Preclinical Drug Research: R&D of a Drug The AUC is directly related to relative
bioavailability. Relative bioavailability compares
the AUC of 2 formulations. For example, if the AUCs
Pharmacokinetic (PK) parameters are 121.7 ± 53.8 for gelatin capsules and 129.0 ±
39.3 µg.h/mL for oral absorption, the relative
bioavailability is close to 100%, indicating no
PK -> “What does the body to the drug?” -> ADME significant difference between the 2 formulations.
Bioequivalence compares the systemic exposure (bioavailability) of a test compound with reference compounds. Typically
to know for example if your drug is better than the one already on the market. This is usually tested in healthy volunteers.
• Bioavailability measures how much of a drug is available to enter the circulation (the bloodstream).
- Relative bioavailability compares the systemic exposure of a drug candidate in different dosage forms (e.g., tablet
vs. liquid). -> AUC !!!
- Absolute bioavailability compares the rule of administration (e.g., oral) to intravenous administration, which is
considered 100% bioavailable.
• Volume of Distribution (Vd) gives an idea of how widely the drug spreads throughout the body once it enters the
bloodstream. -> Vd = Dose / Plasma concentration -> lower plasma conc. => higher Vd -> in liters or L/kg.
Ê Timing plays a major role, as drug concentration in the blood will vary depending on the rate and extent of absorption.
The concentration taken within minutes of administration will be very different to the concentration taken many hours
later, producing completely different Vd values.
Good or Bad absorption? -> Lipinski rule of 5
Ê Factors influencing Vd:
Ê Poor (oral) absorption is more likely when:
- Drug properties: size, charge, pKa, lipid solubility,
- MW > 500 Da
water solubility.
- cLog P > 5 (poor water solubility)
- Patient’s properties: age, gender, body composition - H-bound donors (NH, OH) > 5
(muscle vs. fat), hydration status, water distribution - H-bound acceptors (N, O) > 10
(e.g., oedema, ascites, pregnancy). - Rotable bonds > 10
-> Extended
- PSA > 140 Aº
Excipients are needed to enhance absorption
of drugs with lower water solubility. Ê Reverse for good absorption (good bioavailibility)
The body is usually divided into two spaces, a central compartment (plasma) and a tissue compartment.
Ê A high Vd suggests that the drug does not primarily stays within in the bloodstream but is widely distributed throughout
body tissues. Fat-soluble drugs (high lipid solubility) -> high Vd.
9 However, this can be surprising if you expect the drug to stay in the bloodstream due to high plasma protein binding. Plasma protein
binding refers to how much of a drug binds to proteins (like albumin) in the blood. Normally, we would expect a drug with high protein
binding to remain in the bloodstream en thus have a lower Vd. However, a high Vd can still occur even with high plasma protein binding,
thus the plasma protein binding is not always a good predictor of the Vd.
• Half-life (T ½) refers to the time it takes for the drug concentration in the bloodstream to reduce by half. It is
influenced by clearance (Cl), the drug elimination from the body. Several factors affect clearance and, thus, the half-life:
- Susceptibility of the drug to biotransformation -> metabolization, typically in the liver. Drugs that are easily
metabolized will generally have a shorter half-life and thus a faster clearance. They need a higher dose !
- Susceptibility of the drug to removal by filtration or diffusion -> Drugs that are efficiently removed by the kidneys
will have a shorter half-life.
- Active excretion of the drug (its removal by active transport mechanisms) Longer half-lives mean
- Functional status of clearance organs (e.g., liver and kidneys) less frequent dosing !
(e.g., once a day)
- Blood delivery to organs of clearance
Ê Clearance is a first-order process, the amount of drug removed depends on the serum drug concentration.
-> It’s directly proportional !
• In most cases, we want to achieve a steady state (Css) with a drug. A steady state occurs when the rate of drug
administration equals the rate of drug elimination (rate in = rate out), resulting in a constant drug concentration in the
bloodstream over time. This is important because it ensures that the drug maintains a therapeutic effect without
fluctuating too high (causing toxicity) or too low (losing effectiveness).
Ê The time required to reach steady state is approximately 4 to 5 half-lives.
- If it takes too long to reach the steady state, like with certain pain killers, you can give a loading dose.
,• pKa: = drug's dissociation constant, which tells how ionized the drug is in different pH environments (like stomach vs. blood).
So, it allows to determine the charge on a molecule at any given pH.
• LogP = Partition coefficient = measures how well a substance partitions between a lipid (e.g., octanol) and water. -> Thus
indicates the lipophilicity. The higher the LogP, the more lipophilic, the lower the water solubility and thus a poor (oral)
absorption and bioavailability.
• LogDpH 7.4 = Distribution coefficient = a measure for lipophilicity at physiological pH.
> 5 => strongly lipophilic => poor (oral) absorption !!!
-> Remember: lipophilicity is needed to pass membranes (including the BBB), hydrophilic is needed for transport through the blood.
-> Balans between lipophilicity & hydrophilicity !!!
--------------------------------------------------------------------------------------------------------------------------
Bioavailability is influenced by membrane permeability, transporters, and metabolic stability.
â Membrane permeability
A drug must pass through cellular membranes to enter the bloodstream. Drugs with good membrane permeability can be
absorbed more easily.
Ê PAMPA and Caco-2 tests are used to evaluate membrane permeability of drugs.
- PAMPA (Parallel Artificial Membrane Permeability Assay) uses an artificial lipid membrane to measure passive diffusion of a
drug. It is quick, simple, and inexpensive, but does not include active transport.
- Caco-2 test is a widely used method that utilizes a tumor cell line to simulate the intestinal barrier. It evaluates both passive
and active transport, including efflux mechanisms, making it more biologically relevant. However, it is more expensive and time-
consuming.
Membrane permeability of a drug is influenced by several factors:
• Lipophilicity: a fat-soluble drugs has a high lipophilicity ( LogP) and tend to cross cell membranes more easily.
• Aqueous Solubility: a good aqueous solubility is necessary for absorption and determines the bioavailability of the drug,
but too much solubility can hinder membrane passage.
• Molecular weight (MW): smaller molecules (< 500 Da) are generally considered to have better membrane permeability and
pass the membranes more easily.
• PSA (Polar Surface Area): indicates how much of the molecule's surface area is polar (hydrophilic) and thus capable of
forming hydrogen bonds. High PSA usually lowers membrane permeability and transport.
• Number of Hydrogen Bond Acceptors and Donors: too many hydrogen bond donors (NH, OH; > 5) and acceptors (N, O > 10)
can reduce the ability of the drug to cross the lipid membrane, as these interactions increase aqueous solubility.
• Number of Rotatable Bonds: affects the drug's flexibility. A large number of rotatable bonds increases the complexity of
the molecular shape and may hinder membrane permeability.
â Transporters
Active transporters can help drugs cross membranes. They are divided into:
1. Influx transporters: Facilitate the uptake of drugs into cells, improving absorption and distribution.
2. Efflux transporters: Actively pump drugs out of cells, limiting absorption.
• Example: P-glycoprotein (Pgp), which plays an important role in the blood-brain barrier (BBB) where it functions as a protective
efflux transporter, by actively pumping toxins, and harmful substances, but also drugs, back into the bloodstream. This limits
drug penetration into the brain, which can reduce the effectiveness of treatments targeting the CNS.
â Metabolic stability
Drugs are often metabolized in the liver or intestines before they reach the bloodstream (first-pass metabolism). A drug
that is metabolized quickly will have lower bioavailability, as less of it reaches circulation in its active form.
Ê Metabolization of a drug occurs in two key phases:
, - Phase I: This phase includes oxidation, reduction, and hydrolysis reactions, where enzymes such as CYP450 play a
critical role. These reactions can inactivate the drug, activate it (as seen with prodrugs), or produce toxic
metabolites, such as NAPQI in the case of paracetamol overdose.
- Phase II: This phase involves conjugation reactions, such as acetylation and methylation. The primary goal is to
transform the lipid-soluble drug into a more water-soluble form for easier elimination.
Most abundant CYP-enzymes in liver
Ê CYP2D6 is polymorphic, meaning it causes variations in drug metabolism between individuals, resulting in fast,
intermediate, and slow metabolizers.
- Fast metabolizers have lower drug concentrations in the body due to increased CYP2D6 activity, often requiring a higher drug
dose for therapeutic effectiveness.
- Slow metabolizers have higher drug concentrations in the body because of reduced CYP2D6 activity, requiring a lower drug dose
to avoid toxicity.
Ê CYP3A4, on the other hand, is inducible, meaning its activity can be increased by certain drugs, foods, or environmental
factors. By taking the drug, CYP3A4 activity increases, resulting in faster drug metabolism, lower concentrations of
the drug in the body, and potentially necessitating a dose adjustment.
Preclinical in vitro assays for studying drug metabolism include:
1. Liver Microsomes (S9)
Basic Characteristics: Fractionated from subcellular organelles via ultracentrifugation, primarily isolating the SER.
Advantages:
- High concentration of CYP enzymes, along with UGT and other Phase II enzymes present.
- Highly suitable for Phase I and II metabolism assays, particularly useful for evaluating CYP enzyme activity.
- Easy to obtain, commercially available, and relatively inexpensive.
Disadvantages:
- Requires ultracentrifugation, lacks nuclear components, and does not allow for induction studies.
Use Cases: Phase I/II drug metabolism studies, CYP enzyme activity assays.
Availability: Relatively accessible, with samples sourced from organ transplants or commercial suppliers.
2. cDNA CYPs
Basic Characteristics: Individual CYP enzymes are produced via gene expression (e.g., in bacteria, yeast, mammalian cells).
Advantages:
- Ideal for high-throughput screening of specific CYP enzymes, providing highly reproducible results.
Disadvantages:
- Contains only individual enzymes, lacking the full metabolic context found in whole cells or tissues.
- Poor mimic of complex in vivo systems.
Use Cases: Screening for specific CYP450 involvement in drug metabolism.
Availability: = Good -> commercially available.
3. Permanent Cell Lines
Basic Characteristics: exhibit variable enzyme expression levels, lacking a full complement of the relevant metabolic enzymes.
Advantages:
- Supernatant can be used to study cytosolic enzyme activity.
- More accessible and easier to use compared to primary hepatocytes.
Disadvantages:
- Poor expression of relevant enzymes and lacks nuclear components, preventing induction studies.
Use Cases: Studying cytosolic enzyme activity in drug metabolism.
Availability: Available on request, but only few characterized cell lines exist.
bioavailability. Relative bioavailability compares
the AUC of 2 formulations. For example, if the AUCs
Pharmacokinetic (PK) parameters are 121.7 ± 53.8 for gelatin capsules and 129.0 ±
39.3 µg.h/mL for oral absorption, the relative
bioavailability is close to 100%, indicating no
PK -> “What does the body to the drug?” -> ADME significant difference between the 2 formulations.
Bioequivalence compares the systemic exposure (bioavailability) of a test compound with reference compounds. Typically
to know for example if your drug is better than the one already on the market. This is usually tested in healthy volunteers.
• Bioavailability measures how much of a drug is available to enter the circulation (the bloodstream).
- Relative bioavailability compares the systemic exposure of a drug candidate in different dosage forms (e.g., tablet
vs. liquid). -> AUC !!!
- Absolute bioavailability compares the rule of administration (e.g., oral) to intravenous administration, which is
considered 100% bioavailable.
• Volume of Distribution (Vd) gives an idea of how widely the drug spreads throughout the body once it enters the
bloodstream. -> Vd = Dose / Plasma concentration -> lower plasma conc. => higher Vd -> in liters or L/kg.
Ê Timing plays a major role, as drug concentration in the blood will vary depending on the rate and extent of absorption.
The concentration taken within minutes of administration will be very different to the concentration taken many hours
later, producing completely different Vd values.
Good or Bad absorption? -> Lipinski rule of 5
Ê Factors influencing Vd:
Ê Poor (oral) absorption is more likely when:
- Drug properties: size, charge, pKa, lipid solubility,
- MW > 500 Da
water solubility.
- cLog P > 5 (poor water solubility)
- Patient’s properties: age, gender, body composition - H-bound donors (NH, OH) > 5
(muscle vs. fat), hydration status, water distribution - H-bound acceptors (N, O) > 10
(e.g., oedema, ascites, pregnancy). - Rotable bonds > 10
-> Extended
- PSA > 140 Aº
Excipients are needed to enhance absorption
of drugs with lower water solubility. Ê Reverse for good absorption (good bioavailibility)
The body is usually divided into two spaces, a central compartment (plasma) and a tissue compartment.
Ê A high Vd suggests that the drug does not primarily stays within in the bloodstream but is widely distributed throughout
body tissues. Fat-soluble drugs (high lipid solubility) -> high Vd.
9 However, this can be surprising if you expect the drug to stay in the bloodstream due to high plasma protein binding. Plasma protein
binding refers to how much of a drug binds to proteins (like albumin) in the blood. Normally, we would expect a drug with high protein
binding to remain in the bloodstream en thus have a lower Vd. However, a high Vd can still occur even with high plasma protein binding,
thus the plasma protein binding is not always a good predictor of the Vd.
• Half-life (T ½) refers to the time it takes for the drug concentration in the bloodstream to reduce by half. It is
influenced by clearance (Cl), the drug elimination from the body. Several factors affect clearance and, thus, the half-life:
- Susceptibility of the drug to biotransformation -> metabolization, typically in the liver. Drugs that are easily
metabolized will generally have a shorter half-life and thus a faster clearance. They need a higher dose !
- Susceptibility of the drug to removal by filtration or diffusion -> Drugs that are efficiently removed by the kidneys
will have a shorter half-life.
- Active excretion of the drug (its removal by active transport mechanisms) Longer half-lives mean
- Functional status of clearance organs (e.g., liver and kidneys) less frequent dosing !
(e.g., once a day)
- Blood delivery to organs of clearance
Ê Clearance is a first-order process, the amount of drug removed depends on the serum drug concentration.
-> It’s directly proportional !
• In most cases, we want to achieve a steady state (Css) with a drug. A steady state occurs when the rate of drug
administration equals the rate of drug elimination (rate in = rate out), resulting in a constant drug concentration in the
bloodstream over time. This is important because it ensures that the drug maintains a therapeutic effect without
fluctuating too high (causing toxicity) or too low (losing effectiveness).
Ê The time required to reach steady state is approximately 4 to 5 half-lives.
- If it takes too long to reach the steady state, like with certain pain killers, you can give a loading dose.
,• pKa: = drug's dissociation constant, which tells how ionized the drug is in different pH environments (like stomach vs. blood).
So, it allows to determine the charge on a molecule at any given pH.
• LogP = Partition coefficient = measures how well a substance partitions between a lipid (e.g., octanol) and water. -> Thus
indicates the lipophilicity. The higher the LogP, the more lipophilic, the lower the water solubility and thus a poor (oral)
absorption and bioavailability.
• LogDpH 7.4 = Distribution coefficient = a measure for lipophilicity at physiological pH.
> 5 => strongly lipophilic => poor (oral) absorption !!!
-> Remember: lipophilicity is needed to pass membranes (including the BBB), hydrophilic is needed for transport through the blood.
-> Balans between lipophilicity & hydrophilicity !!!
--------------------------------------------------------------------------------------------------------------------------
Bioavailability is influenced by membrane permeability, transporters, and metabolic stability.
â Membrane permeability
A drug must pass through cellular membranes to enter the bloodstream. Drugs with good membrane permeability can be
absorbed more easily.
Ê PAMPA and Caco-2 tests are used to evaluate membrane permeability of drugs.
- PAMPA (Parallel Artificial Membrane Permeability Assay) uses an artificial lipid membrane to measure passive diffusion of a
drug. It is quick, simple, and inexpensive, but does not include active transport.
- Caco-2 test is a widely used method that utilizes a tumor cell line to simulate the intestinal barrier. It evaluates both passive
and active transport, including efflux mechanisms, making it more biologically relevant. However, it is more expensive and time-
consuming.
Membrane permeability of a drug is influenced by several factors:
• Lipophilicity: a fat-soluble drugs has a high lipophilicity ( LogP) and tend to cross cell membranes more easily.
• Aqueous Solubility: a good aqueous solubility is necessary for absorption and determines the bioavailability of the drug,
but too much solubility can hinder membrane passage.
• Molecular weight (MW): smaller molecules (< 500 Da) are generally considered to have better membrane permeability and
pass the membranes more easily.
• PSA (Polar Surface Area): indicates how much of the molecule's surface area is polar (hydrophilic) and thus capable of
forming hydrogen bonds. High PSA usually lowers membrane permeability and transport.
• Number of Hydrogen Bond Acceptors and Donors: too many hydrogen bond donors (NH, OH; > 5) and acceptors (N, O > 10)
can reduce the ability of the drug to cross the lipid membrane, as these interactions increase aqueous solubility.
• Number of Rotatable Bonds: affects the drug's flexibility. A large number of rotatable bonds increases the complexity of
the molecular shape and may hinder membrane permeability.
â Transporters
Active transporters can help drugs cross membranes. They are divided into:
1. Influx transporters: Facilitate the uptake of drugs into cells, improving absorption and distribution.
2. Efflux transporters: Actively pump drugs out of cells, limiting absorption.
• Example: P-glycoprotein (Pgp), which plays an important role in the blood-brain barrier (BBB) where it functions as a protective
efflux transporter, by actively pumping toxins, and harmful substances, but also drugs, back into the bloodstream. This limits
drug penetration into the brain, which can reduce the effectiveness of treatments targeting the CNS.
â Metabolic stability
Drugs are often metabolized in the liver or intestines before they reach the bloodstream (first-pass metabolism). A drug
that is metabolized quickly will have lower bioavailability, as less of it reaches circulation in its active form.
Ê Metabolization of a drug occurs in two key phases:
, - Phase I: This phase includes oxidation, reduction, and hydrolysis reactions, where enzymes such as CYP450 play a
critical role. These reactions can inactivate the drug, activate it (as seen with prodrugs), or produce toxic
metabolites, such as NAPQI in the case of paracetamol overdose.
- Phase II: This phase involves conjugation reactions, such as acetylation and methylation. The primary goal is to
transform the lipid-soluble drug into a more water-soluble form for easier elimination.
Most abundant CYP-enzymes in liver
Ê CYP2D6 is polymorphic, meaning it causes variations in drug metabolism between individuals, resulting in fast,
intermediate, and slow metabolizers.
- Fast metabolizers have lower drug concentrations in the body due to increased CYP2D6 activity, often requiring a higher drug
dose for therapeutic effectiveness.
- Slow metabolizers have higher drug concentrations in the body because of reduced CYP2D6 activity, requiring a lower drug dose
to avoid toxicity.
Ê CYP3A4, on the other hand, is inducible, meaning its activity can be increased by certain drugs, foods, or environmental
factors. By taking the drug, CYP3A4 activity increases, resulting in faster drug metabolism, lower concentrations of
the drug in the body, and potentially necessitating a dose adjustment.
Preclinical in vitro assays for studying drug metabolism include:
1. Liver Microsomes (S9)
Basic Characteristics: Fractionated from subcellular organelles via ultracentrifugation, primarily isolating the SER.
Advantages:
- High concentration of CYP enzymes, along with UGT and other Phase II enzymes present.
- Highly suitable for Phase I and II metabolism assays, particularly useful for evaluating CYP enzyme activity.
- Easy to obtain, commercially available, and relatively inexpensive.
Disadvantages:
- Requires ultracentrifugation, lacks nuclear components, and does not allow for induction studies.
Use Cases: Phase I/II drug metabolism studies, CYP enzyme activity assays.
Availability: Relatively accessible, with samples sourced from organ transplants or commercial suppliers.
2. cDNA CYPs
Basic Characteristics: Individual CYP enzymes are produced via gene expression (e.g., in bacteria, yeast, mammalian cells).
Advantages:
- Ideal for high-throughput screening of specific CYP enzymes, providing highly reproducible results.
Disadvantages:
- Contains only individual enzymes, lacking the full metabolic context found in whole cells or tissues.
- Poor mimic of complex in vivo systems.
Use Cases: Screening for specific CYP450 involvement in drug metabolism.
Availability: = Good -> commercially available.
3. Permanent Cell Lines
Basic Characteristics: exhibit variable enzyme expression levels, lacking a full complement of the relevant metabolic enzymes.
Advantages:
- Supernatant can be used to study cytosolic enzyme activity.
- More accessible and easier to use compared to primary hepatocytes.
Disadvantages:
- Poor expression of relevant enzymes and lacks nuclear components, preventing induction studies.
Use Cases: Studying cytosolic enzyme activity in drug metabolism.
Availability: Available on request, but only few characterized cell lines exist.
