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COMPLETE PHARMACOLOGY EXAM GUIDE

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1. TRANSPORT OF DRUGS ACROSS BIOLOGICAL MEMBRANES

The Biological Membrane

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Classification of Drug Transport Mechanisms

DRUG TRANSPORT
│
├── 1. Passive Transport (No energy required)
│   ├── A. Simple (Passive) Diffusion
│   └── B. Filtration (Aqueous diffusion)
│
├── 2. Specialized Transport (Carrier-mediated)
│   ├── A. Facilitated Diffusion
│   ├── B. Active Transport
│   └── C. Endocytosis / Exocytosis
│
└── 3. Ion Pair Transport

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A. Simple (Passive) Diffusion

• Most common mechanism for drug absorption
• Drug moves from high concentration → low concentration (down concentration gradient)
• No carrier, no energy (ATP) required
• Obeys Fick's Law of Diffusion

Rate of diffusion = (C₁ - C₂) × SA × P × Kp / d

• C₁ - C₂ = Concentration gradient
• SA = Surface area of membrane
• P = Permeability coefficient of drug
• Kp = Partition coefficient (lipid/water)
• d = Thickness of membrane

1. Lipid solubility (lipophilic drugs pass easily)
2. Small molecular size
3. Non-ionized form (uncharged)

For weak acids: pH - pKa = log [ionized] / [non-ionized] For weak bases: pH - pKa = log [non-ionized] / [ionized]

• Aspirin (weak acid, pKa 3.5) - absorbed in stomach (pH 1-2, non-ionized form favored)
• Morphine (weak base) - better absorbed in intestine (alkaline pH)
• Ethanol, steroids, O₂, CO₂ - all diffuse passively

• Weak acid drug overdose (aspirin) → alkalize urine (NaHCO₃) → ionizes drug → traps it in urine → accelerates excretion
• Weak base drug overdose (amphetamine) → acidify urine → promotes excretion

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B. Filtration (Aqueous Diffusion / Paracellular transport)

• Movement through aqueous channels (pores) between or within cells
• Driven by hydrostatic or osmotic pressure gradient
• Limited to small, water-soluble molecules (<200 Da)
• Glomerular filtration of drugs (e.g., inulin, mannitol) is a key example
• Capillary endothelium has large pores → allows most drugs through
• Blood-brain barrier (BBB) has tight junctions → blocks paracellular transport

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C. Facilitated Diffusion

• Carrier-mediated transport down the concentration gradient
• No energy required, but uses a specific carrier protein
• Shows saturation kinetics (Km, Vmax) and competitive inhibition (like enzymes)
• Stereoselective - specific for certain molecular shapes

• Glucose transport into RBCs (GLUT transporters)
• Vitamin B12 absorption (intrinsic factor)

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D. Active Transport

• Movement against concentration gradient (low → high)
• Requires energy (ATP)
• Uses specific carrier proteins (pumps)
• Shows saturation, specificity, and inhibition
• Can be primary active (directly uses ATP) or secondary active (co-transport)

• Na⁺/K⁺-ATPase pump
• Levodopa - absorbed via amino acid active transporter (L-amino acid transporter, LAT1)
• 5-Fluorouracil - enters cells via active transport
• P-glycoprotein (P-gp) - efflux pump; pumps drugs OUT of cells (responsible for MDR - Multi-Drug Resistance)
  • Found in gut, liver, kidney, BBB
  • Inhibited by verapamil → increases drug bioavailability
• Organic Anion Transporters (OATs), Organic Cation Transporters (OCTs) - in kidney tubules

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E. Endocytosis and Exocytosis

• Endocytosis: Cell membrane engulfs drug by invagination → forms vesicle inside cell
  • Pinocytosis: Fluid-phase (non-specific)
  • Receptor-mediated endocytosis: Specific (e.g., LDL-cholesterol, insulin)
• Exocytosis: Reverse process - vesicle fuses with membrane → releases contents outside
  • e.g., neurotransmitter release (ACh, norepinephrine)

• Vitamin B12 - receptor-mediated endocytosis in ileal cells
• Large proteins/nanoparticle drug delivery systems

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Summary Table: Types of Drug Transport

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2. FACTORS AFFECTING DRUG ABSORPTION

A. Physicochemical Factors (Drug Properties)

• More lipophilic → better passive diffusion → better absorption
• Measured by partition coefficient (P) or log P
• Log P >1 = good oral absorption; too high → poor water solubility

• Only non-ionized form crosses membranes
• Weak acids (ASA) absorbed better in acidic pH (stomach)
• Weak bases (morphine, atropine) absorbed better in alkaline pH (intestine)
• Henderson-Hasselbalch equation governs this

• <200 Da → filtration possible
• 200-500 Da → passive diffusion

• 500 Da → poor oral absorption; may need special transport

• Solution > suspension > capsule > tablet > coated tablet (in terms of dissolution rate)
• Particle size: smaller particles → larger surface area → faster dissolution → faster absorption
• Salt form: affects solubility (e.g., sodium salts dissolve faster)
• Polymorphism: different crystal forms have different solubilities

• Acid-labile drugs (e.g., penicillin G, erythromycin) are degraded in stomach acid → require enteric coating or parenteral route

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B. Physiological Factors (Patient Factors)

• IV > inhalation > sublingual > IM/SC > oral > rectal > topical
• IV = 100% bioavailability (no absorption step)

• Gastric emptying rate: Faster emptying → drug reaches small intestine faster → faster absorption (small intestine is the main absorptive site due to large surface area - villi, microvilli)
  • Factors that SLOW gastric emptying: food (fatty meals), anticholinergic drugs, opioids
  • Factors that SPEED gastric emptying: metoclopramide, prokinetics
• Intestinal motility: Too fast → reduced contact time → reduced absorption
• pH of GI tract: Stomach pH 1-3; small intestine pH 5-7; large intestine pH 7-8
• Surface area: Small intestine has maximum surface area (due to villi and microvilli = "brush border") → primary site of absorption
• Splanchnic blood flow: Reduced blood flow (shock, CCF) → reduced absorption
• First-pass metabolism: Drugs absorbed from gut pass through liver via portal vein BEFORE reaching systemic circulation. Liver enzymes metabolize drug → reduced bioavailability
  • High first-pass drugs: morphine, propranolol, nitroglycerin, lidocaine, aspirin, verapamil
  • To bypass first-pass: sublingual (NTG), transdermal, rectal, IV

• Fatty food increases absorption of lipophilic drugs (griseofulvin, itraconazole)
• Calcium in dairy/antacids chelates tetracyclines and fluoroquinolones → reduced absorption
• Food can delay gastric emptying → delays absorption peak (Tmax) but may not affect total absorption (AUC)

• Neonates: reduced gastric acid → higher pH → alkaline pH → affects weak acid absorption
• Elderly: reduced GI motility, reduced splanchnic blood flow

• Malabsorption (Crohn's disease, celiac): reduced absorption
• Achlorhydria: reduced dissolution of acidic drugs

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3. VOLUME OF DISTRIBUTION (Vd)

Definition

Vd = Amount of drug in body / Plasma drug concentration Vd = D / Cp

• Units: Liters (L) or L/kg

Significance of Vd Values:

Factors Affecting Vd:

• Highly protein-bound drugs → remain in circulation → small Vd
• Acidic drugs bind to albumin (warfarin, aspirin, phenytoin)
• Basic drugs bind to alpha-1 acid glycoprotein (propranolol, lidocaine)
• Only free (unbound) drug is pharmacologically active, can cross membranes, and can be metabolized/excreted

• Strong tissue binding → drug moves out of plasma → large Vd
• Chloroquine accumulates in liver, kidney, lung
• Digoxin binds to Na/K ATPase in cardiac muscle

• Lipophilic drugs → cross BBB, accumulate in fat → large Vd
• Hydrophilic drugs → remain in plasma/interstitial fluid → small Vd

• Obesity → increased Vd for lipophilic drugs
• Edema/ascites → increased Vd for hydrophilic drugs
• Neonates: more total body water → larger Vd for water-soluble drugs

Clinical Use of Vd:

• Loading dose calculation:

Loading Dose = Vd × Target plasma concentration / Bioavailability (F) or: LD = Vd × Cp(target)

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4. KEY DEFINITIONS

BIOAVAILABILITY (F)

F = AUC(oral) / AUC(IV) × 100%

• AUC = Area Under the Curve (plasma concentration vs. time) - represents total drug exposure
• IV administration: F = 100% (reference)
• Oral: F < 100% due to incomplete absorption and first-pass metabolism

1. First-pass metabolism (most important for oral drugs)
2. Incomplete absorption
3. Gut wall metabolism (CYP3A4 in enterocytes)
4. P-glycoprotein efflux
5. Interaction with food/other drugs
6. Chemical instability in GI tract

• Morphine: F = 25-30%
• Propranolol: F = 25%
• Nitroglycerin: F = <1% oral → given sublingual or transdermal
• Lidocaine: F = <35% → given IV only
• Verapamil: F = 20-35%

• Diazepam: F = 100%
• Theophylline: F = ~100%
• Prednisolone: F = ~80%

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BIOEQUIVALENCE

• AUC and Cmax of the test product must be within 80-125% of the reference product (90% confidence interval)
• Tmax should be similar (acceptable range)

• Pharmaceutical equivalents: Same active ingredient, same dose form, same strength
• Bioequivalent: Same pharmacokinetic profile
• Therapeutic equivalents: Same clinical efficacy and safety (both pharmaceutical equivalents AND bioequivalent)

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PRODRUG

1. Improve oral bioavailability (bypass first-pass problem)
2. Improve stability
3. Improve taste/tolerability
4. Reduce toxicity
5. Achieve site-specific delivery (targeted therapy)
6. Overcome poor solubility

• Hepatic enzymes (CYP450, esterases)
• Gut wall enzymes
• Plasma esterases
• Target organ enzymes

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HOFMANN ELIMINATION (Hoffman Elimination)

• The drug undergoes a chemical (non-biological) β-elimination reaction at body temperature (37°C) and physiological pH (7.4)
• The drug degrades into inactive breakdown products spontaneously in plasma

• Drugs eliminated by Hofmann elimination are safe in patients with hepatic or renal failure
• No dose adjustment needed in liver/kidney disease
• Also safe in patients with abnormal plasma cholinesterase (pseudocholinesterase)

1. Atracurium - neuromuscular blocking agent (NMB)
2. Cisatracurium - more potent isomer of atracurium; cleaner Hofmann profile
   • Also undergoes ester hydrolysis by plasma esterases
   • Produces laudanosine (a metabolite that can cause CNS stimulation at very high levels - seizures in experimental animals)

• Mivacurium and succinylcholine are metabolized by plasma pseudocholinesterase (NOT Hofmann)
• Deficiency of pseudocholinesterase → prolonged neuromuscular blockade with succinylcholine/mivacurium → dibucaine number used to test pseudocholinesterase activity

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HALF-LIFE (t½)

t½ = 0.693 × Vd / CL

• 0.693 = natural log of 2 (ln 2)
• Vd = Volume of distribution
• CL = Total clearance

• Half-life is constant for first-order kinetics (independent of dose/concentration)
• After 4-5 half-lives: ~94-97% of drug eliminated → considered clinically eliminated
• After 4-5 half-lives with repeated dosing: steady-state (Css) is reached
• Half-life determines dosing interval and duration of action

1. First-order kinetics (most drugs): A constant FRACTION of drug is eliminated per unit time → t½ is constant
   • Rate of elimination ∝ drug concentration
2. Zero-order kinetics (saturation kinetics): A constant AMOUNT is eliminated per unit time → t½ NOT constant; increases with dose
   • Occurs when elimination mechanisms are saturated
   • Examples: Phenytoin, Alcohol (ethanol), Aspirin (high doses), Theophylline (high doses)
   • Michaelis-Menten kinetics applies

Css = Dose × F / (CL × Dosing interval) or Css = (F × Dose/τ) / CL

• Loading dose can achieve Css quickly when t½ is very long (e.g., amiodarone, digoxin)

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5. COMPETITIVE vs. NON-COMPETITIVE ANTAGONISM

Competitive Antagonism (Surmountable Antagonism)

• Shifts dose-response curve to the RIGHT (parallel shift)
• Maximum response (Emax) is preserved - can be overcome by increasing agonist concentration
• Affinity (EC50/KD) increases (higher concentration of agonist needed to achieve same effect)
• Efficacy/Emax is NOT reduced
• Reduction in potency (higher EC50) but same maximum

Response (%)
100 |────────────────────────────────
    |         ----      -----
    |      --              --
    |    -    (Agonist alone)   - (Agonist + competitive antag)
    |  -
    |_________________________________
         Log[Agonist dose]
         ← shift right, same Emax

1. Atropine vs. Acetylcholine (at muscarinic receptors) - classic example
2. Naloxone vs. Opioids (at opioid mu receptors) - reverses opioid overdose; can overcome with more opioid
3. Propranolol vs. Epinephrine (at β-adrenergic receptors)
4. Cimetidine vs. Histamine (at H2 receptors in stomach)
5. Neostigmine (indirectly) - can overcome competitive NMB (e.g., pancuronium, vecuronium) by increasing ACh
6. Flumazenil vs. Benzodiazepines (at GABA-A receptor benzodiazepine site)
7. Prazosin vs. Norepinephrine (at α1-adrenergic receptors)

• Reversible competitive: Classic, most common (above)
• Irreversible competitive (non-equilibrium competitive): Antagonist binds covalently → cannot be overcome by increasing agonist → Emax is reduced at high antagonist concentrations
  • e.g., Phenoxybenzamine (α-blocker, covalent bond) - though some classify separately

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Non-Competitive Antagonism (Insurmountable Antagonism)

• Reduces Emax (maximum response) - cannot be overcome by increasing agonist
• Dose-response curve shifts right AND plateau is lowered
• Potency of agonist may or may not change
• Binding is often irreversible (covalent) or the antagonist blocks a step downstream

Response (%)
100 |──────────────────────────
    |    ─────── (Agonist alone)
 60 |         ───────── (+ non-competitive antag; lower Emax)
    |
    |_________________________________
         Log[Agonist dose]
         Emax is reduced

1. Phenoxybenzamine (irreversible α-blocker) - classic non-competitive / insurmountable
2. Ketamine (NMDA receptor - open channel block, prevents agonist effect even at high glutamate)
3. Aspirin (irreversible COX inhibitor - not a receptor antagonist per se, but shows insurmountable blockade of prostanoid synthesis)
4. Organophosphates (irreversible acetylcholinesterase inhibitors) - cannot overcome with more ACh
5. Allosteric modulators that reduce receptor coupling

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Comparison Table: Competitive vs. Non-Competitive

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6. AGONIST, PARTIAL AGONIST, INVERSE AGONIST

Receptor Activation Concepts

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Full Agonist

• Intrinsic efficacy = 1 (maximum)
• Both high affinity AND high efficacy

• Morphine (full µ-opioid agonist)
• Adrenaline/Epinephrine (full α and β agonist)
• Isoproterenol (full β-agonist)
• Salbutamol (albuterol) - full β2 agonist

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Partial Agonist

• Has BOTH agonist and antagonist properties depending on context
• In the absence of a full agonist: it acts as an agonist (produces partial response)
• In the presence of a full agonist: it acts as an antagonist (displaces full agonist, net effect is LESS than full agonist alone)
• Ceiling effect (plateau) below 100% Emax

• Built-in "ceiling" - safer overdose profile
• Buprenorphine - ceiling on respiratory depression → safer than full opioid agonists

1. Buprenorphine (partial µ-opioid agonist) - used in opioid dependence treatment; also in chronic pain
2. Buspirone (partial 5-HT1A agonist) - anxiolytic; no dependence
3. Pindolol (partial β-agonist - has intrinsic sympathomimetic activity/ISA)
4. Aripiprazole (partial D2 and 5-HT1A agonist) - antipsychotic
5. Clomiphene (partial estrogen agonist/antagonist) - used for ovulation induction

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Inverse Agonist

• R (inactive state) ⇌ R* (active state) → some constitutive activity exists

1. Beta-carboline compounds (inverse agonist at GABA-A benzodiazepine site) - cause anxiety, seizures
2. Ro 15-4513 (inverse agonist at benzodiazepine site)
3. Propranolol - has some inverse agonist activity at β-receptors (not just neutral antagonist)
4. Mifepristone (RU-486) - considered inverse agonist at progesterone receptor in some contexts
5. Histamine H3 receptor inverse agonists - some antihistamines

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7. ADVERSE DRUG REACTIONS (ADR)

Classification of ADRs

Type A - Augmented (Pharmacological) Reactions

• Most common (~80% of all ADRs)
• Predictable, dose-dependent
• Related to the pharmacological action of the drug (exaggerated normal effect)
• Low mortality
• Managed by dose reduction
• Examples:
  • Bradycardia with beta-blockers
  • Hypoglycemia with insulin
  • Hemorrhage with anticoagulants (warfarin)
  • Dry cough with ACE inhibitors (bradykinin accumulation)

Type B - Bizarre (Idiosyncratic) Reactions

• Unpredictable, dose-independent
• NOT related to pharmacological action
• High mortality (rare but severe)
• Includes immunological reactions and genetic polymorphisms
• Examples:
  • Anaphylaxis with penicillin
  • Malignant hyperthermia with succinylcholine/halothane (due to ryanodine receptor mutation)
  • Stevens-Johnson Syndrome (SJS) with carbamazepine, allopurinol, sulfonamides
  • Aplastic anemia with chloramphenicol
  • Hemolytic anemia with primaquine in G6PD deficiency

Type C - Chronic / Continuous

• Long-term, dose-related
• Associated with prolonged use
• Examples:
  • Osteoporosis with long-term corticosteroids
  • HPA axis suppression with steroids
  • Tardive dyskinesia with long-term antipsychotics (D2 receptor upregulation)
  • Renal papillary necrosis with long-term NSAIDs

Type D - Delayed

• Occur after long latency period
• Examples:
  • Carcinogenicity: alkylating agents → secondary malignancies
  • Teratogenicity: thalidomide (phocomelia), phenytoin (fetal hydantoin syndrome), valproate (neural tube defects)
  • Tardive dyskinesia: antipsychotics

Type E - End-of-Use (Withdrawal)

• Occurs when drug is abruptly stopped after chronic use
• Examples:
  • Opioid withdrawal syndrome
  • Benzodiazepine withdrawal → seizures
  • Clonidine withdrawal → hypertensive crisis ("rebound hypertension")
  • Corticosteroid withdrawal → adrenal crisis

Type F - Failure of Therapy (Unexpected Failure)

• Drug fails to produce the expected therapeutic effect
• Often due to drug interactions, resistance, poor compliance
• Example: Oral contraceptive failure with enzyme inducers (rifampicin, carbamazepine)

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Additional Classification of ADRs by Mechanism:

• Dose-related, e.g., digoxin toxicity (arrhythmias, visual disturbances), opioid overdose

• Type I (IgE-mediated): Immediate hypersensitivity - anaphylaxis (penicillin)
• Type II (cytotoxic): Drug-induced hemolytic anemia (methyldopa, penicillin)
• Type III (immune complex): Serum sickness (penicillin, sulfonamides)
• Type IV (cell-mediated/delayed): Contact dermatitis (topical antibiotics), SJS

• Genetic polymorphisms: G6PD deficiency → hemolysis with oxidant drugs
• CYP2D6 poor metabolizers → codeine toxicity (in certain populations, ultra-rapid metabolizers convert codeine → morphine too quickly)

• IgE-independent mast cell degranulation
• e.g., Aspirin/NSAID-induced urticaria/bronchospasm (via leukotriene excess)
• Radiocontrast media, vancomycin (Red Man Syndrome)

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Severity Classification:

• Mild: No antidote needed; resolves spontaneously (e.g., GI upset)
• Moderate: Requires change in therapy (e.g., rash requiring drug discontinuation)
• Severe: Life-threatening; requires hospitalization (anaphylaxis, SJS, TEN)
• Fatal: Death (e.g., agranulocytosis from clozapine/carbimazole)

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ADR Monitoring: Pharmacovigilance

• Yellow Card Scheme (UK) / MedWatch (FDA) - spontaneous reporting
• Phase IV post-marketing surveillance - detects rare/late ADRs
• Causality assessment: WHO-UMC scale (Certain, Probable, Possible, Unlikely, Unclassified, Unassessable)
• Naranjo Scale - probability assessment for ADR causality

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8. G-PROTEIN COUPLED RECEPTORS (GPCR)

Structure of GPCRs

• 7-Transmembrane (7-TM) receptors
• Heptahelical receptors
• Serpentine receptors (because the polypeptide chain crosses the membrane 7 times)

• Single polypeptide chain with 7 hydrophobic transmembrane alpha-helical segments (TM1-TM7)
• N-terminus: Extracellular (glycosylated)
• C-terminus: Intracellular (phosphorylation sites)
• 3 extracellular loops (EL1, EL2, EL3) - form drug binding pocket
• 3 intracellular loops (IL1, IL2, IL3) - couple to G-proteins; IL3 is the largest
• Orthosteric binding site: Between TM helices (for classical ligands)
• Allosteric binding sites: Other locations

G-Protein Structure

• Gα (alpha) - binds GTP/GDP; has GTPase activity; determines downstream effect
• Gβ (beta) + Gγ (gamma) - dimer; anchors complex to membrane; also has signaling functions

GPCR Signal Transduction - Step by Step

1. Agonist binds to GPCR → receptor conformational change
2. Receptor activates Gαs by promoting GDP → GTP exchange (Gαs-GTP released from Gβγ)
3. Gαs-GTP activates Adenylyl Cyclase (AC)
4. AC converts ATP → cAMP
5. cAMP activates Protein Kinase A (PKA)
6. PKA phosphorylates target proteins → cellular response (e.g., glycogenolysis, lipolysis, cardiac +inotropy/chronotropy)
7. Termination: Gα hydrolyzes GTP → GDP (intrinsic GTPase); cAMP is degraded by Phosphodiesterase (PDE) → AMP
8. PDE inhibitors (e.g., sildenafil, theophylline, milrinone) prolong cAMP/cGMP signals

1. Agonist → Gαq activation
2. Gαq activates Phospholipase C-β (PLC-β)
3. PLC-β cleaves PIP2 → IP3 + DAG
4. IP3 (Inositol trisphosphate): Goes to ER → opens IP3-gated Ca2+ channels → ↑ intracellular Ca2+ → activates calmodulin → smooth muscle contraction, enzyme activation
5. DAG (Diacylglycerol): Stays in membrane → activates Protein Kinase C (PKC) → various cellular effects

1. Agonist → Gαi activation
2. Gαi inhibits adenylyl cyclase → ↓ cAMP → ↓ PKA activity
3. Gβγ subunit: Opens K+ channels (GIRK) → hyperpolarization; Closes Ca2+ channels
4. Example: M2 muscarinic receptor in heart → ↓ HR, AV node slowing

Desensitization/Down-regulation of GPCRs:

1. Homologous desensitization: GPCR kinases (GRKs) phosphorylate activated receptors → β-arrestin binds → receptor uncoupled from G-protein
2. β-arrestin: Promotes receptor internalization (clathrin-mediated endocytosis), also initiates independent signaling
3. Receptor downregulation: Prolonged agonist exposure → decreased receptor number
4. Clinical relevance: Tachyphylaxis with β-agonists (salbutamol), tolerance to opioids

Toxins Acting on GPCRs:

• Cholera toxin: ADP-ribosylates Gαs → locks Gαs in active state → persistent ↑cAMP → massive Cl⁻ secretion in gut → "rice-water" diarrhea
• Pertussis toxin: ADP-ribosylates Gαi → prevents Gi activation → cannot decrease cAMP; also relevant in whooping cough pathology
• Gs mutations: Gain of function → McCune-Albright syndrome; Loss → pseudohypoparathyroidism (Albright hereditary osteodystrophy)

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9. JAK-STAT SIGNALING PATHWAY

Overview

JAK Family (4 members):

1. JAK1 - widely expressed; IFN-γ, IL-2, IL-4, IL-6, IL-10
2. JAK2 - hematopoiesis; Erythropoietin (EPO), Thrombopoietin (TPO), GH, Prolactin; JAK2 V617F mutation → Polycythemia Vera, ET, MF (myeloproliferative neoplasms)
3. JAK3 - mainly lymphocytes; IL-2, IL-4, IL-7, IL-9, IL-15, IL-21 (common γ chain cytokines); JAK3 loss → SCID
4. TYK2 - Type I interferons, IL-12, IL-23

STAT Family (7 members):

• STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6
• STAT3 - commonly hyperactivated in cancer (many solid tumors, lymphomas)
• STAT5 - activated by EPO, GH, Prolactin; JAK2 V617F constitutively activates STAT5
• STAT6 - activated by IL-4/IL-13 → drives Th2 immunity, allergic responses

JAK-STAT Mechanism (Step by Step):

1. Cytokine binds to receptor subunits → receptor dimerization/oligomerization
2. Receptor dimerization → brings JAKs into proximity → trans-phosphorylation (JAKs activate each other)
3. Activated JAKs phosphorylate tyrosine residues on the receptor (create docking sites)
4. STAT proteins bind to phosphotyrosine docking sites via SH2 domains
5. JAKs phosphorylate STATs → pSTAT forms
6. pSTATs dimerize (via SH2-pTyr interactions)
7. pSTAT dimer translocates to nucleus
8. Binds GAS elements (Gamma Activation Sequences) in DNA
9. Activates target gene transcription
10. Negative regulators terminate signaling:
    - SOCS (Suppressors of Cytokine Signaling) - inhibit JAKs (feedback inhibition)
    - PIAS (Protein Inhibitor of Activated STATs) - inhibit STAT DNA binding
    - Protein tyrosine phosphatases (PTPs) - dephosphorylate JAKs/STATs

Key Cytokine-JAK-STAT Pairs:

JAK Inhibitors (JAKinibs) - Drugs:

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10. RECEPTOR THEORIES

1. Occupancy Theory (Clark's Theory, 1926)

Effect = Emax × [D] / (KD + [D])

• [D] = drug concentration
• KD = Dissociation constant (= EC50 in this theory)
• Emax = Maximum effect

1. Drug-receptor interaction is reversible
2. Response is proportional to receptor occupancy
3. Maximal response occurs when all receptors are occupied
4. One drug molecule binds to one receptor

• Cannot explain partial agonism (even 100% occupancy by partial agonist → submaximal effect)
• Cannot explain spare receptors
• Does not account for intrinsic efficacy

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2. Rate Theory (Paton's Theory, 1961)

• Agonists: high rate of association AND dissociation (frequent making/breaking) → sustained stimulation
• Antagonists: high affinity (strong binding), LOW rate of dissociation → occupy receptor → no stimulation
• Drug stimulation = "each binding event produces a quantum of response"

• Less widely accepted than occupancy theory
• Cannot fully explain all pharmacological phenomena

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3. Induced Fit Theory (Koshland, 1958 - adapted for pharmacology)

• Agonist: induces active R* conformation
• Antagonist: induces inactive R conformation or occupies without producing active conformational change

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4. Spare Receptor Theory (Stephenson, 1956)

Response = f(e × [DR] / [R]total)

• For some systems, maximum effect is achieved when only a FRACTION of receptors are occupied - remaining receptors are "spare" or "silent"
• e.g., in some tissues, only 1-5% of total receptors need to be occupied to achieve Emax

• Provides sensitivity buffer - even low drug concentrations can achieve maximal response
• Protects against receptor downregulation
• Explains why partial agonists with low efficacy may not achieve Emax even when all receptors are occupied

• Irreversible blockade (phenoxybenzamine) at low concentrations → doesn't reduce Emax (spares are still enough), but shifts dose-response curve rightward
• At higher blockade → Emax begins to fall (spare receptors used up)

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5. Two-State (Ternary Complex) Model / Extended Ternary Complex Model

• R (inactive state) - couples poorly to G-protein
• R (active state)* - couples efficiently to G-protein

• Full agonist: Preferentially binds and stabilizes R* → shifts equilibrium to R* → maximum response
• Partial agonist: Binds both R and R*; less selective for R* → partial shift
• Neutral antagonist: Binds equally to R and R* → does not shift equilibrium, just blocks other ligands
• Inverse agonist: Preferentially binds and stabilizes R (inactive) → shifts equilibrium toward R → reduces constitutive activity below baseline

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6. Operational Model (Black and Leff, 1983)

• Transducer ratio (τ): Represents the coupling efficiency between receptor and response
• Can explain partial agonism, spare receptors, and system-dependent variations in drug effects
• Biased agonism (functional selectivity): A drug can preferentially activate one downstream pathway over another at the same receptor (e.g., biased toward G-protein vs. β-arrestin signaling)
  • Clinical importance: β-arrestin-biased opioid agonists (TRV130/oliceridine) → analgesia without respiratory depression

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11. DRUG DISCOVERY AND DEVELOPMENT PROCESS

Overview

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Stage 1: Target Identification and Validation

• Identify a molecular target (receptor, enzyme, transporter, ion channel, DNA) relevant to the disease
• Tools: Genomics, proteomics, bioinformatics, GWAS (Genome-Wide Association Studies), knockout animal models, RNAi screens

• Confirm that modulating the target produces the desired therapeutic effect
• Validate using: knock-out animals, siRNA knockdown, selective tool compounds, patient genetic data (Mendelian randomization)
• Example: PCSK9 identified as target for LDL reduction based on human loss-of-function mutations

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Stage 2: Hit Identification

1. High-Throughput Screening (HTS): Screen large chemical libraries (millions of compounds) against target using automated assays
2. Fragment-Based Drug Discovery: Screen small chemical fragments (~200 Da), then link/grow them
3. Virtual screening / In silico screening: Computational docking of compounds against target structure
4. Natural products: Plants, bacteria, fungi (e.g., penicillin from Penicillium, taxol from yew tree, morphine from opium poppy)
5. Biologics: Monoclonal antibodies, proteins
6. Me-too drugs: Modify existing drugs
7. Phenotypic screening: Screen for desired effect in cells/animals without knowing target

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Stage 3: Hit-to-Lead Optimization (Lead Optimization)

• Hit: Compound that shows activity in primary screen
• Lead compound: Optimized hit with better potency, selectivity, and drug-like properties
• Lead optimization using Medicinal Chemistry:
  • Structure-Activity Relationship (SAR) studies
  • Optimize ADMET: Absorption, Distribution, Metabolism, Excretion, Toxicity
  • Improve potency (IC50, EC50 reduction)
  • Reduce off-target effects
  • Apply Lipinski's Rule of Five (for oral bioavailability):
    1. MW ≤ 500 Da
    2. Log P ≤ 5 (lipophilicity)
    3. H-bond donors ≤ 5 (OH + NH groups)
    4. H-bond acceptors ≤ 10 (N + O atoms)
    5. Rotatable bonds ≤ 10 (some versions)
  • Apply Veber's Rules (for oral bioavailability): Rotatable bonds ≤ 10, TPSA ≤ 140 Ų

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Stage 4: Preclinical Testing

• Enzyme inhibition/activation assays
• Cell-based assays (receptor binding, functional assays)
• ADMET profiling (Caco-2 cells for absorption, liver microsomes for metabolism)
• Genotoxicity: Ames test (bacterial mutagenicity), chromosomal aberration test

• Pharmacological efficacy studies (animal models of disease)
• Safety pharmacology: Cardiovascular (hERG channel - QT prolongation risk), CNS, respiratory effects
• Acute toxicity: Single dose; LD50 determination
• Subacute/Chronic toxicity: 2 weeks to 2 years depending on intended clinical use
• Special toxicity studies:
  • Genotoxicity/Mutagenicity (Ames test, micronucleus test)
  • Carcinogenicity (2-year rat/mouse studies)
  • Reproductive and developmental toxicity (Segment I, II, III studies)
    • Segment I: Fertility and reproductive performance
    • Segment II: Embryo-fetal development (teratogenicity) - organogenesis period
    • Segment III: Perinatal and postnatal studies
  • Local tolerance
  • Immunotoxicity

• Filed with regulatory authority (FDA/EMA/CDSCO)
• Contains: preclinical data, proposed clinical protocol, CMC (Chemistry, Manufacturing, Controls) data
• Approval to start human trials

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Stage 5: Clinical Trials (Human Studies)

Phase I Trials

• Subject: Healthy volunteers (20-80 subjects); occasionally patients (in oncology, HIV)
• Purpose:
  • Safety (adverse effects, maximum tolerated dose - MTD)
  • Pharmacokinetics (ADME: absorption, distribution, metabolism, excretion)
  • Pharmacodynamics (dose-response)
  • Dose range finding
  • Drug-drug interaction potential
• Duration: Few months to 1 year
• Design: Open-label, dose escalation (starting at 1/10th of NOAEL in most sensitive animal species)
• First-in-human dose: Based on NOAEL (No Observed Adverse Effect Level) from preclinical studies using safety factors

Phase II Trials

• Subject: Patients with target disease (100-300)
• Purpose:
  • Preliminary efficacy (proof of concept)
  • Safety and tolerability in patients
  • Dose optimization (Phase IIa: dose finding; Phase IIb: dose confirmation)
  • Define optimal dose regimen for Phase III
• Duration: Several months to 2 years
• Design: Often randomized, double-blind, placebo-controlled

Phase III Trials

• Subject: Large number of patients (hundreds to thousands; typically 1,000-5,000+)
• Purpose:
  • Definitive efficacy vs. placebo or active comparator
  • Safety (detect less common adverse effects)
  • Establish benefit-risk profile
  • Confirm dosing regimen
• Duration: 2-5 years
• Design: Randomized Controlled Trial (RCT), multicenter, international
• Pivotal trials - form the basis for regulatory approval

• New Drug Application (NDA) to FDA or Marketing Authorization Application (MAA) to EMA
• Contains all clinical, preclinical, and manufacturing data
• Review takes 6-12 months (standard) or 6 months (Priority Review)

Phase IV Trials (Post-Marketing Surveillance)

• After drug approval and market launch
• Purpose:
  • Monitor long-term safety (pharmacovigilance)
  • Detect rare ADRs (1 in 10,000+ patients) not seen in Phase III
  • Compare with other marketed drugs
  • New indications, populations (pediatric, elderly), formulations
  • Cost-effectiveness studies
• Tools: Yellow Card reporting, registries, electronic health records, prescription event monitoring (PEM)
• Can lead to: Drug withdrawal (e.g., rofecoxib-Vioxx withdrawn due to cardiovascular risk detected in Phase IV; thalidomide withdrawn due to teratogenicity)

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Summary Timeline:

TARGET          HIT/LEAD        PRECLINICAL         CLINICAL TRIALS            MARKET
IDENTIFICATION  IDENTIFICATION  TESTING     Phase I → Phase II → Phase III → NDA → Phase IV
│──────────────│───────────────│───────────│──────────────────────────────────│────────
  2-4 years      1-2 years      1-2 years           6-8 years               Post-market
                                                    
                 ← ─ ─ ─ ─ ─ ─ ─  10-15 years total ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ →

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Regulatory Bodies:

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Special Drug Approval Pathways (FDA):

1. Fast Track: Serious conditions + unmet medical need
2. Breakthrough Therapy Designation: Preliminary evidence shows substantial improvement over available therapy
3. Accelerated Approval: Based on surrogate endpoints (e.g., tumor response rate) for serious conditions
4. Priority Review: 6-month review instead of 12 months for significant improvement

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Drug Development Failure Rates:

• Of 10,000 compounds entering discovery: ~250 enter preclinical → 5 enter clinical trials → 1 reaches market
• Phase I failure (mostly safety): ~30%
• Phase II failure (mostly efficacy): ~60%
• Phase III failure (efficacy + safety + commercial): ~30%
• Overall attrition: >90% fail

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QUICK REVISION SUMMARY TABLE

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What You Got - Quick Index:

• Fick's Law: Rate ∝ (C₁-C₂) × SA × Kp / d
• Henderson-Hasselbalch: pH - pKa = log [ionized]/[non-ionized] (weak acid)
• Bioavailability: F = AUC_oral / AUC_IV × 100%
• Vd = Dose / Plasma Concentration
• t½ = 0.693 × Vd / Clearance
• Loading Dose = Vd × Target Cp