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Transcript
Cytochrome P450 Cytochrome P450 (abbreviated CYP, P450, infrequently CYP450) is a very large and diverse superfamily of hemoproteins found in all domains of life.[1] Cytochromes P450 use a plethora of both exogenous and endogenous compounds as substrates in enzymatic reactions. Usually they form part of multicomponent electron transfer chains, called P450containing systems. The most common reaction catalysed by cytochrome P450 is a monooxygenase reaction, e.g. insertion of one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to water: RH + O2 + 2H+ + 2e– → ROH + H2O CYP enzymes have been identified from all lineages of life, including mammals, birds, fish, insects, worms, sea squirts, sea urchins, plants, fungi, slime molds, bacteria and archaea. More than 7700 distinct CYP sequences are known (as of September 2007; see the web site of the P450 Nomenclature Committee for current counts).[2] The name cytochrome P450 is derived from the fact that these are colored ('chrome') cellular ('cyto') proteins, with a "pigment at 450 nm", so named for the characteristic Soret peak formed by absorbance of light at wavelengths near 450 nm when the heme iron is reduced (often with sodium dithionite) and complexed to carbon monoxide. Nomenclature The current nomenclature guidelines suggest that members of new CYP families share >40% amino acid identity, while members of subfamiles must share >55% amino acid identity. There is a Nomenclature Committee that keeps track of and assigns new names. Mechanism The P450 catalytic cycle The active site of cytochrome P450 contains a heme iron center. The iron is tethered to the P450 protein via a thiolate ligand derived from a cysteine residue. This cysteine and several flanking residues are highly conserved in known CYPs and have the formal PROSITE signature consensus pattern [FW] - [SGNH] - x - [GD] - {F} - [RKHPT] - {P} - C [LIVMFAP] - [GAD].[4] Because of the vast variety of reactions catalyzed by CYPs, the activities and properties of the many CYPs differ in many aspects. In general, the P450 catalytic cycle proceeds as follows: 1: The substrate binds to the active site of the enzyme, in close proximity to the heme group, on the side opposite to the peptide chain. The bound substrate induces a change in the conformation of the active site, often displacing a water molecule from the distal axial coordination position of the heme iron[5], and sometimes changing the state of the heme iron from low-spin to high-spin[6]. This gives rise to a change in the spectral properties of the enzyme, with an increase in absorbance at 390 nm and a decrease at 420 nm. This can be measured by difference spectrometry and is referred to as the "type I" difference spectrum (see inset graph in figure). Some substrates cause an opposite change in spectral properties, a "reverse type I" spectrum, by processes that are as yet unclear. Inhibitors and certain substrates that bind directly to the heme iron give rise to the type II difference spectrum, with a maximum at 430 nm and a minimum at 390 nm (see inset graph in figure). If no reducing equivalents are available, this complex may remain stable, allowing the degree of binding to be determined from absorbance measurements in vitro[7] 2: The change in the electronic state of the active site favors the transfer of an electron from NAD(P)H via cytochrome P450 reductase or another associated reductase[8]. This takes place by way of the electron transfer chain, as described above, reducing the ferric heme iron to the ferrous state. 3: Molecular oxygen binds covalently to the distal axial coordination position of the heme iron. The cysteine ligand is a better electron donor than histidine, with the oxygen consequently being activated to a greater extent than in other heme proteins. However, this sometimes allows the bond to dissociate, the so-called "decoupling reaction", releasing a reactive superoxide radical, interrupting the catalytic cycle[5]. 4: A second electron is transferred via the electron-transport system, either from cytochrome P450 reductase, ferredoxins, or cytochrome b5, reducing the dioxygen adduct to a negatively charged peroxo group. This is a short-lived intermediate state. 5: The peroxo group formed in step 4 is rapidly protonated twice by local transfer from water or from surrounding amino-acid side chains, releasing one water molecule, and forming a highly reactive iron(V)-oxo species[5]. 6: Depending on the substrate and enzyme involved, P450 enzymes can catalyse any of a wide variety of reactions. A hypothetical hydroxylation is shown in this illustration. After the product has been released from the active site, the enzyme returns to its original state, with a water molecule returning to occupy the distal coordination position of the iron nucleus. S: An alternative route for mono-oxygenation is via the "peroxide shunt": interaction with single-oxygen donors such as peroxides and hypochlorites can lead directly to the formation of the iron-oxo intermediate, allowing the catalytic cycle to be completed without going through steps 3, 4 and 5[7]. A hypothetical peroxide "XOOH" is shown in the diagram. C: If carbon monoxide (CO) binds to reduced P450, the catalytic cycle is interrupted. This reaction yields the classic CO difference spectrum with a maximum at 450 nm. Because most CYPs require a protein partner to deliver one or more electrons to reduce the iron (and eventually molecular oxygen), CYPs are properly speaking part of P450containing systems of proteins. Five general schemes are known: CPR/cyb5/P450 systems employed by most eukaryotic microsomal (i.e., not mitochondrial) CYPs involve the reduction of cytochrome P450 reductase (variously CPR,POR, or CYPOR) by NADPH, and the transfer of reducing power as electrons to the CYP. Cytochrome b5 (cyb5) can also contribute reducing power to this system after being reduced by cytochrome b5 reductase (CYB5R). FR/Fd/P450 systems which are employed by mitochondrial and some bacterial CYPs. CYB5R/cyb5/P450 systems in which both electrons required by the CYP come from cytochrome b5. FMN/Fd/P450 systems originally found in Rhodococcus sp. in which a FMNdomain-containing reductase is fused to the CYP. P450 only systems, which do not require external reducing power. Notably these include CYP5 (thromboxane synthase), CYP8, prostacyclin synthase, and CYP74A (allene oxide synthase). P450s in humans Human CYPs are primarily membrane-associated proteins, located either in the inner membrane of mitochondria or in the endoplasmic reticulum of cells. CYPs metabolize thousands of endogenous and exogenous compounds. Most CYPs can metabolize multiple substrates, and many can catalyze multiple reactions, which accounts for their central importance in metabolizing the extremely large number of endogenous and exogenous molecules. In the liver, these substrates include drugs and toxic compounds as well as metabolic products such as bilirubin (a breakdown product of hemoglobin). Cytochrome P450 enzymes are present in most other tissues of the body, and play important roles in hormone synthesis and breakdown (including estrogen and testosterone synthesis and metabolism), cholesterol synthesis, and vitamin D metabolism. Hepatic cytochromes P450 are the most widely studied. The Human Genome Project has identified 57 human genes coding for the various cytochrome P450 enzymes.[9] Drug metabolism CYPs are the major enzymes involved in drug metabolism, accounting for ∼75% of the total metabolism.[10]. Cytochrome P450 is the most important element of oxidative metabolism (also known as phase I metabolism). (Metabolism in this context is the chemical modification or degradation of drugs.) Drug interaction Many drugs may increase or decrease the activity of various CYP isozymes in a phenomenon known as enzyme induction and inhibition. This is a major source of adverse drug interactions, since changes in CYP enzyme activity may affect the metabolism and clearance of various drugs. For example, if one drug inhibits the CYP-mediated metabolism of another drug, the second drug may accumulate within the body to toxic levels, possibly causing an overdose. Hence, these drug interactions may necessitate dosage adjustments or choosing drugs which do not interact with the CYP system. Such drug interactions are extra important to take into account when using drugs of vital importance to the patient, drugs with important side effects and drugs with small therapeutic windows, but any drug may be subject to an altered plasma concentration due to altered drug metabolism. A classical example includes anti-epileptic drugs. Phenytoin, for example, induces CYP1A2, CYP2C9, CYP2C19 and CYP3A4. Substrates for the latter may be drugs with critical dosage, like amiodarone or carbamazepine, whose blood plasma concentration may decrease because of enzyme induction. Interaction of other substances In addition, naturally occurring compounds may cause a similar effect. For example, bioactive compounds found in grapefruit juice and some other fruit juices, including bergamottin, dihydroxybergamottin, and paradisin-A, have been found to inhibit CYP3A4mediated metabolism of certain medications, leading to increased bioavailability and thus the strong possibility of overdosing.[11] Because of this risk, avoiding grapefruit juice and fresh grapefruits entirely while on drugs is usually advised. Other examples: Saint-John's wort, a common herbal remedy induces CYP3A4. Tobacco smoking induces CYP1A2 (example substrates are clozapine/olanzapine) CYP Families in Humans Humans have 57 genes and more than 59 pseudogenes divided among 18 families of cytochrome P450 genes and 43 subfamilies.[12] This is a summary of the genes and of the proteins they encode. See the homepage of the Cytochrome P450 Nomenclature Committee for detailed information.[9] Family Function Members Names drug and steroid CYP1 (especially estrogen) metabolism 3 subfamilies, 3 genes, 1 pseudogene CYP1A1, CYP1A2, CYP1B1 drug and steroid CYP2 metabolism 13 subfamilies, 16 genes, 16 pseudogenes CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1 drug and steroid CYP3 (including testosterone) metabolism 1 subfamily, 4 genes, 2 pseudogenes CYP3A4, CYP3A5, CYP3A7, CYP3A43 arachidonic acid or fatty CYP4 acid metabolism CYP4A11, CYP4A22, CYP4B1, 6 subfamilies, 11 CYP4F2, CYP4F3, CYP4F8, genes, 10 CYP4F11, CYP4F12, CYP4F22, pseudogenes CYP4V2, CYP4X1, CYP4Z1 CYP5 thromboxane A2 synthase 1 subfamily, 1 gene CYP5A1 bile acid biosynthesis 7CYP7 alpha hydroxylase of steroid nucleus 2 subfamilies, 2 genes CYP7A1, CYP7B1 CYP8 varied 2 subfamilies, 2 genes CYP8A1 (prostacyclin synthase), CYP8B1 (bile acid biosynthesis) CYP11 steroid biosynthesis 2 subfamilies, 3 genes CYP11A1, CYP11B1, CYP11B2 1 subfamily, 1 gene CYP17A1 steroid biosynthesis: CYP19 aromatase synthesizes estrogen 1 subfamily, 1 gene CYP19A1 CYP20 unknown function 1 subfamily, 1 gene CYP20A1 CYP21 steroid biosynthesis 2 subfamilies, 2 genes, 1 pseudogene CYP21A2 CYP24 vitamin D degradation 1 subfamily, 1 gene CYP24A1 CYP26 retinoic acid hydroxylase 3 subfamilies, 3 genes CYP26A1, CYP26B1, CYP26C1 CYP17 steroid biosynthesis, 17alpha hydroxylase CYP27 varied 3 subfamilies, 3 genes CYP27A1 (bile acid biosynthesis), CYP27B1 (vitamin D3 1-alpha hydroxylase, activates vitamin D3), CYP27C1 (unknown function) CYP39 7-alpha hydroxylation of 1 subfamily, 1 24-hydroxycholesterol gene CYP39A1 CYP46 cholesterol 24hydroxylase 1 subfamily, 1 gene CYP46A1 CYP51 cholesterol biosynthesis 1 subfamily, 1 gene, 3 pseudogenes CYP51A1 (lanosterol 14-alpha demethylase) References 1. ^ International Union of Pure and Applied Chemistry. "cytochrome P450". Compendium of Chemical Terminology Internet edition. Danielson P (2002). "The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans". Curr Drug Metab 3 (6): 561–97. doi:10.2174/1389200023337054. PMID 12369887. 2. ^ "Dr. Nelson Lab Website". Retrieved on 2007-11-19. 3. ^ "NCBI sequence viewer". Retrieved on 2007-11-19. 4. ^ PROSITE consensus pattern for P450 5. ^ a b c Bernard Meunier, Samuël P. de Visser and Sason Shaik (2004). "Mechanism of Oxidation Reactions Catalyzed by Cytochrome P450 Enzymes". Chemical Reviews 104 (9): 3947–3980. doi:10.1021/cr020443g. 6. ^ Thomas L. Poulos, Barry C. Finzel and Andrew J. Howard (1987). "High-resolution crystal structure of cytochrome P450cam". Journal of Molecular Biology 195 (3): 687–700. doi:10.1016/0022-2836(87)90190-2. 7. ^ a b P.R. Ortiz de Montellano (Ed.) (1995). Cytochrome P450 : structure, mechanism, and biochemistry, 2nd ed.. New York: Plenum. 8. ^ S. G. Sligar, D. L. Cinti, G. G. Gibson and J. B. Schenkman (1979). "Spin state control of the hepatic cytochrome P450 redox potential". Biochemical and Biophysical Research Communications 90 (3): 925–932. doi:10.1016/0006-291X(79)91916-8. 9. ^ a b ""P450 Table"". 10. ^ F. Peter Guengerich (2008). "Cytochrome P450 and Chemical Toxicology". Chemical Research in Toxicology 21: 70–83. doi:10.1021/tx700079z. 11. ^ Bailey DG, Dresser GK (2004). "Interactions between grapefruit juice and cardiovascular drugs". Am J Cardiovasc Drug 4 (5): 281–297. doi:10.2165/00129784-200404050-00002. PMID 15449971. 12. ^ Nelson D (2003). Cytochrome P450s in humans. Retrieved May 9, 2005. Phases of Drug Metabolism Phase I - Oxidation / Reduction /Hydrolysis - Phase II Conjugation Drug Interactions due to Hepatic Metabolism Nearly always due to interaction at Phase I enzymes, rather than Phase II i.e. commonly due to interaction at cytochrome P450 enzymes…some of which are genetically absent Phase I Drug Oxidation Cytochrome P450 Isoforms CYP1A2 CYP2A6 CYP2B6 CYP3A4/5 CYP2C9 CYP2C19 CYP2D6 CYP2E1 Cytochrome P450 Nomenclature e.g. for CYP2D6 CYP= cytochrome P450 2= genetic family D=genetic sub-family 6=specific gene NOTE that this nomenclature is genetically based: it has NO functional implication. Role of Cytochrome P450 and P-glycoprotein in PK-interactions Substrates, Inducers & Inhibitors of Human CYPs I) Drug-Drug Interactions of Immunosuppressants drugs 1) Methylprednisolone +CsA: Mutual inhibition of metabolism occurs with concurrent use of CsA and methylprednisolone, therefore it is possible that adverse events (convulsions) to occur. 2)Steroids and TAC: Glucocorticoids are inducers of CYP3A and P-gp. The higher the steroid dosage, the higher the dosage of TAC needed to achieve target trough levels . After cessation of concomitant corticosteroid treatment, exposure to TAC inc by 25% 3) CsA+Sirolimus: AUC and Cmax of sirolimus increase by 100% relative to Sirolimus alone, as CsA inhibits P-gp, CYP3A4 when taken with sirolimus. while spacing 4hrs, increase in AUC, Cmax of sirolimus by 30-40%. 4)MMF+Tacrolimus Trough level and AUC of MMF is higher when MMF is used as a part of a Tacrolimus-based regimen (Tacrolimus inhibits glucouronidation of MPA and thus increase conc. of the active form of the drug ) 5)MMF+CSA: CsA inhibits enterohepatic circulation of MPAG and thus decrease blood conc. of MPA. II) Drug Interactions of Immunosupressants and Anti-infectives. A) Antifungals: 1) Amphotericin B+ immunosupressants:(PD) a.Conventional form: additive nephrotoxicity b.liposomal form: these combinations show less additive nephrotoxicity and more neurotoxicity. 2)Azole antifungals + immunosupressants:(PK) Generally inhibit CYP3A, P-gps. Increase concentrations of immunosuppressants in blood AZOLES Immunosupressive drugs Interventions Ketoconazole Cyclosporin ↓ dec.dose by Nizoral ® 80%. Sirolimus Contraindicated Voriconazole Cyclosporin dec.dose by 50 % vefend® Tacrolimus dec.dose by 2/3 Sirolimus Contraindicated Itraconazole Cyclosporin dec..dose sporanox ® Tacrolimus by 50% Fluconazole Diflucan ® Cyclosporin <200mg/day Tacrolimus >200mg/day No dose adjustments Adjust acc.to TDM B)Drug Interactions of Immunosupressants and Antibiotics 1) Metronidazole or fluoroquinolones +MMF:(PD) Result in elemination in intestinal flora and are associated with 35-45% reduction in MPA bioavailability (AUC). 2) Imipenem + CsA or TAC:(PD) Neurotoxicity may be increased when imipenem is adminstered to CsA or TAC treated patients . Meropenem may be a safer carbapenem for transplant patient receiving either CsA or TAC. 3) Sulfonamides (TMP-SMX)+ CsA: (PD) Additive nephrotoxicity 4) Macrolides + CsA, TAC, SIR: (PK) Erythromycin and Clarithromycin inhibit metabolism of CsA, TAC, SIR via CYP3A4 inhibition (grade 1,A) Azithromycin and Dirithomycin (may increase CsA concentration) can be used safely (grade 2,3-C) 5)Aminoglycosides + CsA,TAC: (PD) Gentamicin,Tobramycin, Amikacin have additive nephrotoxicity (grade 2,B) . 6)Rifampin + CsA, TAC, SIR: (PK) (grade 1,A) Rifampin induces metabolism of CsA,TAC, SIR. 7) Fluoroquinolones: (PK) Ofloxacin ( grade 2,B) , Ciprofloxacin (grade 3,B) and Norfloxacin→ increase risk of nephrotoxicity with CsA. C)Drug Interactions of Immunosupressants and Antivirals(PD) 1) Acyclovir, Valacyclovir : CsA,TAC, SIR →Additive neurotoxicity in the presence of renal insufficiency. False decrease in CsA blood levels as a result of interference of Acyclovir with the assay. 2) Ganciclovir : Aza, SIR → Exacerbation of hematological toxicity. 3)Foscarnet, Cidovir: CsA,TAC → Additive nephrotoxicity ,electrolyte abnormalities, Hypocalcemia, hypomagnesemia for foscarnet) III) Drug Interactions of Immunosupressants and Antihypertensives 1) Calcium Channel Blockers: (PK) Dihydropyridine group (eg: nifedipine, amlodipine and felodipine) Nondihydro-pyridine group (verapamil and diltiazem) Both types cause slowing of the cytochrome P-450, causing marked increase in the levels of CsA, TAC. The most clinically important examples of the reduced P-450 activity occurs with the nondihydropyridine gp Amlodipine has lower affinity to the enzyme, co-administration with CsA increase gingival overgrowth. 2)β-Blockers: (PD) Inrease risk of hyperkalemia, incidence of D.M esp.with tacrolimus. 3) ACEIs and ARABs(PD): • Generally increase risk of hyperkalemia, reduced GFR with CsA and TAC. • ACEIs + Azathioprine or MMF : Exacerbation of hematological toxicity (anemia). 4) Diuetics(PD): • K-sparing diuretics increase risk of hyperkalemia with CsA, TAC, SIR. • Loop diuretics exacerbate hypomagnesemia with CsA, TAC, SIR. 5)others: Clonidine : Limited data suggests that CsA conc increase dramatically in some cases when clonidine is added. IV) Interactions of Immunosupressants and lipid lowering Agents 1) Statins (PK): •Atrovastatin, Cerivastatin, lovastatin and Simvastatin are all substrates for CYP3A4 and most of them are subject to extensive pre-systemic drug metabolism. CSA (and perhaps TAC) inc. bioavailability, dec. clearance of statins. → Accumulation of statins, inc. risk of Myositis and Rhabdomyolysis. •In contrast Fluvastatin (metabolized mainly by CYP2C9) and Pravastatin (eliminated by other metabolic routes) are less likely to be involved in this type of interaction. V) Drug Interactions of Immunosupressants and Antidepressants •Most SSRIs such as Fluvoxamine (Faverin ®), Fluxoetine (Prozac ®) Sertaline (lustral®) and Escitalopram (cipralex®) are weak inhibitors of CYP3A4. •Sertaline and Escitalopram are considered the antidepressant of choice due to minimal drug interactions and side effects. • Nefazodone (Serzone®) mixed SSRI and norepinephrine reuptake inhibitor ,is a potent CYP3A4 inhibitor and should be avoided in transplant patients due to the potential for severe toxicity. • St.John’s wort (Safamood ®) is a potent inducer of CYP 3A4 and causing a decrease in the levels of cyclosporin and tacrolimus. VI) Drug Interactions of Immunosupressants and Anti-ulcer Medications (PK) 1) H2-Receptor Antagonist: Cimitidne (Tagamet ®) inhibits various CYP450 isoenzymes while Ranitidine (Zantac®), Famotidine (Servipep ®), Nizatidine (Nizatine ®) do not. Ranitidine, Cimitidne potentiate nephrotoxicity of CsA and TAC. 2) Proton Pump Inhibitors: Omperazole (losec ®) inhibit CYP3A and increases TAC levels. 3) Antacids and Sucralfate: Separate administration by 2 hrs. VII) Antidiabetics Glibenclamide (Doanil®): Glibenclamide is a substrate and inhibitor of CYP450→ Increase CsA levels. Troglitazone : Decrease CsA levels by induction of CYP450 3A. Pharmacogenetics During the past 50 years pharmacogenetics has focussed on drug metabolizing enzymes. Firstly, Drug metabolizing enzymes may vary more in function and expression than most other pharmacokinetic or pharmacodynamic targets Secondly, If the elimination of drug depends on one enzyme, then the drug level is a very specific in vivo marker of the enzyme in question. AUC & steady-state concentration of the drug is a very specific marker of drug elimination Pharmacogenetic testing by either: - Phenotype testing Metabolic ratio = Drug unchanged Metabolite Poor Metabolizer: Metabolic ratio higher than normal Extensive Metabolizer: metabolic ratios below normal values - Genotype testing by microarray