PHAR 401 Revision III - Protein Therapeutics

PHAR 401 Revision III - Part A

• Course: Pharmacy 401
• Revision: III - Part A
• Instructor: Shahira AbdelSalam ElBanna, PhD
• Email: [email protected]

Introduction to Protein Therapeutics

• Subject: Protein Therapeutics
• Course: Pharmacy 401

Large Molecule vs. Small Molecule Drugs

• Small molecule drugs:
  • Size: <1000 Da
  • Synthesis complexity: Simple chemical synthesis
  • Stability: Stable
  • Administration routes: All routes
  • Immunogenicity: Usually not a problem
  • Cost: Lower Cost
• Biological drugs:
  • Size: ~>1500 Da
  • Synthesis complexity: Complex through recombinant technology
  • Stability: Sensitive to storage & handling conditions
  • Administration routes: Parenteral routes
  • Immunogenicity: Immunogenic
  • Cost: Expensive

Protein Synthesis and Folding

• Protein Folding:
  • Primary Structure: Amino acid sequence
  • Secondary Structure: Alpha helix and beta-pleated sheets
  • Tertiary Structure: 3-D folded protein structure
  • Quaternary Structure: Multiple polypeptide chains interacting
  • Biological activity tied to correct folding
  • Protein formulations often not 100% correctly folded
  • Non-covalent interactions define biological activity (True)

Levels of Protein Structure

• Primary structure:
  • Peptide bonds between amino acids
• Secondary structure:
  • Alpha helices and beta-pleated sheets; stabilized by hydrogen bonding
• Tertiary structure:
  • 3D structure; results from folding due to interactions among R groups
• Quaternary structure:
  • Two or more polypeptides; interactions form the final 3D structure

Types of bonds and interactions share in protein structure formation

• Covalent bonds:
  • Peptide bonds
  • Disulfide bonds
• Non-covalent bonds:
  • Hydrogen bonds
  • Hydrophobic bonds
  • Electrostatic bonds
  • Van der Waals forces

Intramolecular interactions maintain protein tertiary structure

• Electrostatic forces: Attraction between opposite charges
• Hydrogen bonds: Hydrogen shared between electronegative atoms
• Van der Waals forces: Fluctuations in electron clouds
• Hydrophobic forces: Hydrophobic groups interact unfavorably with water

Regulation of protein folding in the endoplasmic reticulum

• Newly synthesized proteins: Are translocated to the ER and fold into 3D structures with chaperones
• Correctly folded proteins: Are transferred to the Golgi complex then to the extracellular environment
• Incorrectly folded proteins: Are identified by quality control systems, ubiquitinated and degraded.

Challenges with Protein-based Therapeutics

• Stability: Proteins are large and unstable molecules. Tertiary structure can be disrupted by mild storage conditions. Proteins can be degraded in the body and eliminated by rapid clearance.

Aggregates and Particles

• Factors leading to aggregation: Agitation, temperature, extremes of pH and high concentration. Manufacturing, storage and handling also affect aggregation.

Immunogenicity

• Anti-drug antibody (ADA) response: Formation of antibodies against therapeutic protein
• Clinical consequences:
  • Loss of efficacy
  • Neutralization of endogenous protein
  • Immune reaction (allergy, anaphylaxis)

Immunogenicity: Some causes

• Protein aggregation
• Sequence variation and glycosylation
• Contaminants and impurities
• Dose and dosing frequency
• Length of treatment
• Route of administration (especially subcutaneous)
• Individual patient features and unknown factors

Year 4: Monoclonal Antibodies in the Treatment of Cancer (1)

Hematopoiesis

• Process where pluripotent stem cells give rise to different blood cell types.

Effector Antibodies

• Direct effect:
  • Neutralization (soluble or insoluble Ag/toxins/viruses).
• Indirect effect:
  • Blockade (antigenic receptors)
  • Antibody-dependent cellular cytotoxicity (ADCC)
  • Complement-dependent cellular cytotoxicity (CDC)

The diversity of antibody functions/ therapeutic use of MAbs

• Neutralizing mediators of disease.
• Neutralizing infectious agents.
• Receptor binding.
• Initiation of effector mechanisms.
• Delivery of toxic or modulatory payload

Pharmacokinetics of MAbs

• Antibodies eliminated by excretion or catabolism.
• MAbs too large to be filtered by kidneys (except in pathologic cases).
• Full antibodies display long half-lives based partly on size & resistance to proteases.
• Fc region largely responsible for prolonged circulation.

Fc receptors

• Proteins on immune effector cells (like macrophages) that bind to the Fc region of an antibody.
• Interaction with antibody-coated target cell results in immune cell activation or inhibition.

Pharmacokinetics of MAbs is complex

• Albumin and IgG bind to FcRn non-competitively.

FcRn extends IgG half-life

• Principle: FcRn extends IgG half-life by reducing lysosomal degradation in endothelial cells.
• Mechanism:
  • IgG binds to Fc receptor FcRn (Brambell) on endothelial cells
  • Uptake of IgG during internalization (endocytosis)
  • Dissociation at the physiological pH of the extracellular environment.
  • Antibodies are recycled from the endosome.
  • Other proteins don't bind to FcRn and are degraded in the lysosome

Rituximab (MabThera or Rituxan)

• Mode of action: ADCC, CDC, apoptosis
• Mainstay to treat: Non-Hodgkin's lymphomas (NHLs), chronic lymphocytic leukemia (CLL), and other B-cell triggered conditions (e.g. transplant rejection, autoimmune disorders - rheumatoid arthritis).

Rituximab (MabThera or Rituxan): Adverse Reactions (ARS)

• Rapid depletion of CD20+ B cells in the peripheral blood
• Infusion-related reactions (IRRs) include fever, chills, myalgias, and hypersensitivity reactions
• Tumor lysis syndrome (TLS)
• Late onset neutropenia (LON)

RtUXimab (MabThera or Rituxan) Additional ARS

• To minimize IRRs:
  • Increase infusion rates slowly (50 mg/h up to 400 mg/h every 30 minutes), Infusion times 3-6 hours.
  • Pre-medication with antihistamine and paracetamol
• Hepatitis B reactivation

RtUXimab (MabThera or Rituxan): Tumor Lysis Syndrome (TLS)

• Potentially deadly complication of tumors or treatment.
• Acute metabolic syndrome caused by the release of nucleic acids, proteins, and intracellular metabolites of lysed cancer cells.
• Hyperuricemia, hyperkalemia, hyperphosphatemia.

RtUXimab (MabThera or Rituxan): Hepatitis B Reactivation

• Viral reactivation.
• Hepatitis B testing for anti CD20 monoclonal antibody patients.
• Prophylactic antiviral therapy for HBsAg or HBcAb positive patients.

Year 4: Monoclonal Antibodies in the Treatment of Cancer (2)

Tumor-specific IgG (Trastuzumab & Pertuzumab)

• HER2 receptors on HER2-expressing breast cancer cells can dimerize with themselves or other HER receptors in ligand-dependent or ligand-independent manners.

Trastuzumab (Herceptin)

• Binds to HER2 domain IV to prevent ligand-independent dimerization.
• Inhibits intracellular signaling resulting in tumor arrest.
• Stimulates immune system to recognize and eliminate HER2 overexpressing cells (Ab-ADCC, NK & monocytes effects).
• Cardiotoxicity.

Pertuzumab (Perjeta)

• Binds to domain II on HER2.
• Prevents HRG-mediated HER2/HER3 dimerization and signalling along with inhibiting HER2 dimerization with other HER receptors.
• Used in combination with trastuzumab and docetaxel for HER2-positive metastatic breast cancer

Antibody Drug Conjugates (ADC) A. Brentuximab vedotin (Adcetris)

• Binding of the ADC to CD30-expressing cells, internalization of the ADC-CD30 complex, release of MMAE after proteolytic cleavage, MMAE binds to tubulin, causing G2/M arrest and apoptotic death.

Antibody Drug Conjugates (ADC) B. Trastuzumab emtansine (Kadcyla)

• Proteolytic degradation inside the target cell
• Releases Emtansine
• EMTANSINE Mechanism of action: Binds to microtubules and inhibits their polymerization, causing cell-cycle arrest and cell death
• Approved for metastatic breast cancer (in patients resistant to trastuzumab alone)
• Adverse reactions include cardiotoxicity

ADC Toxicities

• Target-induced toxicity
• FcR-driven toxicity
• Pinocytosis driven toxicity
• Bystander toxicity

Immune Checkpoint Inhibitors

• Immune checkpoints are inhibitory pathways expressed by T cells that limit T cell activation to maintain self-tolerance.
• Checkpoint blockade inhibits these negative signals allowing for a more robust T cell response.
• Two important inhibitory receptors: Cytotoxic T lymphocyte—associated protein 4 (CTLA-4), and programmed death-1 protein (PD-1)

CTLA-4 - Ipilimumab

• Cytotoxic T-lymphocyte antigen 4 (CTLA4) is unregulated on T cells during activation
• CTLA4 downregulates T-cell activity to maintain normal immunological homeostasis
• CTLA4 downmodulates the amplitude of T-cell activation to avoid T-cell activation against cancer cells
• Blocking CTLA4 with a specific antibody maintains T-cell stimulation and was approved as a treatment for melanoma

PD-1 - Nivolumab

• PD1 (programmed death-1) is a receptor on T cells
• Inhibiting the interaction between PD-1 and its ligand on the tumor cell unleashes T cells against tumor cells
• As a first line treatment monotherapy for inoperable/metastatic melanomas

Immune Checkpoint Inhibitors: Additional Information

• Checkpoint blockade therapies can allow T cells to attack normal organs.
• Patients can develop life-threatening diabetes, cytokine storm, or autoimmune diseases.

Angiogenesis inhibition (Bevacizumab)

• Blood vessel development is due to the VEGF family of proteins and their receptors.
• VEGF-A is a direct-acting angiogenic protein that is expressed in most tumors and reflects the extent of angiogenesis.

Rationale for Anti-Angiogenesis Therapy

• Angiogenesis and neovascularization allow the tumor to progress to an invasive state.
• Endothelial cells are genetically more stable than tumor cells, thus reducing the likelihood of drug resistance
• Easier access to tumor vasculature by drugs
• One drug can work against multiple tumors, as all solid tumors depend on vasculature.

Monoclonal antibodies targeting TNF-α to treat inflammation

Autoimmune Inflammatory Diseases

• Rheumatoid Arthritis (RA): Joint inflammation, with TNF-α targeting treatment.
• Psoriasis: Chronic skin disorder with several anti-TNF-a treatments (etanercept, adalimumab, infliximab).
• Inflammatory bowel disease (IBD): Crohn's disease (CD), and ulcerative colitis (UC).
• Systemic lupus erythematosus (SLE): Widespread tissue inflammation, with immunosuppressive treatment but not anti-TNF-a.

TNF-α

• TNF-α is released into serum from different cells (macrophages, T cells, natural killer cells) as a soluble cytokine (homo-trimer of 17 kDa monomer).
• Produced after TACE cleavage of tmTNF-α ( a homo-trimer of 26 kDa monomers)
• Involved in systemic inflammation and immune cell function.
• Binds to TNF-R1 (p55) and TNF-R2 (p75) which plays a role in apoptosis or NF-kB activation and inflammatory gene activation.

TNF-α is pleiotropic

• TNF-α is produced at high concentrations in RA, Crohn's disease, and Psoriasis.
• Other MAbs in inflammation use targets besides TNF-α (e.g., rituximab for CD20).

Anti-TNF-α therapy

• Infections can occur due to inhibition of protective cytokine functions like TNF-a
• Serious infections due to bacterial, mycobacterial, invasive fungal, or viral infections.
• Patients need close monitoring for infection symptoms.

MAb for immunity-mediated inflammatory diseases

• Various monoclonal antibodies that target TNF-α
• Recombinant fusion protein targeting TNF-R2,
• Other antibodies targeting different targets (e.g., rituximab).

Mode of Action of Anti TNF-a Drugs

• ADCC (Antibody-dependent cellular cytotoxicity) on NK cells;
• CDC (Complement-dependent cytotoxicity) on immune cells through C3.

Monoclonal anti-TNF antibody: Infliximab & Adalimumab

• Infliximab (Remicade®) is a mouse/human chimeric monoclonal anti-TNF antibody of IgG1 isotype.
• Adalimumab (Humira®) is a human IgG1 antibody

Certolizumab (Cimzia)

• PEGylated Fab' fragment of a humanized IgG1 monoclonal anti-TNF-a antibody.
• Neutralizes TNF-α activity by binding to it.

Etanercept (Enbrel)

• Dimeric fusion protein with extracellular ligand-binding portion of human 75 kDa (p75) TNF-receptor linked to the Fc portion of human IgG1.
• Inhibits TNF-a and TNF-β binding to their receptors, rendering TNF biologically inactive

Other drugs used in Inflammatory diseases A) Rituximab

• Second-line biologic therapy for RA.
• B cells involved in RA pathogenesis.

B) Abatacept

• Activated T lymphocytes implicated in RA.
• A fusion protein; extracellular domain of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) linked to the Fc portion of human IgG1.
• Binds to CD80 and CD86 on APCs to block T-cell activation.
• Not to be given with TNF antagonists concurrently due to severe adverse reactions

Cytokines; alpha-helical barrel proteins (IFN-α2) & PEG-IFN-α2

Transmission of HCV

• Injection and illegal drug use
• Transfusion and organ transplantation
• Hemodialysis
• Healthcare workers (needle stick injury/blood splash to the eye)
• Sexual activity
• Tattooing/body piercing
• Vertical Transmission (5%-6% among infants born to infected women

HCV Genotypes

• Single-stranded RNA virus that replicates extremely rapidly (10^12 virions/day.)
• Six genetically distinct groups (genotypes 1-6) have been identified.
• Genotype does not change during infection; important for evaluation of chronic HCV infection.
• Strongest predictor of response to treatment.
• Higher cure rates with PEG-IFN/ribavirin for genotypes 2 & 3; treatment duration shorter than genotype 1.

HCV Treatment

• PEGylated interferon-alpha 2+ ribavirin therapy (50% cure rate)
• Low MW direct-acting antivirals (DAAs), including viral protease, NS5A, & polymerase inhibitors.
• Fixed dose combinations (FDCs) of DAAs

General Characteristics of Cytokines

• Mod. low MW proteins.
• Potent—active at very low concentrations
• Produced by multiple cell types.; secretion brief & self-limited; autocrine, paracrine & endocrine action.
• Multiple cell types, pleiotropic.
• Actions are redundant—they affect synthesis & action of other cytokines

Cytokines & Chemokines

• Cytokine producing cells secrete cytokines with different effects.
• Cytokines can have autocrine, paracrine, and endocrine actions.

Cytokines of Therapeutic Relevance

• Interleukins (ILs): secreted by leukocytes, affect hematopoietic cell growth and differentiation
• Hematopoietic growth factors (HGFs): include erythropoietin, G-CSF, and thrombopoietin
• Interferons (IFNs): proteins produced in response to viral infections, tumors, etc; promote an antiviral state and regulate the immune system.

Interferons (IFNs)

• Produced by eukaryotic cells in response to viral infections, tumors, etc.
• Promote an antiviral state and help regulate immune system function.

Native Interferon & Viral Infection

• Cells respond to viral infection by producing IFN.
• IFN receptors activate antiviral proteins (ISGs) in the infected cell.

Interferon therapy: Interferon-alpha 2 (IFN-α2)

• Type 1 interferon, several isoforms exist.
• Induced by viral infection & other pathogens
• Mechanism: binds to dimeric cell surface receptor (e.g. JAK-STAT)
• Increases the expression of IFN stimulated genes (ISGs) for anti-viral, anti-proliferative & immunoregulatory states
• Stimulates macrophages & NKs

Limitations of Native IFN-a2 Therapy

• Rapid absorption: After subcutaneous injection
• Vast systemic distribution & high renal clearance: Widespread distribution & rapid removal.
• Short serum half-life: Short time in the body
• Wide fluctuation in serum concentration: Serum levels fluctuate greatly, irregular responses
• Inadequate antiviral coverage: Limited range of viruses affected
• Severe ARS: Include influenza-like symptoms.
• Low compliance/Poor adherence: Difficulty in keeping up the treatment schedule.
• Reduction of activity: Observed in some instances

IFN-α2 Therapeutics

• Produced in E. coli
• Approved for HBV, HCV, Kaposi sarcoma, renal cell carcinoma, melanoma, etc.
• PEG-IFN (Pegasys) are biobetter versions designed for sustained virologic response (SVR). (undetectable HCV RNA 12 weeks post-treatment)

HCV treatment II Development of Direct Acting Antivirals (DAAs)

The pros and cons of DAAs

• Challenges: Some viral resistance, side effects (fever, headache, nausea, and fatigue), and high cost
• Despite these drawbacks: Better efficacy, improved adherence, lower incidence/severity of ARs.
• Sovaldi (sofosbuvir): Novel direct-acting antiviral, pangenotypic activity (treating many HCV genotypes), higher barrier to resistance.

HCV Treatment III Fixed Dose Combinations of DAAs

• Fixed dose combinations (FDCs) with PEG-IFN/ribavirin for HCV
• Several FDCs are already approved, under clinical development, or discontinued.

Cytokines 2: alpha-helical barrel proteins (IFN-β and Growth Hormone)

• (IFN-beta) & Growth Hormone are alpha-helical barrel proteins

Multiple Sclerosis

• Chronic progressive CNS disorder characterized by inflammation, demyelination, and neurodegeneration.
• Primarily affects females.
• Commonly presents as relapsing-remitting MS (RRMS).
• Multifactorial and highly individual disease in each patient; autoimmune response to self-antigens.

MS: A Complex Condition

• Symptoms are highly variable in severity/duration,
• Include fatigue, visual impairment, spasticity, ataxia, weakness, bladder/bowel dysfunction, sexual dysfunction, and cognitive impairment

Rationale for Early Treatment of MS

• Current therapies seem to address only early stages of the disease.
• Early damage is caused by an immune attack with T and B cells and macrophages infiltrating the CNS.
• Immune-modulating treatments affect inflammation, which predominates in the early stage.
• Early disease actively drives long-term disability.
• Relapse frequency correlates with accumulating disability during the relapsing-remitting phase of MS.

Interferon-Beta (IFN-β)

• Mechanism of Action: Thought to be primarily through anti-viral action and antagonism of pro-inflammatory cytokines and class II MHC antigen expression, to reduce relapse rate, prevent fixed disability, and provide symptomatic management of fixed neurological deficits.
• IFN-β-1b: E. coli-produced, non-glycosylated.
• IFN-β-1a (Avonex): Glycosylated, independently developed from CHO cells
• PEG-IFNβ-1a: PEGylated version of Avonex; approved in 2014

Growth hormone

• Essential hormone for normal growth and development.
• Isolated in the 1950s from pituitary glands, obtained from cadavers and patients.
• Affects lipolysis, stimulates protein synthesis, and affects glucose metabolism.
• First clinical use was in hypopituitary children in 1957.
• Recombinant GH was reported in 1982.

Control of GH secretion

• Secreted from somatotrophs in anterior pituitary.
• Regulate GH release (GHRH and somatostatin)
• Circulating GH binds to GH receptor on peripheral tissue (muscle, liver, bone) inducing secretion of IGF-1 to promote growth.
• Secretion changes during life (greatest during gestation and puberty), it demonstrates a pulsatile characteristic with major surges at sleep onset—slow wave sleep.

Insulin-like Growth Factor 1 (IGF-1)

• Hormone similar to insulin in structure. Important for childhood growth and anabolic effects in adults.
• Primarily produced by the liver.
• Involved in the growth and having important metabolic effects throughout life.
• Found in all tissues; tyrosine kinase receptor activation causes proliferation.
• Biomarker for GH activity in humans

Clinical Use of HGH

• Growth promotion:
  • Growth hormone deficiency in children
  • Idiopathic short stature (ISS)
  • Growth failure with chronic renal insufficiency in children
  • Growth failure in children born small for gestational age (SGA)
• Modulation of metabolism:
  • Long-term replacement therapy in adults with GH deficiency (childhood or adult onset), AIDS wasting, and in short bowel syndrome.

Efforts to extend the duration of action of GH

• Several methods to extend GH action.
• PEGylated GH; Jintrolong
• SC administration once weekly; mimics normal pulse pattern.

Acromegaly

• Growth disorder, characterized by abnormal enlargement of hands, feet, and skull
• Often caused from pituitary adenoma leading to GH overproduction.

EPO to treat Anemia

• Subject: Erythropoiesis, blood, hematopoietic growth factors
• Anemia: decreased Red Blood Cell (RBC) count,
• EPO (Erythropoietin): Glycoprotein produced mostly by kidney cells (and some in liver in fetus). Regulates erythropoiesis (RBC production) in mammals
• Factors affecting EPO production:
  • Tissue oxygen tension -Oxygen demand of producer cells -Relative oxygen supply

Physiological role of EPO

• EPO increases committed progenitor cells,
• causing them to proliferate and differentiate
• Moves reticulocytes from bone marrow to peripheral circulation
• Tissue hypoxia stimulates production of EPO (Often due to anemia)

Anemia of Renal Disease

• Anemia is common in Chronic Kidney Disease (CKD) and Chronic Renal Failure (CRF), caused by decreased erythropoietin production.
• Recombinant human EPO (rhEPO) is used to treat anemia in CKD, by stimulating erythropoiesis (RBC production)

Some EPO side effects

• Escalation of existing hypertension,
• Clotting at injection sites - Joint pain
• Flu-like symptoms
• Rare side effect is pure red cell aplasia (PRCA), triggered by antibodies formed to epoetins, leading to antibody formation in re-exposure.
• Strategies to reduce PRCA: using longer-acting EPO medications (like darbepoetin and Mircera).

Glycosylated & PEGylated EPO

• Additional glycosylation or PEGylation for longer half-life, modified EPO formulations for improved activity/safety

Hematopoietic Growth Factors (HGFs): G-CSF & Coagulation Factors

G-CSF Therapeutics

• Filgrastim (rhG-CSF): absorbed quickly (3.5 hrs) upon subcutaneous or IV administration
• Pegfilgrastim (PEG-GCSF): Non-Specific PEGylation with 15-80h half-life via SC administration (non-linear pharmacokinetics for cancer patients)
• Lipegfilgrastim: Site-specific PEGylation; Improvement on pharmacokinetics over pegfilgrastim

Summary of HGF indications

• EPO: Treatment of anemia associated with chronic renal failure
• G-CSF:
  • Treat chemotherapy-induced neutropenia.
  • Prevent infections in HIV/AIDS patients.
  • Support hematopoiesis after bone marrow transplantation
• Pharmaceutical issues: Recombinant HGFs need to stay frozen; avoid shaking, inspect for particulate matter before administration

Hematopoietic Growth Factors (HGFs): Thrombopoetin

• Thrombopoietin (TPO) is a glycoprotein hormone, secreted by the liver and kidney.
• Regulates platelet production and differentiation of megakaryocytes.

Three major events happen to rapidly stop the loss of blood

• 1. Vasoconstriction: Reduce blood flow to the damaged area
• 2. Clumping of blood platelets: To form a platelet plug
• 3. Aggregation of fibrin: Form an insoluble clot over the rupture to stop blood loss (Fibrin mesh).

Clot formation

• Two initial pathways for clot formation: Intrinsic & extrinsic. Both pathways activated when clotting factors make contact with exposed surface molecules when a blood vessel is damaged.
• Final steps are shared by both pathways—supplying the protease (Factor Xa) which activates thrombin to activate fibrin.

Simplified version of the coagulation pathway

• Intrinsic pathway (Initiating factor activation with exposed membrane matrix proteins)
• Extrinsic pathway (Initiating factor activation by trauma outside blood vessel causing tissue damage) and Factors acting in both pathways.

Hereditary genetic disorders of coagulation factors (Hemophilia)

• Hemophilia: hereditary genetic disorders that impair the body's ability to control blood clotting (when a blood vessel is broken down.)
• Hemophilia A (factor VIII deficiency): Common form
• Hemophilia B (factor IX deficiency): Rare form
• Complications include deep muscle bleed, joint damage, infection, pain, and even death. Recombinant coagulation factors improve treatment.

Factor VIII as a medicine for Hemophilia A

Normal FVIII plasma concentration— ~200 ng/mL FVIII - large precursor protein devoid of activity—serves as cofactor in FX activation by FXa. It works in the presence of calcium ion and phospholipid (PL) Recombinant FVIII—avoids use of blood-extracted FVIII, (risk of blood-borne diseases) Patients often develop antibodies to FVIII

Factor VIII as a medicine for Hemophilia A: Additional Treatment

• Administration of other blood factors (especially FVII) from intrinsic pathways.
• Administration of VIII and an immunosuppressive agent

Factor VIII, Anti-hemophilic Factor (coagulation factors are complex proteins)

• Factor VIII is synthesized primarily, from liver as a large glycoprotein (approximately ~300 kda), composed of three distinct domains in arrangement (ABC)
• Factor VIII activity NOT controlled by B Domain, large B Domain can be removed w/o loss of FVIII activity
• Refacto (B-domain deleted rhFactor VIII): lacks B domain, essentially identical to full length native FVIIIa after proteolytic cleavage.

Recombinant FIX to treat Hemophilia B

• FIX is activated by FXa (intrinsic pathway)
• Activated FIX, with FVIIla, activates FX for converting Prothrombin to thrombin
• Thrombin converts fibrinogen to fibrin, which forms clots.

Development of Inhibitors (Antibodies) in Hemophilia

• Some develop antibodies/inhibitors against clotting factors, resulting in ineffective treatment.
• Bypass agents (e.g., rFVIIa), products mimicking factor VIII (e.g., emicizumab), high-dose clotting factor concentrates, and ITI therapy (Immune tolerance induction or therapy) are used to overcome this.

Other causes & solutions of bleeding disorder

• Low platelet count (thrombocytopenia): Solutions include blood transfusion, frozen platelets, and Recombinant Thrombopoietin (TPO)
• Vitamin K deficiency: Vit K
• Calcium (Ca): Ca
• Decreased coagulation factors in liver disease: Blood transfusion of clotting factors and platelets; TPO if produced by liver.

Endogenous sources of hematopoietic growth factors (HGFs)

• Hematopoietic growth factors with sources (G-CSF, EPO, and TPO)

Thank you

This quiz focuses on essential topics in Pharmacy 401, specifically regarding protein therapeutics. It covers the distinction between large and small molecule drugs, including their properties, synthesis, and costs. Additionally, it touches on protein synthesis processes, such as folding and structure.