Pharmacology and Drug Development: From Discovery to Market

Posted on Jan 19, 2025 in Biotechnology

Drug Discovery

Target Profile – Intended therapeutic site of action and clinical outcome

Lead Identification – Identified candidate compounds with potential drug activity commensurate with profile from a library of actives (hits)

Lead Optimization – Identification / modification of lead compounds for best action / least side effects, etc.

Combinatorial Chemistry – Generation of active compounds (hits) from a library of building blocks

Structure-Activity Relationship – Determination of the relationship between a specific chemical structure and a pharmacological action

Clinical Investigation

US: IND – Investigational New Drug (Application)

EU: CTA / CTX – Clinical Trial Authorization/Clinical Trials Exemption

Marketing Approval

US: NDA (New Drug Application); ANDA (Abbreviated New Drug Application); BLA (Biologic License Application)

EU: MAA (Marketing Authorization Application); CTD (Common Technical Document; common format for organization of information in marketing authorization (registration) applications. Format for CTD acceptable in three regions (US, Europe, Japan). Content requirements are not fully harmonized and there are differences between the three regions.)

Label

Label—The label is the document physically attached directly to the packaging materials that are in direct contact with the excipient, drug substance, or drug product.

Labeling—Labeling includes the label and the documents included with, but not attached to, the packaging materials that are in direct contact with the excipient, drug substance, or preparation (e.g., package insert).

Clinical Pharmacology

Broad Categories of Pharmacology

Pharmacodynamics (How the drug affects the body)

Pharmacokinetics (How the body affects the drug): ADME (Absorption, distribution, metabolism, excretion), Clearance

Pharmacodynamic Interactions

Drug-receptor effects (Increased effect); Enhancement by occupancy: Diazepam and zopiclone (Reduced/blocked effect); Competitive antagonism: salbutamol and propranolol; Enhanced therapeutic effects (Alcohol and sedatives); Side effects (Aspirin and diclofenac (both acting on cytoprotective pathways))

Pharmacokinetic Interactions: Metabolism

Phase I metabolism (1. Convert parent compound into a more polar (=hydrophilic) metabolite by adding or unmasking functional groups (-OH, -SH, -NH2, -COOH, etc.), 2. Often these metabolites are inactive, 3. May be sufficiently polar to be excreted readily)

Phase II metabolism (1. Conjugation with endogenous substrate to further increase aqueous solubility, 2. Conjugation with glucuronide, sulfate, acetate, amino acid, 3. Phase I usually precede phase II reactions)

Many organs, systems (gut, lungs, skin and kidneys) are involved but the liver is the principal site of drug metabolism

Drug Metabolism

Phase I Metabolism: Functions

Tend to make drugs (1. More water soluble, 2. Less active, 3. Less toxic)

Prepares drugs for greater metabolic conversion and clearance

Phase I Metabolism: Reactions

Oxidation (1. Cytochrome P450 (CYP), 2. Cytoplasmic), Alcohol dehydrogenase, Xanthine oxidase, Monoamine oxidase, Reduction (CYP in liver, flora in gut), Hydrolysis (CYP, Other (e.g. cholinesterases))

Phase I Metabolism: Reactions

Oxidation, Reduction, Hydrolytic cleavage, Alkylation (Methylation), Dealkylation, Ring cyclization, N-carboxylation, Dimerization, Transamidation, Isomerization, Decarboxylation

Phase I Reactions: Oxidations

Two types of oxidation reactions: 1. Oxygen is incorporated into the drug molecule (e.g. hydroxylation); 2. Oxidation causes the loss of part of the drug molecule (e.g. oxidative deamination, dealkylation)

Microsomal Mixed Function Oxidases (MFOs):

  • Microsomes (form in vitro after cell homogenization and fractionation of ER): 1. Rough microsomes are primarily associated with protein synthesis; 2. Smooth microsomes contain a class of oxidative enzymes called.
  • Mixed Function Oxidases or Monooxygenases: These enzymes require a reducing agent (NADPH) and molecular oxygen (one oxygen atom appearing in the product and the other in the form of water)
  • MFO consists of two enzymes: 1. Flavoprotein, NADPH-cytochrome c reductase (One mole of this enzyme contains one mole each of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); Enzyme is also called NADPH-cytochrome P450 reductase)/ 2. Cytochrome P450 (named based on its light absorption at 450 nm when complexed with carbon monoxide; is a hemoprotein containing an iron atom which can alternate between the ferrous (Fe++) and ferric (Fe+++) states; Electron acceptor; Serves as terminal oxidase; its relative abundance compared to NADPH-cytochrome P450 reductase makes it the rate-limiting step in the oxidation reactions)
  • Humans have 18 families of cytochrome P450 genes and 43 subfamilies

CYP Families

  • Multiple CYP gene families have been identified in humans, and the categories are based upon protein sequence homology
  • Most of the drug metabolizing enzymes are in CYP 1, 2, & 3 families
  • CYPs have molecular weights of 45-60 kDa
  • Frequently, two or more enzymes can catalyze the same type of oxidation, indicating redundant and broad substrate specificity
  • CYP3A4 is very common to the metabolism of many drugs; its presence in the GI tract is responsible for poor oral availability of many drugs

Phase I Reactions: Oxidations

  • Induction of P450 enzymes: 1. PPAR (peroxisome proliferator activated receptor) ligands (e.g.clofibrate); 2. CYP1 family are induced by aromatic hydrocarbons (cigarette smoke; charred food); 3. CYP2E enzymes induced by ethanol; 4. CYP2B enzymes induced 40-50 fold by barbiturates
  • Polymorphisms cause differences in drug metabolism: 1. CYP2C19 has a polymorphism that changes the enzyme’s ability to metabolize mephenytoin (a marker drug). In Caucasians, the polymorphism for the poor metabolizer phenotype is only seen in 3% of the population. However, it is seen in 20% of the Asian population.
  • P450s and drug interactions:
    1. Barbiturates induce CYP2B => increased metabolism of other drugs
    2. Antifungals (e.g. ketoconazole) inhibit fungal CYP51 and unintentionally also human CYP3A4 => reduced metabolism of other drugs
    3. Grapefruit juice contains a CYP3A4 inhibitor =>12 fold increase in some drug concentrations
      • CYP3A4 Substrates: • Acetaminophen (Tylenol) • Codeine (narcotic) • Cyclosporin A (immunosuppressant), • Diazepam (Valium) • Erythromycin (Antibiotic) • Lidocaine (local anesthetic), • Lovastatin (HMGCoA reductase inhibitor), • Taxol (cancer drug), • Warfarin (anticoagulant).
  • Drug oxidation requires:
    1. Cytochrome P450
    2. Cytochrome P450 reductase
    3. NADPH
    4. Molecular oxygen
  • The CYP cycle involves four steps:
    1. Oxidized (Fe3+) cytochrome P-450 combines with a drug substrate to form a binary complex.
    2. NADPH donates an electron to the cytochrome P-450 reductase, which in turn reduces the oxidized cytochrome P-450-drug complex.
    3. A second electron is introduced from NADPH via the same cytochrome P-450 reductase, which serves to reduce molecular oxygen and form an “activated oxygen”-cytochrome P-450-substrate complex.
    4. This complex in turn transfers “activated” oxygen to the drug substrate to form the oxidized product. The potent oxidizing properties of this activated oxygen permit oxidation of a large number of substrates.

Non P450 Mediated Oxidations

  • Flavin containing monooxygenase system
    1. Present mainly in liver but some is expressed in gut and lung
    2. Located in smooth endoplasmic reticulum
    3. Oxidizes compounds containing sulfur and nitrogen
    4. Uses NADH and NADPH as cofactors
  • Alcohol dehydrogenase (cytosol)
    1. Aldehyde oxidation (cytosol)
    2. Xanthine oxidase
    3. Amine oxidases
    4. Monoamine oxidase (nerve terminals, mitochondria)
    5. Diamine oxidase found in liver microsomes
      • Primarily endogenous metabolism

Phase I Reactions: Oxidations

Monoamine Oxidases (MAO):

  • Catalyze oxidative deamination of endogenous catecholamines (epinephrine)
  • Located in nerve terminals and peripheral tissues
  • Substrates for catecholamine metabolism found in foods (tyramine) can cause a drug/food interaction
  • Inhibited by class of antidepressants called MAO inhibitors (Inhibition of MAO isoforms in the CNS also effects levels of serotonin – Tranylcypromine) These drugs can cause severe or fatal drug/drug interactions with drugs that increase release of catecholamines or inhibit their reuptake in nerve terminals (Meperidine, pentazocine, dextromethorphan, SSRI antidepressants)

Phase I Reactions: Summary

  • Almost any drug can undergo modifications by drug-metabolizing enzyme systems
  • Drugs can be subject to several Phase I pathways
  • These reactions create functional groups that place the drugs in a correct chemical state to be acted upon by Phase II conjugative mechanisms
  • Main function of phase I reactions is to prepare chemicals for phase II metabolism and subsequent excretion
  • Phase II is the true detoxification step in the metabolism process.

Phase II Metabolism

Phase II Metabolism: Functions

  • Primary : Conjugation (binding to another molecule)
    1. Bigger than the drug alone
    2. Less lipid soluble
    3. Less able to cross cell membranes
    4. Less likely to reach site of activity
  • More likely to be removed

Phase II Metabolism: Reactions

  • Glucuronidation by UDP-Glucuronosyltransferase: (on -OH, -COOH, -NH2, -SH groups)
  • Sulfation by Sulfotransferase: (on -NH2, -SO2NH2, -OH groups)
  • Acetylation by acetyltransferase: (on -NH2, -SO2NH2, -OH groups)
  • Amino acid conjugation (on -COOH groups)
  • Glutathione conjugation by Glutathione-S-transferase: (to epoxides or organic halides)
  • Fatty acid conjugation (on -OH groups)
  • Condensation reactions

Glucuronidation

  • This reaction involves the addition of sugars to lipids and other apolar xenobiotics and is an important step in the body’s elimination of foreign substances (such as drugs and medications) as well as endogenous substances (including endogenous toxins). UDP-glucuronosyltransferase (UGT) is present in humans, other animals, plants, and bacteria.
  • The glucuronidation reaction consists of the transfer of the glucuronosyl group from uridine 5’-diphospho-glucuronic acid (UDPGA) to substrate molecules that contain oxygen, nitrogen, sulfur or carboxyl functional groups. The resulting glucuronide is more polar (e.g. hydrophilic) and more easily excreted than the substrate molecule. The product solubility in blood is increased allowing it to be eliminated from the body by the kidneys.
  • The substances resulting from glucuronidation are known as glucuronides (or glucuronosides) and are typically much more water-soluble than the non-glucuronic acid-containing substance from which they were originally synthesized. The human body uses glucuronidation to make a large variety of substances more water-soluble, and, in this way, allow for their subsequent elimination from the body upon urination. Hormones may also be glucuronidated to allow for easier transport around the body. Pharmacologists have linked drugs to glucuronic acid to allow for more effective delivery of a broad range of substances.
  • The conjugation of xenobiotic molecules with hydrophilic molecular species such as glucuronic acid is known as phase II metabolism.
  • Glucuronidation ( = conjugation to a-d-glucuronic acid)
    1. Quantitatively the most important phase II pathway for drugs and endogenous compounds
    2. Products are often excreted in the bile.
    3. Enterohepatic recycling may occur due to gut glucuronidases
    4. Requires enzyme UDP-glucuronosyltransferase (UGT):
  • Genetic family of enzymes
    1. Metabolizes a broad range of structurally diverse endogenous and exogenous compounds
    2. Structurally related family with approximately 18 isoforms in man
  • Glucuronidation – requires creation of high energy intermediate:

9k=

Glucuronidation Pathway and Enterohepatic Recirculation

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N-glucuronidation:

  • Occurs with amines (mainly aromatic )
  • Occurs with amides and sulfonamides

Z

O-glucuronidation:

  • Occurs by ester linkages with carboxylic acids
  • Occurs by ether linkages with phenols and alcohols

Z

Sulfation

  • Major pathway for phenols but also occurs for alcohols, amines and thiols
  • Energy rich donor required: PAPS (3’-Phosphoadenosine-5’-phosphosulfate)
  • Sulfation and glucuronidation are competing pathways:
    1. Sulfation predominates at low substrate concentrations
    2. Glucuronidation predominates at higher concentrations
    3. There is relatively less PAPS in cell cytosol compared to UDPGA
  • Sulfotransferases (=SULTs) catalyze transfer of sulfate to substrates:
    1. Phenol, alcohol and arylamine sulfotransferases are fairly non-specific
    2. Steroid sulfotransferases are very specific

Acetylation

  • Common reaction for aromatic amines and sulfonamides
  • Requires co-factor acetyl-CoA
  • Responsible enzyme is N-acetyltransferase
  • Takes place mainly in the liver
  • Important in sulfonamide metabolism because acetyl-sulfonamides are less soluble than the parent compound and may cause renal toxicity due to precipitation in the kidney

Fatty Acid Conjugation

  • Stearic and palmitic acids are conjugated to drug by esterification reaction
  • Occurs in liver microsomal fraction
    1. Cannabinols are metabolized in this fashion => long half-life

Other Conjugations

Amino Acid Conjugation

  • ATP-dependent acid: CoA ligase forms active CoA-amino acid conjugates which then react with drugs by N-Acetylation:
    1. Usual amino acids involved are: Glycine. Glutamine, Ornithine, Arginine

Glutathione Conjugation

  • Tripeptide Gly-Cys-Glu; conjugated by glutathione-S-transferase (GST)
  • Glutathione is a protective factor for removal of potentially toxic compounds
  • Conjugated compounds can subsequently be attacked by g-glutamyltranspeptidase and a peptidase to yield the cysteine conjugate => product can be further acetylated to N-acetylcysteine conjugate

Effectors of Drug Metabolism

Inhibitors and Inducers of Microsomal Enzymes

  • INHIBITORS: cimetidine prolongs action of drugs or inhibits action of those biotransformed to active agents (pro-drugs)
  • INDUCERS: barbiturates, carbamazepine shorten action of drugs or increase effects of those biotransformed to active agents
  • BLOCKERS acting on non-microsomal enzymes (MAOI, anticholinesterase drugs)

Factors Affecting Biotransformation

  • Age (reduced in aged patients & children)
  • Sex (women more sensitive to ethanol?)
  • Species (phenylbutazone 3h rabbit, 6h horse, 8h monkey, 18h mouse, 36h man); route of biotransformation can also change
  • Race (fast and slow isoniazid acetylators, fast = 95% Eskimo; 50% British; 13% Finnish; 13% Egyptian.
  • Clinical or physiological condition
  • First-pass (pre-systemic) metabolism

Phase I and II: Summary

  • Products are generally more water soluble
  • These reactions products are ready for (renal) excretion
  • There are many complementary, sequential and competing pathways
  • Phase I and Phase II metabolism are a coupled interactive system interfacing with endogenous metabolic pathways

Excretion

Excretion of Drugs

  • Glomerular filtration allows drugs < 25kDa through
  • Tubular secretion active carrier process for cations and for anions; inhibited by probenecid.
  • Passive re-absorption of lipid soluble drugs back into the body across the tubule cells.
  • Note the effect of pH: in weak acid, drug more present in ionized form than in alkaline pH therefore re-absorbed less and excreted faster; vice-versa for weak bases.

Special Aspects of Excretion

  • In lactating women the drug/metabolites is present in their milk
  • Little excreted in feces unless poor formulation or diarrhea
  • Volatile agents (general anesthetics) via lungs
  • The entero-hepatic shunt glucuronic acid conjugates with MW >300 are increasingly excreted in bile; hydrolysis of say -OH conjugate by beta-glucuronidase in gut will restore active drug which will be reabsorbed and produce an additional effect.

The enterohepatic shunt

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Clearance

  • Removal of drug from the body
  • Parent drug and metabolites have individual clearance characteristics
  • Linked to ADME characteristics of the compound

Types of Clearance

  • Metabolic
    • First pass metabolism e.g. nitrates
    • Mostly liver
    • Other metabolic tissues
  • Renal (urinary)
  • Biliary (fecal)
  • Other (expired air, sweat)

Summary

  • Pharmacodynamic interactions:
    • When drugs have similar (additive) or antagonistic effects
    • (potentiation, or diminution of effect)
  • Pharmacokinetic interactions:
    • When drugs interfere with each other’s mechanisms of clearance
    • (taking one drug in the presence of another causes either accumulation, or greatly expedited metabolism)

Diazepam – Valium

  • Diazepam acts as an allosteric modulator of the GABAA receptor and enhances the inhibitory effect of the neurotransmitter γ-aminobutyric acid (GABA). Diazepam binds as an agonist to the benzodiazepine binding site of this receptor (a chloride ion channel) and causes a conformational change; this increases the receptor’s sensitivity to GABA. Increased GABA activity results in an increased opening rate at the chloride channel and thus in an increased influx of chloride ions into the cell. The increase in the intracellular chloride concentration leads to hyperpolarization and thus to a reduced excitability of the cell.

Zopiclone

  • Zopiclone is a drug used in sleeping pills and is related to benzodiazepines in its effect. It has a plasma half-life of 4 to 6 hours. It is quickly absorbed and distributed in the body. Chemically, zopiclone belongs to the class of cyclopyrrolones. Of the two enantiomers (R) – and (S) -zopiclone, only the (S) -zopiclone (eszopiclone) has a sleep-inducing effect, and to a lesser extent also anxiolytic, anticonvulsant and muscle-relaxing. [2] Pure (S) -zopiclone has been approved as a sleeping pill in the USA since 2005 and is available there under the brand name Lunesta®.
  • The therapeutic pharmacological properties of zopiclone include hypnotic, anxiolytic, anticonvulsant and myorelaxant properties.[60] Both zopiclone and benzodiazepines act indiscriminately at the benzodiazepine binding site on α1, α2, α3 and α5 GABAA containing receptors as full agonists causing an enhancement of the actions of GABA to produce the therapeutic and adverse effects of zopiclone. Like benzodiazepines zopiclone and its active metabolite desmethylzopiclone also inhibit N-methyl-D-aspartate (NMDA) receptors and nicotinic acetylcholine (nAChRs) receptors which might play a role in the addictive properties of these drugs

Salbutamol

  • Salbutamol (INN) or albuterol (USAN) is a short-acting β2-adrenergic receptor agonist used for the relief of bronchospasm in conditions such as asthma and chronic obstructive pulmonary disease.
  • Salbutamol was the first selective β2-receptor agonist to be marketed — in 1968.

Propranolol

  • Propranolol (INN) is a non-selective beta blocker mainly used in the treatment of hypertension. It was the first successful beta blocker developed.
  • As a non-selective beta blocker it blocks the action of epinephrine and norepinephrine on both β1- and β2-adrenergic receptors. It has little intrinsic sympathomimetic activity (ISA) but has strong membrane stabilizing activity (only at high blood concentrations, eg overdosage).
  • Propranolol is often used by musicians and other performers to prevent stage fright. It has been taken by surgeons to reduce their own innate hand tremors during surgery.

Selective Serotonin Reuptake Inhibitor (SSRI)

  • Selective serotonin reuptake inhibitors or serotonin-specific reuptake inhibitor (SSRIs) are a class of compounds typically used as antidepressants in the treatment of depression, anxiety disorders, and some personality disorders. They are also typically effective and used in treating premature ejaculation problems as well as some cases of insomnia.
  • SSRIs increase the extracellular level of the neurotransmitter serotonin by inhibiting its reuptake into the presynaptic cell, increasing the level of serotonin available to bind to the postsynaptic receptor. They have varying degrees of selectivity for the other monoamine transporters, with pure SSRIs having only weak affinity for the noradrenalin and dopamine transporter.
  • The first class of psychotropic drugs to be rationally designed, SSRIs are the most widely prescribed antidepressants in many countries

Selective Serotonin Reuptake Inhibitor (SSRI)

  • SSRIs are believed to act by inhibiting the reuptake of serotonin after being released in synapses. How much an individual will respond to this, however, also depends on genetics. In addition, several other mechanisms are suggested for the desired effect, e.g. neuroprotection and anti-inflammatory and immunomodulatory factors. Taken together, SSRI has several advantages compared with tricyclic antidepressants (TCA)s and 5-HT-prodrugs. However, the latter might be required in addition to SSRIs in certain situations.