ADME metabolism and excretion

properties for drugs which are used for chronic pain

• need to have a long half life

• reduces dosage frequency

what is half life proportional and inversely proportional to ?

• proportional to the volume of distribution

• inversely proportional to the clearance

clearance - CL

the volume of plasma that is effectively cleared of drug by an eliminating organ per unit time

zero order elimination kinetics

• elimination of a constant quantity per time unit of the drug quantity present in the organism

  • not dependent on time like first order

first order elimination kinetics

elimination of a constant fraction per time unit of the drug quantity present in the organism

what is the most common elimination kinetics order for drugs?

1st

what is the calculation for half life?

t1/2 = 0.693 x Vd / CL

where are drugs eliminated

–urine > most common route for water-soluble drugs and metabolites

–bile > most common route for drugs undergoing enterohepatic recycling

–faeces > e.g. unabsorbed drug, bile products

–lungs > e.g. volatile substances

–skin (sweat) > contributes little

glomerular filtration

• occurs in the renal corpuscle

• passive, non selective driven by hydrostatic pressure

• larger than 40KDa cannot move through the capillary fenestrations of the bowmans capsule

• only unbound drugs are filtered

• filtration rate is directly related to glomerular filtration rate

passive reabsorption

• lipid soluble drugs reabsorbed from the nephron into the peritubular capillaries

• pH partition hypothesis - reabsorption affected by pH of urine

• ion trapping

how does pH of urine affect reabsorption of lipid soluble drugs?

• acidic urine promotes the reabsorption of acidic drugs

• alkaline urine promotes the reabsorption of basic drugs

relationship between difference in size of clearance and glomerular filtrate rate

If CL = GFR The drug is filtered only. It is neither secreted nor reabsorbed

If CL < GFR: The drug is being reabsorbed back into the blood (e.g., Glucose or many lipophilic drugs).

If CL > GFR: The drug is being actively secreted into the tubule (e.g., Penicillin).

definition of metabolism

Drug metabolism is the enzyme-mediated conversion of a lipid-soluble compound into a more water-soluble one

how is metabolism a major liability of lead series requiring improvement?

• high clearance

• short half life

• first pass metabolism

• speed of metabolism

• drug drug interactions

• toxicity

reaction sites for metabolism

• liver

• kidney

• lung

• GI tract

• brain

• plasma

• skin

examples of phase 1 reactions

• oxidation

  • most important reactions

  • eg cytochrome P450

• hydrolysis

• reduction

• hydration

Purpose of phase 1 DME reactions

• functionalisation by producing or uncovering reactive functional groups

• makes drugs slightly more polar → water soluble

• preparation for phase 2

Examples of pharmacological activation:

• pro drugs need to be functionalised to have biological activity

• glyceryl trinitrate - NO

• azathioprine - meracaptopurine

• cortisone - hydrocortisone

• codeine - morphone

Which haem proteins mediate most phase 1 dependent reactions

CYP1, 2 and 3

Requirements of CYP450

substrate (DH), P450 enzyme, molecular oxygen, NADPH and NADPH-P450 reductase

What is the outcome of CYPs involved in drug metabolism

hydroxylated product

where do phase 2 dme reactions occur

mainly liver

Requirements for conjugation in phase 2

susceptible to conjugation if OH, SH or amino group present

What are products from Phase 2 DME reactions

• water soluble

• increased MW

• inactive

Pharmacological inactivation

• occurs during phase 2 DME

• decreases receptor affinity

• enhances excretion

Conjugation

• glucuronidation*, sulphation*, amino acids, glutathione, fatty acids, etc.

• involve transferase enzymes

Conjugation of paracetamol

• glucuronidation with UDP-a-glucuronide

• paracetamol → paracetamol glucuronide

enterohepatic circulation

• drug is absorbed in gi tract and into systemic circulation

• drug in liver + glucoronyl transferase —> drug glucoronide

• bile into gi tract

• some excreted as faces

• beta glucoronidase causes hydrolysis of drug to release glucoronide

• drug can be reasborbed into gi tract systemic circulation and liver

• increases reservoir of available drug

outcomes of metabolism

• pharmacological activation or inactivation

• changes in type of pharmacological response

• no change in pharmacological activity

• changes in drug distribution

internal factors affecting drug metabolism

• species, genetic, age, sex - lesser extent

• disease eg hepatic dysfunction

external factors affecting drug metabolism

• lifestyle - cigarette smoking induces metabolism of eg caffeine, haloperidol, proranalol, estradiol

• environment

• induces or inhibitors - effect on half life

• diet eg brussel sprouts inc metabolism, grapefruit dec metabolism

impact of grapefruit juice of presystemic circulation

• dec inhibition of CYP3A4

• inc bioavailability of felodipine

  • due to decrease in metabolism

metabolism assessment

• metabolic stability

• identification of metabolites

• drug drug interactions

• toxicity

limitations of past approach for ADME assessment

• low throughput

• requires re synthesis

• time consuming

• expensive

advantages of current approach for ADME assessment

• high throughput

• relatively rapid

• quality molecules

• less expensive

• more selective

what are the main excretory routes?

• the kidneys

• the hepatobiliary system

• the lungs (important for volatile/gaseous anaesthetics

which 3 fundamental processes account for renal drug excretion

• 1.glomerular filtration

• 2.active tubular secretion

• 3.passive reabsorption (diffusion from the concentrated tubular fluid back across tubular epithelium)

Which of the following statements about the mechanisms of the metabolism of drugs is correct?

• A.The oxidation or hydrolysis of compounds during phase 1 metabolism inactivates the drug.

• B.Phase 2 reactions are catabolic.

• C.The toxicity of racemic mixtures is often due to the differing metabolism of the stereoisomers.

• D.Lipophilic drugs are excreted more readily than polar drugs.

• E.Microsomal enzymes mainly metabolise polar drugs

C

ways to assess metabolic stability

• in silico - soft spots

• reaction phenotyping

• microsomal stability assay

• S9 or hepatocyte stability

• phase 2 stability assay

in vitro assessment of metabolic stability process with liver

1. Perfuse liver with isotonic buffer containing Ca2+ chelating agent to clear the blood and loosen the cell-cell junctions. Perfuse with collagenase solution to dissociate the hepatocytes from the surrounding tissue. Rat liver yields about 1 x109 hepatocytes; human liver yields 50 x 109 hepatocytes.

2. Isolated hepatocytes can be used either as a suspension or as primary cell cultures.

examples of in vitro metabolic stability assessment models

• 2D monolayers

• 3D liver spheroid cultures

limitation and improvement of metabolic stability assessment models

• can lose activity over time

• liver on a chip to improve modelling of the human liver

in vitro model process for hepatocytes or microsomes on a plate

1. 96 well plate containing the test compound and either hepatocytes or microsomes. If microsomes, need to add any required co-factors.

2. Incubate at 37 C.

3. Aliquots can be removed either at the end or multiple time points.

4. The sample is quenched with acetonitrile to inactivate the enzymes and precipitate the microsomal material.

5. The sample is then centrifuged and the supernatant is then assessed using either liquid chromatography or mass spectrometry. These systems can calculate the amount of test compound remaining after incubation.

how are drug drug interactions - induction - tested?

1. Add test chemical to primary hepatocyte culture and culture for 2 days.

2. After removing the test chemical, you can then add substrates that will be selectively metabolised by specific CYP450 isoforms.

3. The production of metabolites can then be measured.  If the test chemical has caused induction of the liver enzymes, then we would expect to see the concentration of metabolites increase with the concentration of inducer.

how are drug drug interactions - inhibition - tested?

1.Add test chemical to primary hepatocyte culture and incubate.

2.Substrate for P450 can then be added and the production of metabolites measured.

3.If the test chemical has caused inhibition of the liver enzymes, then we would expect to see the concentration of metabolites decrease with the concentration of inhibitor.

in vivo for testing metabolic stability for discovery phase

• measure AUC, Cmax, Tmax

  • Cmax = maximum plasma concentration achieved after administration Tmax = time taken to achieve Cmax

• early metabolism profiling

• allometric scaling to predict human dose and PK

in vivo method for assessing metabolic stability during pre clinical phase

• need to understand ADME of parent drug and metabolite before human trials

• testing formulation, safety and toxicology

• use radiolabeled compounds to quantify tissue distribution to track drug for metabolite profiling