Lecture 8: Phase 1 Metabolism The purpose of metabolism is to detoxify a drug, eliminate a drug or activate a drug. In metabolism there are two phases, Phase I and Phase II. Phase I is the introduction of small polar groups usually this terminates pharmacological activity and this has a detoxifying effect. Overall the introduction of small polar groups increases the polarity and the aqueous solubility of the drug. A drug given by injection or otherwise will still be metabolised but will not pass directly to the liver. Xenobiotic metabolism: Xenobiotic is a synonym for a foreign or exogenous chemical. Includes drugs, dietary chemicals, environmental pollutants, industrial chemicals and other poisons. By comparison, an endobiotic is an endogenous chemical or one that has a physiological role in tissues à e.g. vitamins, sex steroids etc. Metabolism and elimination of chemicals: The body treats xenobiotics and endobiotics in similar ways such chemicals must be sufficiently lipid soluble to enter cells. Biotransformation enzymes convert lipid soluble chemicals into more polar products these are more readily removed by elimination. Generally metabolites have decreased pharmacological or toxicological activity (although there are many exceptions). Lipophilic character of drugs: Chemicals partition between phases in an immiscible two-phase system e.g. consisting of non-aqueous and aqueous phases. The partition coefficient (P) increases with lipophilic character (log P often used). P = [non-aqueous phase]/[aqueous phase]. The larger the value of P and the smaller log P, the more water-soluble. The smaller the value of P and the larger log P, the more lipid soluble. Biotransformation serves to decrease lipophilicity. What happens to drugs after absorption: Orally absorbed drugs enter the liver via the portal circulation. Unbound drug in portal blood perfuses through hepatocytes that contain metabolic enzyme. Some metabolism occurs (first pass effect). Unchanged drug and some drug metabolites exit the liver via the central vein. The drug enters the systemic circulation and is delivered to tissues where it exerts its pharmacological effect. Drug metabolites in blood may be excreted in the kidney. Other metabolites may be excreted in bile and are deposited in the intestine. Impact of metabolism on drug concentrations in blood: Following absorption most drugs undergo metabolism prior to elimination. After maximal serum concentrations are achieved elimination occurs rapidly. Biotransformation converts drugs to forms that are more readily eliminated. Phase 1 Reactions: Chemical conversion of a lipophilic chemical into a more polar analogue. Inclusion of a new functional group, usually by oxidation, reduction or hydrolysis. In the case of phenacetin oxidation of the O-ethyl group produces a phenol (paracetamol), with ~10-fold lower lipophilic character. Cytochromes P450: CYP enzymes are the enzymes responsible for Phase I metabolism. CYPs are the major class of Phase I biotransformation enzymes. There are 57 human P450 genes; about 12 mediate drug oxidation.
Unlike most enzymes P450s act on diverse substrates, including the xenobiotics and endobiotics to which we are exposed. Low substrate specificity. However, there are still some drugs and chemicals that are oxidised by a single P450. Factors that affect P450 activities can markedly affect drug elimination. Characteristic P450 reactions: Drugs can form several metabolites because they have more than one substituent that can be oxidised. Appropriate substituents can increase the number of alternate metabolic pathways. 2 or more P450s may oxidise a drug, e.g. tamoxifen is oxidised at an aromatic carbon (4- hydroxylation) and an aliphatic carbon (N-dealkylation). P450 Structure and Function: All P450s have a haem group involved in oxygen activation. Each P450 encoded by a different gene. The polypeptide chain differs between P450s and controls substrate specificity. Properties of P450s that affect drug safety and efficacy: P450s accommodate many substrates. If substrates require the same enzyme for clearance pharmacokinetic drug-drug interactions can occur. Many of these clinically important. Drug clearance decreased, so drug can accumulate and produce toxicity. Some P450s induced by coadministered chemicals. Some P450s are subject to genetic polymorphisms. Important drug metabolising CYPs: CYP3A4 Quantitatively the major enzyme in liver à role in metabolism of ~60% of drugs. Large active site so can accommodate a wide range of substrates as well as very bulky molecules. Some important drug classes e.g. statins, HIV protease inhibitors, benzodiazepines, and calcium channel blockers. Also oxidises sex steroids and environmental chemicals, including aflatoxins and polycyclic aromatic hydrocarbons. Readily inhibited à prone to pharmacokinetic drug interactions e.g. with ketoconazole, ritonavir, diltiazem and grapefruit juice. Most inhibitory interactions with CYP3A4: Simple competition between drugs for enzyme. Short-lived interactions, but can be serious. In previous example increased AUC and longer half-life means more drug stays in the body for longer. Results in exaggerated pharmacological effect. Eventually the problem drug is eliminated and its concentration in the body decreases. Metabolic activity then returns to normal. Grapefruit Juice: Grapefruit juice inhibits intestinal metabolism so more drug is absorbed. Grapefruit juice is preferred by the CYP3A4 enzyme to the drug, preventing metabolism of the drug and allowing a greater amount of the drug to move into the circulation. GFJ contains bergamottin that inactivates intestinal but not liver CYP3A4. Drugs that undergo presystemic oxidation in the intestine are susceptible to the GFJ effect. This is a problem if the drug causes toxicity and has variable oral bioavailability. In effect, a larger proportion of each dose is absorbed if GFJ inactivates intestinal CYP3A4. Also occurs with other bergamottin-containing citrus fruits e.g. seville oranges, limes. Summary: Lipophilic xenobiotics and endobiotics are oxidised by cytochromes P450s. In Phase I metabolism a functional group is introduced into a substrate chemical (including drugs). CYP3A4 is quantitatively most important and metabolises most drugs. Low substrate specificity in CYPs makes them susceptible to drug-drug interactions. The grapefruit juice interaction is an unusual but important example of long-lived CYP inhibition.
Lecture 9: Phase I and Phase II metabolism In Phase I metabolism a functional group is introduced into a substrate chemical to make it easier to eliminate. Induction of CYP genes: CYP genes are activated by exposure to certain drugs and chemicals. In the late 1990s a new nuclear receptor was identified. The pregnane X-receptor (PXR). PXR is a sensor in cells for foreign compounds including antiepileptic drugs, anti-tuberculosis drugs, the HIV drug efavirenz, St John s wort and possibly other herbal agents. PXR then stimulates the production of CYPs. Consequently, PXR stimulates the ability of the body to eliminate a drug more rapidly. CYP3A4 Induction: CYP3A4 metabolises the important anticancer agent imatinib. St Johns wort increases the activity of CYP3A4, breaking down the anti-cancer drug before it has an opportunity to act. Induction of CYP1A2: The CYP1A2 gene is inducible by chemicals in cigarette smoke and other environmental pollutants like dioxins. Mechanism involves activation of the aryl hydrocarbon receptor (AhR). CYP1A2 substrate drugs have significant toxicities, e.g. metabolise clozapine and theophylline CYP1A2 is inducible by cigarette smoke Clinical significance: Many schizophrenia patients are heavy smokers The antipsychotic drug clozapine may be the only effective agent in some patients. Clozapine is metabolised by CYP1A2. Some patients in the community may be stabilised on clozapine and a high level of cigarette use. They may experience toxicity if they are institutionalised - which decreases their smoking behaviour and they continue with the same dose of clozapine. A common scenario: their clozapine is re-established at a lower dose, they leave the institution, resume previous smoking behaviour, and clozapine efficacy is decreased. Higher maintenance doses of clozapine required in smokers. Cessation of smoking leads to increases in plasma clozapine concentrations, confusion, seizures, stupor and even coma. CYPs and active metabolites: Most drug metabolites are pharmacologically less active than the parent drugs. However, some drugs undergo phase I oxidation to active metabolites. Diazepam and its metabolites all have anti- anxiety and tranquilizer activity. CYP2D6 is required for the activity of codeine: A pro-drug must be broken down to reach its active form. Codeine is a pro-drug. CYP2D6 converts codeine to morphine, but only at extremely low percentages. The metabolite morphine is a more potent opioid receptor agonist. Tamoxifen is also a pro-drug: Tamoxifen is also activated by CYP2D6 to 4-hydroxytamoxifen and endoxifen. These are effective estrogen receptor antagonists. The N-desmethyl metabolite and tamoxifen are not potent estrogen receptor antagonists. CYP-dependent activation of toxic hydrocarbons in cigarette smoke: Constituents of soot and tobacco smoke are associated with toxicity and cancer. Identified polycyclic aromatic hydrocarbons (PAH) in these pollutants. Extracts of these pollutants, when administered to rodents, increased the conversion of PAH to DNAbinding species. Phase II Metabolism:
Phase II metabolism makes a substance extremely water soluble to make it easier to eliminate. Phase I metabolites may still be sufficiently lipid soluble that they are still difficult to excrete. In phase II metabolism the phase I metabolites are conjugated with very polar endogenous molecules. Phase I attaches a suitable functional group and Phase II attaches a very polar soluble compound such as a sugar structure. Major Phase II biotransformation pathways: After formation phase II conjugates are excreted rapidly in urine or faeces. Major phase II enzymes include UDP-glucuronosyltransferases, sulfotransferases, N-acetyltransferases and glutathione S-transferases. Quantitatively minor pathways include amino acid conjugation, methylation. Glutathione S-transferases (GSTs): Conjugation of drugs and chemicals with glutathione is mediated by glutathione S-transferases (abbreviation: GSTs). After formation glutathione conjugations are processed further to mercapturic acids. GSTs mainly important for detoxification of environmental compounds. However, small cytotoxic anti-cancer agents, including busulfan are also metabolised by GSTs and excreted as mercapturic acids. Protective role of phase II metabolism against toxic chemicals: PAH are activated to toxic species by CYPs and epoxide hydrolase. Phase II products are less toxic. Greater phase I activity potentially increases toxicity. Greater phase II activity potentially decreases toxicity. Minor conjugation pathways. I: Amino acid conjugation: Benzoic acid conjugated with glycine Hippuric acid isolated from urine of animals that were treated with benzoic acid Minor conjugation pathways. II: Methylation by thiopurine methyltransferase (TPMT) and other methyltransferases. Inhibition and induction of phase II metabolism: Phase II enzymes have a high capacity and there is functional overlap in conjugation of a particular drug, e.g. glucuronidation and sulphation. Therefore competition for conjugation resulting in significant inhibitory interactions is rare. Several phase II genes are inducible by PXR and AhR agonists e.g. rifampicin, St John s wort, environmental chemicals. Exposure to such chemicals can increase phase II gene expression and conjugation activity Summary: CYP induction can increase drug elimination and impair therapy. Some CYP oxidations produce active metabolites and activate prodrugs while others produce toxic intermediates. Major phase II enzymes include UGTs, SULTs, NATs and GSTs. Phase II biotransformation greatly enhances drug clearance and protection against toxic chemicals. Phase II genes are often inducible in parallel with CYP genes. Lecture 10: Phase II Metabolism and Elimination Once formed intracellularly most drug metabolites are highly polar. Problem: metabolites should be more readily excreted but must first cross the cell membrane. Their increased polarity decreases the capacity to move through the plasma membrane. ATP-binding cassette (ABC)-efflux transporters facilitate the initial phase of polar metabolite removal from cells. Unlike influx transporters the efflux transporters require energy expenditure (ATP). ABC-transporters:
Like the influx transporters these proteins span the plasma membrane. P-glycoprotein most intensively studied. Synonyms include multi-drug resistance protein-1 (MDR1); also ABCB1. Present in many tissues: In intestine decreases drug absorption In liver facilitates removal of polar drug metabolites ABC transporter mechanism: ABC transporters exist in the membrane as dimers. In the open dimer form the transporter accepts substrate (inside cell). Binding of ATP produces a conformational shift. The substrate is effluxed and ATP is dephosphorylated to produce ADP. The starting conformation is restored to accept further substrate present in the cell. Inhibition of ABC-transporters: P-glycoprotein accepts many substrates Competition between these substrates can interfere with their clearance. Some drugs are potent inhibitors e.g. verapamil (an antihypertensive drug) Pharmaceutical companies have searched for inhibitors of ABC- transporters because of their role in anticancer drug efflux. Some progress has been made but the best molecules produce toxicity. Several transporter genes are inducible by PXR and AhR agonists. Exposure to particular drugs and chemicals can increase transport activity. Anticancer drug efflux by ABC transporters: Some cytotoxic drugs that are known substrates for P-glycoprotein include etoposide, daunomycin, taxol, vinblastine and doxorubicin. Over-expression of ABC transporters in tumours: ABC transporters are often over-expressed in cancer cells. This enables cancer cells to efficiently eliminate drugs. Prevents the drugs from reaching effective concentrations in cells. Cancer cells are then resistant to these drugs. Some transporters are termed resistance proteins. Therefore interest in pharmacology to identify transporter inhibitors. Routes of elimination: Once a drug has undergone phase I/II metabolism it is ready for elimination. Transporters can direct the drug metabolite back into the systemic circulation (for elimination in urine) or bile (elimination in faeces). Minor elimination pathways à Pulmonary, Breast milk, Sweat, Hair and Saliva. The elimination phase: Three important components: Glomerular filtration à Removal of free drug (not bound to plasma proteins) at the glomerulus. Active tubular secretion à Drug is transported from blood into urine by tubular transporter proteins. Tubular reabsorption à Passive process in which drug in urine diffuses back into blood. Note: common factors influence renal elimination and drug absorption drug ionization and protein binding.
Renal Elimination: Glomerular filtration à Size limit (~10 kda) so only free drug is filtered. Secretion: Specialised anion and cation transporters in tubule (OAT, OCT, etc.) Transport drug from peritubular capillaries into tubule and urine. Reabsorption: Reverse direction: drug moves out of tubule back into plasma à reabsorption favours unionised drug. Therefore urinary ph important Afferent arterioles carry blood from renal artery to the glomerulus. Efferent arterioles carry blood from the glomerulus. Urinary ph affects reabsorption: Weak acids are more dissociated at high ph (ionized, water soluble). Urinary alkalinisers transiently increase ph, which facilitates excretion of weak acids. Sodium bicarbonate used for this purpose. For weak bases ammonium chloride can be used to acidify urine and decrease reabsorption. Non-ionized acidic drug in blood can move across tubular membrane. This is prevented if the urine is made alkaline. Acidic drug is ionized so reabsorption is decreased and elimination is increased. Faecal excretion: Drugs can appear in faeces by two major mechanisms. By not being absorbed into the systemic circulation so drug remains in intestine. By being absorbed, excreted in bile and then being deposited back into the intestine. Transporters efflux drug from hepatocytes. Drug conjugates can be excreted in bile:
Physiological role of bile: Excretion of cholesterol Absorption of lipids Stimulation of intestinal motility Bile produced in liver and may be stored in gall bladder. Drug conjugates formed in liver (phase II metabolites) can be transported into bile and then deposited in the intestine. Intestinal cleavage of phase II drug conjugates: Bacteria in the gut cut off the sugar molecule (glucuronides) that is attached to the drug and consequently make it much less polar. This restores the drug or Phase I metabolite in intestine. The drug or metabolite can be reabsorbed. Enterohepatic recycling: Reabsorption of the drug or metabolite can boost blood levels again. This may produce a secondary phase of therapeutic effect. With further enterohepatic recycling there is gradual loss of drug. Pulmonary elimination: The lung is probably the major organ of excretion for gases and volatile substances that do not require metabolism, e.g. anaesthetics. The breathalyser test quantifies the pulmonary excretion of ethanol. The fate of the methyl group that is lost from some drugs by oxidative metabolism is carbon dioxide. Erythromycin breath test: Small dose of 14C- erythromycin administered. Breath collected at intervals and radioactivity measured. Can be used in patients to measure the activity of liver phase I enzymes. Elimination in breast milk: A large number of drugs are excreted in breast milk. Some are of potential concern, e.g. cytotoxic agents used in cancer chemotherapy, lithium, some antihypertensives, and antiepileptics. However, for many drugs there is little information or the risk appears to be minor. Other pathways of drug elimination: Sweat e.g. THC, benzodiazepine, cocaine, barbiturate, morphine, methadone and cotinine. Hair e.g. amphetamines and other drugs of abuse, some metals. Saliva Wide range of drugs, including antiepileptics and others. Useful in forensic analysis, may also be useful in therapeutic drug monitoring.