advance. 15 Control: Placebo o An inert substance used in clinical trials. o Usually double blinded (neither the participants nor the experimenters know who is receiving a particular treatment) o When to use: no prior drug has been established as standard therapy o Advantage: minimizing bias o Disadvantage Ethical issues: Lack of treatment Patient and physician concerns: withdraw 16 Control: No treatment o Subjects are randomized into test or no treatment groups o Subjects and investigator(s) are not blinded to treatment o Disadvantage: bias 17 Control: Active control o Standard treatment exists o Subjects are randomly assigned to the test treatment or an active control treatment. o When to use: compare “new drug” or “new treatment” with the standard therapy (“as good as” or “superior”) o Usually double-blind 18 Dose-Response Concurrent Control o Objective: to establish a relationship between dose and efficacy and safety o Usually randomized and double blinded o May include other control groups (active or placebo) o Doses may be fixed or gradually raised o If the therapeutic range is not known, the design may be inefficient 19 External control o Compare a group of subjects receiving the test treatment with a group patients external to the study. o The external control can be a group of patients treated at an earlier time (historical control) or a group treated during the same time period but in another setting o Advantages: all patients receive their promising treatments, more attractive to patients and physicians o Disadvantage: cannot be blinded 20 Clinical trial design o Parallel o Cross over o Factorial o Adaptive o Superiority and Non-inferiority o Randomized withdrawal approach 21 Parallel o Subjects are randomized to one of two or more arms o Each arm being allocated a different treatment o Most common used design Test Eligibility Informed Randomization Screening consent Control 22 Cross over o A longitudinal study in which subjects receive a sequence of different treatments (or exposures). Group A Treatment A Treatment A Group B Treatment B Treatment B Washout 23 Cross over o Advantage Each patient has their own control Smaller sample size o Disadvantage Not useful for acute disease Disease must be stable Assumes no period carry over effect 24 Factorial concurrent design o Consists of two or more factors, which allows the investigator to study the effect of each factor on the response variable, as well as the effects of interactions between factors on the response variable. Factor B B1 B2 A1 Group 1 Group 2 Factor A A2 Group 3 Group 4 25 Factorial concurrent design o Advantages Two studies for one Discover interactions o Disadvantages Test of main effect assumes no interaction Often inadequate power to test for interaction Compliance 26 Superiority and Non-Inferiority Trials o Superiority Design: Show that new treatment is better than the control or standard (maybe a placebo) o Non-inferiority: Show that the new treatment is not worse than the standard by more than some margin would have beaten placebo if a placebo arm had been included (regulatory) 27 Equivalence/Non-inferiority Trial o Trial with active (positive) controls o To answer whether new (easier or cheaper) treatment is as good as the current treatment o Must specify margin of “equivalence” or non-inferiority o Can't statistically prove equivalency -- only show that difference is less than something with specified probability o Historical evidence of sensitivity to treatment o Sample size issues are crucial o Small sample size, leading to low power and subsequently lack of significant difference, does not imply “equivalence” 28 Randomized withdrawal/Delayed Start Design approach o Randomized withdrawal All patients receive active drug, then randomized to continue the active drug or placebo (withdrawal of the active drug) o Delayed Start Design Patients are randomized to receive active drug or placebo, then all patients receive active drug o To Maximize the amount of information from the fewest patients Test Active treatment Yes over defined Stable? Randomization time frame Placebo No. Off study 29 Example o Purpose: compare the first occurrence of new ischemic stroke, new myocardial infarction, or other vascular death between Plavix (75 mg daily) and aspirin (325 mg daily). o Study design: double-blind, randomized, parallel, no placebo control o Control: active control (aspirin has well documented benefits) o Sample size: 19,185 o Multicenter: 304 clinical center or hospitals Eligibility Informed 19,185 Randomization Screening consent Data Test: Plavix Control: aspirin collection 9,599 9586 30 Results Plavix Aspirin Patients 9599 9586 Ischemic stroke 438 (4.6%) 461 (4.8%) Myocardial infarction 275 (2.9%) 333 (3.5%) Other vascular death 226 (2.4%) 226 (2.4%) Total 939 (9.8%) 1020 (10.6%) Plavix resulted in overall reduction of outcome events 31 Clinical Trials Purpose Duration Subjects % success 20-100 I Safety and dosage Several months Healthy subjects or ~70% patients Up to several hundred II Efficacy and side effect Months to 2 yrs ~33% Patients Efficacy and 300-3000 III monitoring of adverse 1-4 years ~25-30% Patients reactions Can up to 14 IV* Safety and efficacy Thousands - Years *Post Marketing Surveillance Trial which occurs after a drug is already approved 32 Phase I o Studies designed mainly to investigate the safety/tolerability, identify maximum tolerated dose, PK/PD in humans o A key step in taking potential new medicines from the laboratory to humans. o The tested range of doses will usually be a fraction of the dose that cause harm in animal testing. o Dose escalation to reach maximum tolerated dose (MTD) Dose escalation often based on Fibonacci Series: 1 2 3 5 8 13.... 33 Phase I o These trials are often conducted in an inpatient clinic, where the subjects can be observed by full time staff. o Subjects: Healthy volunteers (most common) Patient volunteers (toxic drug, AIDs therapy) o Volunteers are paid an inconvenience fee for their time spent in the volunteer center. o Limitation Trial restricted to homogenous subjects Performance extrapolated to heterogeneous population 34 Phases II o In patients (therapeutic exploratory trial) o Duration: 6 months to a few years o Subjects: 20-300 patients o To confirm effectiveness, monitor side effects and further evaluate safety o Therapeutic dose: Dose efficacy relationship Dose regimen Duration of therapy Frequency of administration Therapeutic windows 35 Phase III o Therapeutic confirmatory trial o Definitive assessment of how effective the drug is in comparison with current ‘gold standard’ treatment. o To assess overall and relative therapeutic value of the new drug efficacy and safety (benefit/risk). o Are randomized controlled multiple center trials or large patient groups (300-3000) or more depending on the disease/medical condition studied o Duration: much longer (up to 5 years) 36 Phase IV o Post marketing surveillance o No fixed duration/patient population o To detect rare ADRs and DDI o To explore new indications o Harmful effect discovered may results in withdrawal or restriction in certain uses. 37 Summary o Discovery: Target and lead compound o Development: Preclinical (toxicology, PK/PD), Formulation development and manufacture, Clinical trials (I, II, III), Application and marketing o Clinical studies Phase I: Drug is tested on healthy volunteers or patients to determine toxicity relative to dose and to screen for unexpected side effects Phase II: Drug is tested on small group of patients to see if drug has any beneficial effect and to determine the dose level needed for this effect. Phase III: Drug is tested on much larger group of patients and compared with existing treatments and with a placebo Phase IV: Drug is placed on the market and patients are monitored for side effects After the drug has been approved and marketed, the safety and performance of the drug in continually monitored to ensure that is prescribed correctly, and adverse event are reported and investigated. Controls and randomization Clinical trials designs 38 PHMD274B Class activity 1. What is the primary goal during the drug discovery phase? a) To conduct large-scale clinical trials b) To identify and validate biological targets and lead compounds c) To obtain regulatory approval for the drug d) To monitor post-marketing safety 2. Which of the following is NOT typically a component of preclinical studies in drug development? a) Toxicology studies b) Pharmacokinetics and pharmacodynamics (PK/PD) analysis c) Lead compound identification d) Formulation development 3. Which of the following best describes the purpose of randomization in clinical trials? a) To ensure that all participants receive the experimental drug b) To eliminate bias by randomly assigning participants to different treatment groups c) To control the dosage of the drug administered to participants d) To allow subjects to have the flexibility to choose their study groups 4. What is the main objective of dose-response studies in clinical trials? a) To determine the most effective and safe dosage of a drug b) To compare the drug with existing treatments c) To monitor long-term side effects post-marketing  d) To identify the biological target of the drug 5. Which type of control is used when one group of participants receive no treatment? a) Placebo control b) Active control c) Non-treatment control d) External control 6. Which of the following statements of a placebo ARE correct? a) A placebo can enhance the drug's therapeutic effect. b) A placebo is an inert substance used to compare the effect of a new treatment. c) A placebo is often used in double-blinded studies, when no standard therapy is available. d) A substance used to increase the efficacy of the drug under investigation. e) Treatment given to all participants to avoid ethical concerns. 7. In the context of clinical trial designs, what does a "crossover" design imply? a) Participants receive either the experimental drug or a placebo exclusively. b) Participants receive both the experimental drug and the control treatment in a sequential manner. c) The trial is conducted in multiple countries simultaneously. d) The trial adapts its protocols based on interim results. 8. Which phase of clinical trials is a confirmatory trial that involves testing the drug on a larger group of patients and comparing it with existing treatments and a placebo? a) Phase I  b) Phase II c) Phase III d) Phase IV 9. What is the main purpose of Phase IV clinical trials? a) To determine the potential toxicity of the drug in healthy volunteers. b) To assess the drug efficacy in a small patient group. c) To compare the superiority of the new drug with existing treatments in a large population. d) To monitor the drug safety and performance after it has been marketed. 10. Case study: A pharmaceutical company is planning a Bioavailability/Bioequivalence (BABE) study to compare the bioavailability of a generic drug to a reference drug in healthy volunteers. Both drugs are expected to be absorbed similarly. The objective is to assess whether the new generic drug is equivalent to the reference in terms of pharmacokinetic parameters (e.g., Cmax, Tmax, and AUC). Question: (1) This is a typical Phase ___ clinical study. a) Phase I b) Phase II c) Phase III d) Phase IV (2) Which study design would be most appropriate for this BA/BE study? a) Parallel b) Crossover  c) Factorial d) Adaptive e) Superiority (3) Do you think randomization is necessary for this study? Yes No 2 Protein Therapeutics Oct 3, 2024 Kevin Wang, Ph.D. Learning objectives o Define proteins and their therapeutic significance o Explain mechanisms of action in protein drugs o Discuss clinical application of protein-based therapies o Evaluate advantages and limitations of protein drugs o Explore technologies of protein delivery systems 2 Outline o Introduction of protein o Mechanisms of action and clinical application o Advantages and limitations o Route of administration for proteins o Technology of proteins drug development 3 Introduction of proteins 4 Proteins and Peptides o Peptides are short chain of amino acids (AA) joined together by covalent (peptide) bonds (typically < 50 amino acids). o Oligopeptides are relatively short (often ranging from 2 to ~20 AA residues) o Polypeptides are longer chain of amino acids (20-50 AAs, can be even longer but usually < 100). o Proteins are large and complex organic molecules composed of one or more polypeptide chains. https://medium.com/@celinehh/getting-things-into-cells-pt-1-peptides-b9a4d2f41182 5  Types of proteins and peptides Proteins (>50 AA) Peptides (2 - 50 AA) Fibrous Globular Oligomeric Polypeptides Oligopeptides (20-50 AA) (2-20 AA) 6 https://www.differencebtw.com/difference-between-globular-and-fibrous-proteins/ Structure of proteins o Primary: Array of AA in protein structure o Secondary: Spatial array of twisted polypeptide chain (hydrogen bonds) o Tertiary : 3-D structure of functional protein o Quaternary: Composed array of two or more protein/peptide chains (https://www.khanacademy.org/science/biology/macromolecules/proteins-and-amino-acids/a/orders-of- protein-structure) 7 Outline o Introduction of protein o Mechanisms of action and clinical application o Advantages and limitations o Technology of protein drug development 8 Mechanism of action o Binding and Neutralization: o Monoclonal antibodies (mAbs) bind to antigens on cells or pathogens resulting in neutralization of the target for removal by the immune system. o Immune Response Modulation: o Cytokines and mAbs stimulate or suppress immune responses to treat autoimmune diseases or cancer. o Vaccines stimulate the immune system to recognize and generate immunity against specific diseases. o Receptor binding and Cell Signaling: o Cytokines and some mAbs target signaling pathways by binding to specific receptors. o Replacement of endogenous proteins: o Enzyme Replacement Therapies (ERT): replacement of deficient enzymes o Hormones: insulin o Blood Clotting: clotting factors o Others: o Fusion Proteins: combine the actions of two different proteins o Antibody-Drug Conjugates (ADCs): combine monoclonal antibodies with chemotherapy drugs 9 Clinical application of protein therapeutics Reference: Merative Micromedex®; Drugs@FDA Cat# Product Company Administration Mechanism Cancer Herceptin (Trastuzumab) GENENTECH IV infusion Humanized mAB binds HER2 Treatment Rituxan (Rituximab) GENENTECH IV infusion mAB that induces apoptosis in DHL 4 human B cell lymphoma cells Adcetris SEATTLE IV infusion Antibody drug conjugate of binds to CD30 (brentuximab vedotin) GENETICS Autoimmune Humira (Adalimumab) ABBVIE INC SubQ binds TNFα and blocks its interaction with the Diseases (40 to 160 mg) p55 and p75 cell surface TNF receptors. Remicade (Infliximab) CENTOCOR IV infusion mAB (chimeric IgG1 kappa) with specific INC (3 to 10 mg/kg) activity TNFα. Etanercept IMMUNEX SubQ A fusion protein that fuses the TNF receptor to (50 mg, or 0.5 the constant end of the IgG1 antibody mg/kg) neutralizing TNF Hematologic Epogen/Procrit (Epoetin AMGEN SubQ (100 to Glycoprotein that exerts the same biological Disorders alfa) 600 u/kg, 40k to effects as endogenous erythropoietin that is 60k u) produced in the kidney. 10  Cont’d Cat# Product Company Administration Mechanism Hormone Insulin (e.g., (Various) SubQ, inhalation Trigger cellular uptake of glucose by Replacement Humalog, Lantus) binding to insulin receptor Therapy Enzyme Cerezyme GENZYME 2.5 units/kg three times A human enzyme beta-glucocerebrosidase Replacement (Imiglucerase) weekly to 60 units/kg analog, catalyzes the hydrolysis of the lipid Therapy once every 2 weeks via glucocerebroside to glucose and ceramide IV infusion over 1 to 2 which prevents its accumulation in hours macrophages Cardiovascular Activase (Alteplase) GENENTE IV infusion A tissue plasminogen activator enhances Diseases CH the conversion of plasminogen to plasmin by binding to fibrin, initiating fibrinolysis Neurological Tysabri BIOGEN 300 mg, iv infusion Humanized IgG4-kappa mAB, hinders Disorders (Natalizumab) IDEC leukocyte migration by binding to integrins on leukocytes, blocking adhesion to endothelial cells and reducing inflammation 11 Cont’d Cat# Product Company Administration Mechanism Pulmonary Alpha-1 Antitrypsin (Various) 60 mg/kg IV Increase and maintain serum levels Disorders Replacement Therapy infusion once and epithelial lining fluid levels of α1- (Prolastin, Zemaira) weekly proteinase inhibitor Pain Management Botox (Botulinum ALLERGAN IM Counteract botulinum toxins, Toxin) hindering their binding to nerve cells and promoting clearance Infectious Casirivimab and Regeneron IV infusion or mAB specifically directed against Diseases Imdevimab Pharmaceutic SubQ the spike protein of SARS-CoV-2 (REGEN-COV) als, Inc. (600 mg) (EUA) Vaccines influenza, hepatitis B, (Various) SubQ, oral, etc Stimulate immune response and HPV produce memory cells for protection 12 Outline o Introduction of protein o Mechanisms of action and clinical application o Advantages and limitations o Technology of protein drug development 13 Advantage of Protein Therapeutics o High specificity: usually target specific receptors with minimum off-target effects o Less Side Effects: because of their specificity, protein therapeutics tend to have fewer side effects o Biological Similarity: many proteins are structurally and functionally similar to endogenous proteins in the body, making them well-tolerated and compatible. o Long Half-Life: reducing the frequency of administration. o Diverse Applications: rare genetic conditions; orphan diseases o Accommodation to new technology: o Antibody-drug conjugates (ADCs): targeted delivery of drugs to specific cells or tissues o Personalized Medicine based on an individual's genetic and molecular profile o Protein engineering to minimize the risk of an immune response 14 Limitations with proteins for therapeutic use o Very large and unstable molecules o Easy degradation o Low permeability and low bioavailability o Unwanted allergic reactions may develop (even toxicity) o Difficult to obtain in large quantities o High Cost: protein drugs can be expensive to manufacture o Difficult to identify delivery systems 15 Protein stability o In vitro stability of proteins o In vivo (in the body) stability of proteins 16 Protein stability o In Vitro stability of proteins Covalent interactions (chemical) ▫ Deamidation, oxidation, disulfide exchange, and proteolysis Non-Covalent interactions (physical) ▫ Denaturation, aggregation, precipitation, and adsorption 17 Protein stability o In Vitro stability of proteins Covalent interactions (chemical) ▫ Deamidation - conversion of Asn-Gly sequences to a-Asp-Gly or b-Asp-Gly ▫ Oxidation - conversion RSR’ to RSOR’, RSO2R’ or RSO3R’ (Met & Cys) ▫ Disulfide exchange - RS- + R’S-SR’’ goes to RS-SR’’ + R’S- (Cys) ▫ Proteolysis - Asp-Pro, Trypsin (at Lys) or Chymotrypsin (at Phe/Tyr) 18  Non-covalent interactions of proteins Denaturation Adsorption Aggregation Precipitation 19 Improve stability o Using additives/excipients Addition of stabilizing salts or ions (Zn+ for insulin) File:Benchtop freeze dryer.JPG Incorporation of polyols (glycerol or PEG) to improve solubility Addition of sugars or dextran to act as preservative (replace water) Use of surfactants to reduce adsorption and aggregation o Special technology: freeze drying Low temperature reduces microbial growth and metabolism Low temperature reduces thermal or spontaneous denaturation and adsorption Freezing is best for long-term storage (but frequent Freeze/thaw can denature proteins) 20 Protein stability o In Vivo stability of proteins Aggregation at injection sites Environmental Conditions: pH and ionic strength Immune response: elimination by B and T cells Proteolysis by endo/exo proteolytic enzymes (peptidases/proteinase) Small proteins (40 bound to peripheral tissues 9 Elimination o Metabolism o Excretion o Additional pathways 10 Metabolism of Biologics o Biologics are primarily metabolized by proteolytic pathways (lysosomal proteolysis) phagocytosis (reticuloendothelial system, RES) nonspecific endocytosis o Not traditional cytochrome P450 pathways for small molecules. 11 Excretion of Biologics o Kidneys are the major organ for excretion. o Renal elimination is involved in clearing degradation products of biologics. biologics with a molecular weight lower than 30 kDa. o Limited glomerular filtration for biologics due to large size and structural complexity 12 Additional pathway: immunogenicity and Impact on PK o Definition: immune system response to biologic drugs. o Effect on pharmacokinetics: increased clearance, reduced efficacy, and adverse reactions. o Strategies to reduce immunogenicity (humanized antibodies, pegylation). 13 Additional clearance: Targeted mediated clearance o Definition: A clearance mechanism where biologics bind specifically to their target receptors, triggering internalization and degradation. o Process: Binding to target receptors (e.g., cell surface receptors) Internalization via endocytosis Degradation in lysosomes 14  Additional: neonatal Fc receptor (FcRn) o Entry into Cells Abs enter cells through pinocytosis, and then move into acidified endosomes, where they may interact with the FcRn receptor o FcRn Receptor Binding FcRn Bound Abs are protected from degradation. Unbound are transferred to the lysosome for degradation. o Recycling of FcRn-Bound Antibodies FcRn-bound Abs are recycled back to the bloodstream. At physiologic pH, Abs dissociate from FcRn Nature Reviews Immunology 2007: 7, 715-725 15  Pharmacokinetic Parameters o Volume of distribution (Vd): typically low for biologics. o Half-life (t1/2): longer due to slow degradation. Type Drug Vd T1/2 Fusion protein Etanercept (150kDa) ~8L 70 - 100 h Proteins and Peptides Darbepoetin (37 kDa) 3-6L 21 h Monoclonal Ab Herceptin (148 kDa) 2.7 – 3.7 L 5 - 12 days Monoclonal Ab Adalimumab (144 kDa) 4.7- 6 L 10-20 days Antibody-Drug conjugate Brentuximab (153 kDa) 6 - 10 L 4 – 6 days 16 Small chemical drugs ADME Small chemicals Distribution Absorption Liver Tissues Bloodstream Kidneys Intestine Metabolism Feces Excretion 17 Biologics ADME mAb Limited Distribution Liver Tissues GI degradation Bloodstream SC Kidneys Intestine Proteolytic FcRn recycling degradation Feces Excretion 18 Challenges in PK of Biologics o Complexity of biological systems o Difficulties in scaling PK from preclinical to clinical settings o High inter-individual variability 19 Summary o ROA: mainly parenteral (SC or IV) o ADME Absorption ▪ Limited oral bioavailability due to degradation in the GI tract. ▪ Factors influencing absorption: Molecular size, Hydrophilicity (charge, polarity), Gastric degradation, Formulation (stabilizer, permeabilizer, etc) Distribution ▪ Primarily confined to the vascular and interstitial spaces. ▪ Volume of distribution (Vd) of biologics is often small Elimination ▪ metabolism (proteolysis), excretion, targeted mediated clearance, Immunogenicity, ▪ FcRn recycling (longer t1/2) ▪ Usually t1/2 is longer than chemical drugs o Challenges 20 PHMD274B PK and Biologics Class activity 1. Which of the following routes of administration are most commonly used for biologics? a) Oral b) Intravenous c) Intranasal d) Transdermal e) Subcutaneous 2. What is a potential advantage of subcutaneous administration of biologics over IV? a) Faster onset of action b) Avoidance of first-pass metabolism c) Higher patient compliance d) Lower cost compared to intravenous administration 3. Why can insulin not be administered orally? a) It has a very short half-life in the bloodstream b) It is rapidly degraded in the gastrointestinal tract c) It is too hydrophobic to be absorbed in the intestine d) It undergoes extensive hepatic first pass extraction by P450 enzymes 4. Why do biologics typically have a small volume of distribution (Vd)? a) They are highly lipophilic and penetrate deep tissues b) They undergo rapid metabolism in the liver c) They are highly permeable to cell membranes d) They are primarily confined to vascular and interstitial spaces 5. Why do antibodies typically have longer half-lives (t )? 1/2 a) Limited renal excretion b) FcRn recycling and targeted-mediated clearance c) Limited hepatic metabolism via cytochrome P450 d) Confined in the blood vessels 6. Which of the following pathways is primarily responsible for the metabolism of biologics? a) Cytochrome P450 enzyme system b) Lysosomal proteolysis c) Glucuronidation d) Renal excretion 7. How does immunogenicity affect the pharmacokinetics of biologic drugs, and what strategies can be employed to reduce immunogenicity? a) Immunogenicity increases clearance, reduces efficacy, and may cause adverse reactions; strategies include humanized antibodies and pegylation b) Immunogenicity reduces clearance and increases efficacy; strategies include adding PEG to enhance immune tolerance c) Immunogenicity enhances absorption and prolongs half-life; strategies include increasing the molecular size d) Immunogenicity has no significant effect on pharmacokinetics; the main concern is drug formulation 8. Which of the following statements about the renal elimination of biologics is correct? a) All biologics undergo glomerular filtration due to their small size.  b) Renal elimination is only involved in the clearance of biologics with molecular weights greater than 30 kDa. c) Degradation products of biologics are cleared via renal elimination. d) Glomerular filtration is efficient for most biologics regardless of size and structure. 9. Compare the difference between chemical drugs and biologics in terms of PK Chemical Drugs Biologics ROA Absorption Distribution Excretion Metabolism T1/2 Vd 2