Extrapolation of in vitro data to animal and human studies MCQs With Answer

Introduction: Extrapolation of in vitro data to animal and human studies is essential for M.Pharm students to predict in vivo pharmacokinetics, toxicity, and efficacy of new chemical entities. This blog provides focused multiple-choice questions that probe core concepts such as in vitro–in vivo extrapolation (IVIVE), allometric scaling, physiologically based pharmacokinetic (PBPK) modeling, microsomal and hepatocyte assays, enzyme kinetics, transporter effects, protein binding, and species differences. The questions emphasize quantitative scaling factors, sources of variability, and experimental design considerations to help students critically evaluate how lab-based results translate into animal and human outcomes. Answers are provided to reinforce learning and exam preparation.

Q1. Which parameter derived from in vitro hepatic microsomal assays is most directly used to estimate in vivo intrinsic clearance (CLint) for IVIVE?

• Fraction unbound in plasma (fu)
• Apparent permeability (Papp)
• In vitro half-life or substrate depletion rate
• Blood-to-plasma concentration ratio (B/P)

Correct Answer: In vitro half-life or substrate depletion rate

Q2. When scaling microsomal CLint to whole liver, which scaling factor is commonly applied?

• Microsomal protein per gram of liver (MPPGL)
• Hepatocyte viability fraction
• Renal clearance per kilogram
• Blood flow to muscle

Correct Answer: Microsomal protein per gram of liver (MPPGL)

Q3. Which concept describes accounting for species differences in body size and physiology to predict pharmacokinetic parameters across species?

• In vitro permeability scaling
• Allometric scaling
• Michaelis-Menten kinetics
• Freeze-thaw stability

Correct Answer: Allometric scaling

Q4. In PBPK modeling, what primary input distinguishes a perfusion-limited tissue from a permeability-limited tissue?

• Organ blood flow and tissue–plasma partition coefficient
• Renal tubular secretion rate
• Microsomal clearance only
• In vitro transporter ATPase activity

Correct Answer: Organ blood flow and tissue–plasma partition coefficient

Q5. Which in vitro system is preferred to capture both phase I and phase II metabolism for IVIVE of hepatic clearance?

• Recombinant CYP enzymes
• Human liver microsomes
• Isolated mitochondria
• Suspended hepatocytes

Correct Answer: Suspended hepatocytes

Q6. When predicting human hepatic clearance from in vitro data, correcting for plasma protein binding is important because:

• Only unbound drug is available for hepatic uptake and metabolism
• Bound drug is cleared faster by the liver
• Protein binding affects only renal clearance
• Total drug concentration equals unbound concentration

Correct Answer: Only unbound drug is available for hepatic uptake and metabolism

Q7. Time-dependent inhibition (TDI) observed in vitro implies which potential in vivo concern?

• Immediate non-specific toxicity
• Reversible induction of metabolism
• Mechanism-based inhibition leading to prolonged CYP inhibition and drug interactions
• Increased passive diffusion across membranes

Correct Answer: Mechanism-based inhibition leading to prolonged CYP inhibition and drug interactions

Q8. Which correction is essential when using intrinsic clearance from microsomes for whole-liver CLint prediction to account for enzymatic differences?

• Scaling by microsomal protein per gram liver and liver weight
• Scaling by glomerular filtration rate
• Multiplying by fraction unbound in red blood cells
• Dividing by blood viscosity

Correct Answer: Scaling by microsomal protein per gram liver and liver weight

Q9. For drugs with low hepatic extraction ratio, which factor most influences hepatic clearance predicted by IVIVE?

• Hepatic blood flow
• Fraction unbound and intrinsic clearance
• Renal excretion rate
• Splanchnic circulation variability

Correct Answer: Fraction unbound and intrinsic clearance

Q10. Caco-2 permeability assays are primarily used to predict which in vivo property?

• Hepatic intrinsic clearance
• Oral absorption and intestinal permeability
• Plasma protein binding
• Renal clearance mechanisms

Correct Answer: Oral absorption and intestinal permeability

Q11. Which in vitro measurement helps estimate the fraction metabolized by a specific CYP isoform (fm) for DDI risk assessment?

• Metabolic stability in whole blood
• Relative contribution from recombinant CYP phenotyping or chemical inhibition studies
• Passive diffusion coefficient
• Microsomal phospholipid content

Correct Answer: Relative contribution from recombinant CYP phenotyping or chemical inhibition studies

Q12. When scaling clearance between species, which physiological parameter is least informative for small-molecule hepatic clearance differences?

• Liver blood flow
• Body surface area based on weight
• Species-specific enzyme expression and activity
• Species differences in plasma protein binding

Correct Answer: Body surface area based on weight

Q13. A major limitation of using human liver microsomes (HLM) for IVIVE is:

• They contain intact transporters and cell architecture
• They lack phase II enzymes entirely
• They do not capture uptake transporter-mediated clearance and cellular context
• They overpredict biliary excretion

Correct Answer: They do not capture uptake transporter-mediated clearance and cellular context

Q14. Which approach improves IVIVE for compounds with significant active hepatic uptake?

• Use of hepatocytes or sandwich-cultured hepatocytes with transporter activity
• Exclusive reliance on microsomal clearance
• Estimating clearance from logP only
• Measuring only plasma protein binding

Correct Answer: Use of hepatocytes or sandwich-cultured hepatocytes with transporter activity

Q15. The term “unbound intrinsic clearance (CLint,u)” refers to:

• Intrinsic clearance corrected for unbound fraction in the incubation
• Total clearance including renal and biliary routes
• Clearance measured in whole blood without correction
• Clearance normalized to body surface area

Correct Answer: Intrinsic clearance corrected for unbound fraction in the incubation

Q16. When predicting human clearance, which source of variability is often NOT captured by in vitro systems?

• Genetic polymorphisms in CYP enzymes affecting in vivo enzyme abundance
• Intrinsic enzyme kinetics (Km and Vmax)
• Substrate depletion rate in microsomes
• In vitro inhibition constants (Ki)

Correct Answer: Genetic polymorphisms in CYP enzymes affecting in vivo enzyme abundance

Q17. Which best describes the relationship between in vitro Km and in vivo substrate affinity when performing IVIVE using Michaelis–Menten kinetics?

• In vitro Km always equals in vivo Km and requires no scaling
• In vitro Km approximates in vivo Km but transporter context and protein binding can alter apparent affinity
• Km is irrelevant for high-clearance compounds
• In vitro Km must be multiplied by plasma protein binding to get in vivo Km

Correct Answer: In vitro Km approximates in vivo Km but transporter context and protein binding can alter apparent affinity

Q18. Which metric derived from in vitro permeability and solubility experiments helps predict the rate-limiting step for oral absorption?

• Hepatic extraction ratio
• Biopharmaceutics Classification System (BCS) permeability and solubility classification
• Microsomal clearance per mg protein
• Volume of distribution at steady state

Correct Answer: Biopharmaceutics Classification System (BCS) permeability and solubility classification

Q19. In IVIVE, why is the measurement of fraction unbound in microsomal or hepatocyte incubations important?

• It defines the rate of passive renal filtration
• It determines the proportion of drug available for metabolism in the incubation and impacts CLint,u
• It indicates degree of biliary excretion
• It replaces the need for human plasma protein binding data

Correct Answer: It determines the proportion of drug available for metabolism in the incubation and impacts CLint,u

Q20. A compound shows fast in vitro clearance in human hepatocytes but low in vivo clearance in humans. Which factor could explain this discrepancy?

• Negligible plasma protein binding in vivo
• Extrahepatic metabolism or transporter-mediated hepatic uptake differences in vivo
• Perfect correlation between in vitro and in vivo conditions
• Underprediction of in vitro metabolic stability

Correct Answer: Extrahepatic metabolism or transporter-mediated hepatic uptake differences in vivo

Download