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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