Lead Optimization

Question 1

1) What are the key issues to be addressed in lead optimization?

Answer 1

1. Efficacy
2. Potency (target affinity and PK parameters)
3. Adverse effects/toxicity profile
4. Route of administration (stability, absorption, distribution)
5. Onset and duration of action

Question 2

2) What are the key criteria for a lead series?

Answer 2

• Binding/functional potency in primary assay (IC50 < 100nM)
• Potency in secondary assay (cell proliferation GI50 <500nM)
• Meets Lipinski rules (of 5) (MW<500, cLogP<5)
• In vitro ADME liabilities (tó >60min)
• Synthesis in less than 10 steps
• Multiple points of modification
• Patentable

Question 3

3) Explain chemical modification.

Answer 3

The goal of chemical modifications is to determine which functional groups are important for biological activity. The procedure is to alter or remove functional groups using chemical synthesis and test the activity of the altered molecule.

Bioisosteric replacement involves substitution of atoms or groups of atoms in a the parent molecule to produce compounds with broadly similar biological properties to the parent with structural diversity.

Question 4

4) What are the factors that affect absorption?

Answer 4

1. Route of administration: Oral administration is the most convenient and cost-effective. Absorption takes place mostly from the small intestine.
2. Rate of dissolution (tablet, capsule, suspension or solution)
3. Permebility: Dependent on the lipophilicity and extent of ionization of the drug (ionized compounds cannot pass through the lipid bilayer of cells)
4. Drug complex with dissolved food.

Question 5

5) What happens after the drug is absorbed?

Answer 5

It passes through the portal vein and enters the liver, where is may be metabolized.

Question 6

6) What factors affect solubility and stability?

Answer 6

Solubility requires adherence to Lipinski rule of 5. Stability is measured at different pH and temperatures.

Eg. Orally available gemcitabine: prodrug mediates oral-mediated absorption of gemcitabine with less toxicity. Minimal hydrolysis of prodrug to gemcitabine at low pH.

Question 7

7) What is bioavailabilty and why is it important? How is it calculated?

Answer 7

Bioavailabilty is the fraction of unchanged drug that enters systemic circulation. It should be studied as early as possible because a lack of desired response may be due to lack of bioavailability (not reaching the required drug concentration). Compounds can be suitably modified to maximize bioavailability.

F=[AUC(test) x D(iv)]/[AUC(iv) x D(test)] X 100%

Question 8

8) How does drug distribution affect drug response?

Answer 8

A drug can be distributed to tissues/organs from the bloodstream.

Different drug concentrations are attained in different tissues/organs.

A drug may be preferentially distributed to its target tissue/organ or not at all.

Question 9

9) Explain clearance and metabolism.

Answer 9

• Drugs may be eliminated either unchanged (as the parent drug) or as metabolites depending on the lipophilicity
• Most drugs are eliminated through the kidneys which can excrete only relatively polar substances
• Thus lipophilic drugs must be metabolized into more polar metabolites for elimination
• Drugs are metabolized to different extent mostly in the liver
• Metabolism mostly lead to inactivation of a drug but many drugs have active metabolites
• Therefore important to study the metabolism of a drug under development in order to know the impact it may have
• First studied in liver microsomes
• CYP enzymes inhibition – Drug-drug interactions

Question 10

10) What is the process of liver metabolism?

Answer 10

Phase I (Functionalization): Functional groups are altered through monooxygenase reaction via CYP enzymes, leading to a loss of activity. Eg. Paclitaxel undergoes metabolic modifications before it can be renally excreted.

Phase II (Conjugation): Addition of highly polar conjugates to drugs to increase their hydrophilicity. Eg. Irinotecan is metabolized to SN38, an active metabolite. SN-38 is inactivated by UGTs via the addition of glucuronic acid. UGT1A1*38 polymorphism inactivates UGT, making SN-38 difficult to be inactivated, leading to increased toxicity.

Question 11

11) What are the in vitro ADME assays?

Answer 11

1. In vitro: -Microsome metabolism. Incubate animal/human microsome with lead drug candidate, incubate over a timecourse and analyse by LC/MS. Higher percentage of parent compound remaining indicates higher metabolic stability.
   - PAMPA assay: A well within a larger well, lipid membrane in the inner well. Lead molecules in the inner well, identify those that pass through lipid membrane.

Question 12

12) What are the in vivo ADME assays?

Answer 12

1. Animal based models:
   - validate in vivo biomarkers for drug efficacy
   - required for efficacy and toxicity drug evaluation
   - in vivo evaluation of PK/PD in normal/disease animal models
   - Dynamic evaluation of drug efficacy: Histological analysis, tissue sample analysis (RNA,DNA,Protein), in vivo imaging of disease progression.
2. Human chimera mice: These are mice that contain transplanted human hepatocytes. It is a more accurate preclinical model than regular mice in terms of ADME properties. It allows evaluation of disease in human liver model (eg. Hepatitis viral infection) and evaluate new drug efficacy (ie. Antiviral drugs)

Question 13

14) What other evaluations can be done before selection of a preclinical candidate?

Answer 13

Gross pathology, Histopathy, immunohistochemistry, molecular pathology hematology, immunology.

Question 14

12) How can lead optimization be optimized to find the best drug model

Answer 14

• conduct as many secondary assays as possible (proliferation, cell rounding, proliferation, motility, metastasis) with lead series
• conduct secondary assays with organoid spheroids that are better representative of the in vivo cellular environment