Cytochrome P450-mediated metabolism in brain: functional roles and their implications

1. Introduction

2. Human brain cytochromes

P450

3. Brain P450 enzymes in

inflammatory response

4. Summary and conclusions

5. Expert opinion

Review

Cytochrome P450-mediatedmetabolism in brain: functionalroles and their implicationsVijayalakshmi Ravindranath† & Henry W Strobel†Indian Institute of Science, Centre for Neuroscience, Bangalore, India

Introduction: Cytochromes P450 (P450) and associated monooxygenases are a

family of heme proteins involved in metabolism of endogenous compounds

(arachidonic acid, eicosanoids and prostaglandins) as also xenobiotics

including drugs and environmental chemicals. Liver is the major organ

involved in P450-mediated metabolism and hepatic enzymes have been

characterized. Extrahepatic organs, such as lung, kidney and brain have the

capability for biotransformation through P450 enzymes. Brain, including

human brain, expresses P450 enzymes that metabolize xenobiotics and

endogenous compounds.

Areas covered: Anoverviewof P450-mediatedmetabolism inbrain is presented

focusing on distinct differences seen in expression of P450 enzymes, generation

of unique P450 enzymes in brain through alternate splicing and their conse-

quences in terms of metabolism of psychoactive drugs and inflammatory

prompts, such as leukotrienes, thus modulating inflammatory response.

Expert opinion: The brain possesses unique P450s that metabolize drugs and

endogenous compounds through pathways that are markedly different

from that seen in liver indicating that extrapolation directly from liver to

brain is not appropriate. It is therefore necessary to characterize the unique

brain P450s and their ability to metabolize xenobiotics and endogenous

compounds to better understand the functions of this important class of

enzymes in brain, especially human brain.

Keywords: CNS, drug metabolism, inflammation, leukotrienes, psychoactive drugs

Expert Opin. Drug Metab. Toxicol. (2013) 9(5):551-558

1. Introduction

Cytochromes P450 (E.C. 1.14.14.1; P450) and associated monooxygenases [1] existin multiple forms having distinct yet overlapping substrate specificities for drugsand endogenous compounds. P450 enzymes have been classified based on theprimary amino acid sequence. According to this system of classification, twoP450 enzymes that have less than 40% amino acid similarity belong to separategene families, while those that have more than 60% similarity are assigned to thesame gene subfamily. The nomenclature system recommends the use ofthe italicized root symbol ‘CYP’ for human, ‘CYP’ for other animals and ‘cyp’ formouse representing cytochrome P450, followed by arabic number denoting thefamily. Next to that is a letter designating the sub-family (when two or more exist)and an arabic numerical representing the individual gene within the subfamily. The‘CYP’ tag is used for the genes and the proteins are named P450. On the basis ofthis, 36 gene families have been described of which 14 families exist inmammals [2,3]. This nomenclature is followed in the subsequent discussion.

Cytochromes P450 are well known for their ability to metabolize therapeuticdrugs, inactivating many but activating some for their therapeutic effect, fromextensive work done using liver tissue, microsomes or P450 isolated or purified

10.1517/17425255.2013.759208 © 2013 Informa UK, Ltd. ISSN 1742-5255, e-ISSN 1744-7607 551All rights reserved: reproduction in whole or in part not permitted

Exp

ert O

pin.

Dru

g M

etab

. Tox

icol

. Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f L

imer

ick

on 0

4/16

/13

For

pers

onal

use

onl

y.

from liver tissue. While P450 families 1, 2, 3 and 4 have beenshown to catalyze the metabolism of therapeutic drugs to agreater or lesser extent, two human P450 enzymes,CYP2D6 and CYP3A4 stand out above others for their abilityto catalyze the metabolism of the greatest number ofpharmacologically active compounds [4]. Human CYP1Aand CYP4F stand out as the best known examples for thewide range of polycyclic hydrocarbons [5] and eicosanoids/prostaglandins/long chain fatty acids [6], respectively,metabolized in brain [7].The cytochromes P450 are distributed differently in each

tissue and are dependent on species as well [8]. Therefore, thereis no certain association of catalytic activity with sequencehomology across species or tissue. Equally interestingly, wheremembers of the P450 superfamily have been crystallized fromdifferent species, the size and shape of the active site variesquite considerably. The sequence, active site shape, regulationof substrate specificity and the ‘role/roles’ of P450 enzymes indrug metabolism have been characterized [9]. This has resultedin enhanced knowledge of the role of cytochromesP450-mediated activities in the regulation of endogenouspathways involving eicosanoids/prostaglandin signaling [9,10],biosynthesis of cholesterol and hormones; in addition to theirwell-known role in the activation/inactivation of xenobioticsincluding drugs.Hepatic P450s remain the best characterized, since liver is

the major site of drug metabolism. Several hepatic P450sfrom rodent and human liver have been studied extensivelyand the recombinant proteins are used in drug discoveryresearch. Although the amount of P450 present in extrahepaticorgans, such as the lung, kidney and brain is a fraction of thatin the liver [11], their importance was realized after the discov-ery that the P450 enzymes were localized within specific cellsin these organs rendering these cells with significant capabilityto carry out P450-mediated metabolism. Thus, minorpathways of P450-mediated biotransformation can produce

significant effects if they take place at the site of action [12,13].Therefore, P450-mediated metabolism selectively in specificcells could potentially have long-range implications both interms of bioactivation of xenobiotics to toxic metabolites andmetabolism of drugs to active and inactive metabolites. Thebioactivation of ipomeanol, a naturally occurring toxic furanin fungus-infected sweet potato, in situ, in the Clara cells inthe lungs leading to potent pulmonary toxicity [14] remainsan outstanding example. In this study, Boyd demonstratedthat P450 metabolized ipomeanol in the lung to toxicelectrophilic metabolite(s) that bound covalently to cellularmacromolecules selectively in Clara cells leading to pulmonarytoxicity. Normally, hepatic P450-mediated hydroxylation ofdrugs leads to formation of hydrophilic metabolites, whichare excreted by the kidney. However, if the more hydrophilichydroxylated metabolites are formed in the brain, it wouldresult in a longer half-life in the brain due to the presence ofthe blood--CSF (cerebrospinal fluid) barrier [15,16].

Sasame et al. demonstrated the presence of P450 in ratbrain [17] for the first time, when they showed that the levelswere 1/30th of that in the liver. Subsequently, several investi-gators went on to demonstrate the presence of severalP450 enzymes, such as P4502B1/B2 [18], P4501A1/1A2 [5],P4502D [19] and P4502E [20] in rodent brain and also theirability to catalyze the biotransformation of a variety of drugsand other xenobiotics. The presence of P450 in human brainwas first demonstrated in 1990 [21] and Ravindranath and col-laborators went on to establish that several P450 enzymes,such as P4501A1 [5], P4502B [22], P4502D [23], P4502E [24]

and P4503A4 [25] were present in human brain with theirlocalization predominantly in the neurons and also purifiedseveral P450 enzymes from a human brain [26,27]. Thesestudies formally established the presence of P450 in the brain,including human brain, its ability to metabolize variety ofsubstrates and the induction of specific P450 enzymes bydrugs and chemicals, such as phenobarbital, ethanol,etc. [20,28,29]. More recently, several distinct properties of brainP450 have been described. These include generation ofunique P450 enzymes in the brain, but not in the liver byalternate splicing [13,30], differential distribution of P450enzymes [12] leading to altered metabolite profile in the brainand the ability of CYP4F enzymes that normally mediate themetabolism of endogenous compounds to metabolize psycho-active drugs. The implication of these discoveries is describedin detail in the following section.

2. Human brain cytochromes P450

Elegant studies on the localization of P450 enzymes in humanbrain using in situ hybridization and immunohistochemistryhave shown the predominant localization of P450 mRNAand protein in the neurons. Thus, CYP1A1 [5], 2D6 [23] and3A4 [25] are localized in cortical neurons, Purkinje and granulecells in the cerebellum, pyramidal neurons of CA1, CA2 andCA3 areas of hippocampus and reticular neurons in brain

Article highlights.

. Brain is actively involved in P450-mediated metabolismof xenobiotics and endogenous compounds.

. Several P450 enzymes, such as P4501A1, P4502B,P4502E1, P4502D6, P4503A4 and 4F11 are expressed inhuman brain, and are predominantly localizedin neurons.

. Human brain generates unique P450s with distinctfunctional properties through alternate splicing that arenot expressed in other organs.

. P450 enzymes are differentially expressed in brain andliver resulting in unique biotransformation of drugs notseen in liver.

. There is a need to characterize human brainP450 enzymes, specifically those generated throughalternate splicing.

This box summarizes key points contained in the article.

V. Ravindranath & H. W. Strobel

552 Expert Opin. Drug Metab. Toxicol. (2013) 9(5)

Exp

ert O

pin.

Dru

g M

etab

. Tox

icol

. Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f L

imer

ick

on 0

4/16

/13

For

pers

onal

use

onl

y.

stem. Presence of P450 was also seen in spinal cord of bothrodent and humans [31]. The distinct localization ofP450 enzymes in the neurons raises the possibility that metab-olism of psychoactive drugs that act on the brain could occurat site of action of the drugs, namely the neurons.

The presence and activity of CYP3A4 and other family mem-bers, CYP3A43 and CYP3A5, have been noted and described inhuman brain [12,32]. These P450 isoforms, especially CYP3A4,show the same broad range of substrate activity seen in humanliver and gut [33]. Table 1 shows the wide range of drugs, whichare metabolized by CYP3A4. This list includes specifically thoseCYP3A4 substrates, which are active in onemanner or another inthe brain. The list of all CYP3A4 substrates is much greaterindeed comprising greater than 50% of the drugs metabolizedby P450-dependent drug metabolism system.

Cytochrome P450 family 2 is represented in human brainsignificantly by CYP2D6 found in all regions of thebrain [23,34]. CYP2D6 is reported to account for 20 to 30%of the metabolism of therapeutic compounds. Table 2 liststhe range of brain-active drugs metabolized by CYP2D6. Aswith Table 1, Table 2 focuses on drugs affecting brain functionwhile the total list of CYP2D substrate is larger. A comparisonof Tables 1 and 2 reveals a considerable overlap of substratespecificities for brain-active drugs.

The second CYP2 subfamily that is of interest is CYP2E1.CYP2E1 is constitutively expressed in human brain and isof special interest for drug metabolism concerns since itmetabolizes ethanol, a ‘recreational’ drug of choice [35].Chronic alcohol use induces an increase in CYP2E1 proteinlevels [24,36] which is prominently expressed in neurons ofthe cortex, hippocampus and substantia nigra [24,37]. Caffeine,a stimulant, is also metabolized by CYP2E in liver. Othersubstrates of hepatic CYP2E1, which have a primary effectin the brain are listed in Table 3.

This list of substrates for hepatic CYP enzymes has beenabbreviated for emphasis on brain active substrates and hasbeen done at the expense of many other CYP enzymes presentin brain which can metabolize drugs but at lower rate or affin-ity for these substrates than the major forms described in thissection. While all of these forms are important and may play acritical though yet unrecognized role in the metabolism of aspecific substrate, the major forms described comprise mostof the metabolism of drugs in brain. This seems a fair, thoughperhaps ungenerous generalization.

2.1 Differential distribution of hepatic and human

brain P450 enzymesAs seen from above, while the hepatic metabolism of thedrugs that act on brain has been characterized well, little isknown about the metabolism of drugs in situ, in the brain.In order to investigate if brain P450-mediated biotransforma-tion could impact pharmacodynamics of the drug at the siteof action, the authors studied the metabolism of the anxiolyticdrug, alprazolam (ALP), which is metabolized to the a- and4-hydroxylated forms by liver P450. Of these, a-hydroxyalprazolam (a-OH ALP) exhibits ~77% of the intrinsic phar-macological activity, while 4-hydroxy alprazolam (4-OHALP) has only about 14%. Since the plasma levels of thehydroxylated ALP are substantially low in patients, theywere not considered to play a role in the anxiolytic effects ofALP, which are mediated in the brain. Studies from theauthors’ laboratory using rat and human brain showed thata-OH ALP is formed in relatively far higher amounts thanin the liver. Thus, 4-OH ALP formed in rat brain is 3.2%of the concentration in liver but a-OH ALP concentrationis 75% of the corresponding level in the liver. This indicated adifference in the metabolite profiles between the brain and liver,wherein 4-OHALP is themajormetabolite in liver whilea-OHALP is the major metabolite in brain [25]. Hydroxylatedmetabo-lites of ALP can be formed directly at the site of action in thebrain where they can exert prolonged pharmacological actionsince hydroxylated metabolites would have longer half-life dueto reduced clearance from brain. Using recombinant P450s, itwas found that CYP3A43 metabolizes ALP to both a-OHALP and 4-OH ALP, while CYP3A4-mediated metabolismpredominantly leads to the formation of the inactive metabolite,4-OHALP [12]. Very small amounts of a-OH ALP is formed inthe liver sinceCYP3A43 is expressed in very low amounts in liverwhileCYP3A4 is the predominant enzyme and 4-OHALP is themajor metabolite formed. When the expression of these geneswas examined in human brain, it was found that CYP3A43 isexpressed robustly in human brain and in higher levels in someindividuals compared to CYP3A4 [12]. Consequently in humanbrain, a-OH ALP is formed in substantial amounts indicatingthat metabolism of ALP to pharmacologically active metaboliteoccurs in the brain potentially increasing the half-life of thedrug at the site of action [12].

The above observations point to the fact that pharmacody-namic profile of metabolites could differ substantially between

Table 1. Examples of brain-active drugs metabolized

by CYP3A4.

Antidepressants DextromethorphanCitalopramClomipramineVenlafaxineAmitriptyline

Antipsychotic agents AripiprazoleHaloperidolCodeine

Analgesics EthylmorphineMethadoneBuprenorphine

Sedative agents AlprazolamMidazolamDiazepamNordazepamZolpidem

Anticonvulsant Carbamazepine

Selected from KEGG website of CYP 3A4 substrates http://www.genome.jp/

dbget-bin/get_linkdb?-t+4+hsa:1576.

Cytochrome P450-mediated metabolism in brain: functional roles and their implications

Expert Opin. Drug Metab. Toxicol. (2013) 9(5) 553

Exp

ert O

pin.

Dru

g M

etab

. Tox

icol

. Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f L

imer

ick

on 0

4/16

/13

For

pers

onal

use

onl

y.

http://www.genome.jp/dbget-bin/get_linkdb?-t+4+hsa:1576


organs, such as liver and brain because of the varied expressionof P450 enzymes across tissues. Further, because of its lowexpression in liver, CYP3A43 has been thought to haveinsignificant effect on drug metabolism. However, it hassubstantial presence in the brain [20] and could potentiallyplay an important role in generating pharmacologically activemetabolites with increased half-lives. Thus, direct extrapola-tion of information on drug metabolism from the liver tothe brain could lead to erroneous conclusions.

2.2 Brain-specific alternate splicing and novel

P450 isoformsAn interesting feature of the studies on CYP1A1 in humanbrain was the discovery of an alternate spliced CYP1A1 withdeletion of 87bp of exon 6 [5]. Out of a total of 25 human

brain samples examined, exon 6 del CYP1A1 was seen in allthe samples except one, which expressed the wild-typeCYP1A1. Of course, the samples were from south of Indiaand need to be examined in other population groups, sinceexpression of CYP genes is known to vary across populations.Further, this splice variant was present only in brain but notin liver, kidney, lung and heart from the same individual indi-cating brain-specific splicing events. Nervous system has apropensity to generate alternately spliced forms [38] and thisis well exemplified by the diversity seen in receptor subtypesgenerated through alternate splicing. The P450 enzymes alsogenerate diverse proteins in the human brain through alter-nate splicing, potentially leading to novel biotransformationthat may not be seen in the liver [39]. The generation ofalternate spliced RNA may not be related to differences ingenomic sequence and could be regulated by spliceosomalcomplexes and RNA binding proteins, which are yet to bewell understood.

The alternate spliced CYP1A1 with deletion of 87bp ofexon 6 results in a functional enzyme that is quite differentfunctionally from the wild-type CYP1A1 [30]. Wild-typeCYP1A1 metabolizes polycyclic aromatic hydrocarbons, suchas benzo(a)pyrene and 3-methyl cholanthrene to genotoxicmetabolites that covalently bind to DNA leading to carcino-genesis, while the alternate spliced variant does not form anyDNA-binding metabolites [5]. Thus, in individuals expressingthis variant, bioactivation of polycyclic aromatic hydrocar-bons would not occur to a substantial extent. The structuralmodeling of the exon 6 del CYP1A1 [5] indicates alterationin the substrate access channel [40] such that there is lessrestriction of access and an altered angle of approach as com-pared with wild-type CYP1A1. A broader substrate accesschannel would permit free movement of the substrate withinthe access channel. This could be the reason exon 6 delCYP1A1 does not bioactivate benz(a)pyrene to DNA bindingadducts and that it also efficiently metabolizes pentoxy andbenzyloxy resorufin, which are classical substrates for anotherP450 enzyme, namely CYP2B.

Similarly, a splice variant was also found in CYP2D7 (apseudogene) wherein a non-functional enzyme was convertedto a functional form through the partial inclusion of intron 6.Interestingly, using recombinant alternately spliced P4502D7it has been shown that it metabolizes codeine (prodrug)mostly to morphine (active drug) unlike the recombinanthepatic CYP2D6, which metabolizes codeine to predomi-nantly to norcodeine and very small amounts of morphine [13].Thus, the alternate splicing of CYP2D7 that occurs only inthe human brain but not in the liver generates a functionalenzyme that completely alters the metabolite profile ofcodeine and generates the active drug morphine in the brain,at the site of action [5,13]. The human brain thus generatesunique P450 enzymes through alternate splicing in a histio-specific manner and these unique enzymes can modify drugaction or genotoxic potential of procarcinogens by alteringtheir biotransformation.


by CYP2D6.

Tricyclic antidepressants NortriptylineMianseronAmitriptylineDesipramineClomipramineThioridazine

Antipsychotic agents RisperidoneThioridazineAripiprazolePerphenazinePhenothiazineHaloperidolChlorpromazine

Serotonin reuptake inhibitors ParoxetineFluvoxamineFluoxetineVenlafaxine

Opioids TramadolEthylmorphine

Antihypertensive agent DebrisoquineCentral nervous system stimulant AmphetamineAntitussive agent DextromethorphanAnalgesic Codeine

Selected from KEGG website of CYP2D6 substrates http://www.genome.jp/



by CYP2E1.

Anesthetic agents HalothaneEnfluraneMethoxyfluraneIsoflurane

Inebriant AlcoholAnalgesic AcetaminophenMuscle relaxant ChlorzoxazoneStimulant Caffeine

Selected from KEGG website of CYP2E1 substrates http://www.genome.jp/




Exp

ert O

pin.

Dru

g M

etab

. Tox

icol

. Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f L

imer

ick

on 0

4/16

/13

For

pers

onal

use

onl

y.





3. Brain P450 enzymes in inflammatoryresponse

The authors now turn to a specific focus onwhat was heretoforeone of those ‘unrecognized’ CYP players, the CYP4F family. Itis the authors’ opinion that for the human brain, this familyand specifically CYP4F11 has a significant role in brain drugmetabolism and in the regulation of inflammation in brain.

CYP4F isoforms are uniquely distributed among the organsof rats, mice and humans. In the rodent species, all forms ofCYP4F are present in at least some level in all the tissues.For instance, in rat the predominant form in liver and kidneyis CYP4F1 whereas CYP4F5 is present in only small amounts.CYP4F6 is the predominant form in lung with CYP4F4expressed in low levels and CYP4F1 essentially absent [41].A similar tissue-specific distribution of cyp4F form is seenfor mouse tissues as well. The CYP4F subfamily in humansconsists of seven members distributed generally throughoutthe body. CYP4F2 has been shown to be expressed in liver,kidney, skin and other tissues [7,42]. CYP4F3 is expressedstrongly in liver and CYP4F8 is expressed in seminal vesiclesas well as in hair follicles, sweat glands, proximal renal tubulesand the epithelial of gut and urinary tract [43]. CYP4F11 hasbeen reported to be present in human liver, kidney, heart,brain and skeletal muscle [44], while CYP4F12 has been shownin liver, kidney, seminal vesicles, small intestine, prostate glandand ovarian follicles [45-47]. CYP4F22 has been demonstratedat lower expression in placenta, bone marrow, small intestine,liver, skeletal muscle, brain and kidney. It has been implicatedin causing lamellar ichthyosis type 3 [48]. The distribution ofCYP4F forms in rat, mouse and human brains are comparedin Table 4. About 50% of CYP4F expression in rat brain isCYP4F6 and about 37% is CYP4F1. The remaining 14% isdivided between CYP4F4 and CYP4F5. In mouse brain cortexcyp4F15 is expressed at the highest level and cyp4F13 andcyp4F14 with close but lower expression levels. cyp4F16and cyp4F18 are expressed at much lower levels [49,50].A remarkably different picture is seen in human brain (cortex)where CYP4F11 is expressed at 88% in vast excess over otherforms. CYP4F3A is expressed at 8%, 4F12 at 3%, 4F2 at < 1%and 4F3B at 1%. Thus, for human brain CYP4F-dependentmetabolism, CYP4F11 seems the major catalyst.

The cytochromes P450 4F subfamily was identified by itsability to catalyze the w-hydroxylation of leukotriene B4 byKikuta et al. in 1993 [50]. While all the members of the CYP4Fsubfamily catalyze the hydroxylation of leukotriene B4 andarachidonic acid, some do so with greater activity than others.For instance,CYP4F3A has a high Vmax value and lowKm valuefor leukotriene B4 whereas CYP4F3B has a lower Vmax andhigher Km for leukotriene B4 but CYP4F3B has greater activitywith arachidonic acid as a substrate. CYP4F11, on the otherhand, has low activity toward leukotriene B4 and arachidonicacid. However, of all the human CYP4Fs, CYP4F11 has strongactivities with drug substrates [51].

Erythromycin is utilized as a marker drug for CYP3A4activity. Yet CYP4F11 has an equivalent activity for thissubstrate. The list of substrates in Table 5 represents a widerange of therapeutic agents likely to be used in brain injuryor brain crisis cases. CYP4F has marked activity towardmost of the listed drugs. This was an unexpected finding giventhat the rest of P450 enzymes in humans as well as other spe-cies have in general much better biotransformation capabilitywith eicosanoids, prostaglandins and some long chain fattyacids than they exhibit toward drugs. CYP4F11 seems anexception to this rule.

In mouse models of inflammation, such as those seen aftersystemic administration of LPS [49], or infection with Japaneseencephalitis virus [52], cyp4Fs, particularly 4F15, play a veryimportant role in the metabolism of the potent inflammatorymolecule, leukotriene B4 to 20-hydroxy leukotriene B4.

4. Summary and conclusions

In the last three decades, it has been irrevocably demonstratedthat P450 enzymes are present in both rodent and humanbrains in adequate amount and they posses the capability tobiotransform a variety of drugs, especially psychoactive drugsas well as endogenous compounds, which are important forregulation of inflammatory response. The P450 enzymes arelocalized preferentially in the neuronal cells in different brainregions and therefore can potentially impact drug action andinflammatory responses. It is also known that the brainP450 enzymes are localized in endoplasmic reticulum, mito-chondria as well as the plasma membrane. The human brainalso expresses unique P450 enzymes that are generatedthrough alternate splicing and these are of special interestsince they would directly impact the biotransformation ofdrugs, in situ at the site of action. The weaknesses in theresearch done so far mainly reside in the fact that a large num-ber of studies have used antibodies to P450 enzymes that maypotentially lack specificity. Thus, the identification andquantitation of different P450 enzymes in the brain usingimmunoblotting and immunohistochemistry need to bere-examined. There is divergence between mRNA and proteinlevels of P450 enzymes. We see large amounts of mRNAspecies and it remains to be examined whether these translateinto functional P450 enzymes. Further, the informationavailable on human brain is rather limited and future researchneeds to be focused, since this knowledge could impact futuredrug development and deeper understanding of inflammatoryresponse in brain.

The ultimate goal in this field would be to identify themajority of P450 enzymes that are expressed in human brainand understand their functional properties through cloningand expression. Once this information is available, we willbe in a position to utilize this knowledge in drug discoveryand development so that we are able to predict the biotrans-formation pathways that potentially could take place in thebrain. Of course, this requires access to human brain tissue



Exp

ert O

pin.

Dru

g M

etab

. Tox

icol

. Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f L

imer

ick

on 0

4/16

/13

For

pers

onal

use

onl

y.

that can be obtained from postmortem samples representingdifferent anatomical nuclei in the brain. An additional lacunais the lack of complete understanding of the regulation ofalternate splicing, which precludes us from predicting theunique P450 enzymes that could be expressed in the humanbrain using in silicomethods. The generation of antibodies spe-cific to these unique P450 enzymes is also a time-consumingfactor since the availability of commercial antibodies is limited.The field is moving rather slowly and it is hoped that more

interest would be generated to improve the momentum in thecoming years. The current need obviously is to identify andfunctionally characterize the unique P450 enzymes in thebrain and also study their contribution to the biotransforma-tion of drugs and endogenous substrates. This is also excitingsince a deeper understanding of the unique P450 enzymesgenerated in brain through alternate splicing would alsoprovide an insight into the diversity of proteins that aresynthesized through alternate splicing mechanisms.

5. Expert opinion

The focus of drug metabolism research, particularly new drugdiscovery has been on hepatic P450 enzymes. This is justifi-able since liver is the major site of drug action. However,metabolism in the target organ within specialized cells wouldhave tremendous impact on the pharmacodynamics of the

drug and thereby on the therapeutic outcome. Metabolismwithin the central nervous system is an excellent illustration ofthe case in point as can be discerned from above. Not only isthe brain an organ that is actively involved in the metabolismof a variety of foreign compounds including drugs but alsoendogenous compounds, which impact on physiologicalresponses, such as inflammation. The P450 enzymes arelocalized in specialized cells and the distribution of the P450enzymes are highly heterogeneous pointing to their potentialimpact on drug metabolism and therapeutic outcome. Addingto this complexity is the fact that the human brain generatesunique P450s that are not expressed in other organs, throughalternate splicing, thus generating enzymes with distinct func-tional properties. These enzymes can alter the pharmacodynam-ics of drugs through novel biotransformation pathways that arenot known to occur in the liver. Histio-specific isoforms ofP450 enzymes that are generated by alternate splicing couldpotentially mediate novel biotransformation of drugs and pro-drugs within the brain, to generate active drugs and metabolite(s) that can have tremendous impact on drug action. Therefore,preclinical drug discovery based on hepatic P450-mediatedbiotransformation could potentially disregard important drugmetabolism pathways that could occur in the brain, the site ofaction of several psychoactive drugs and thus impact drug action.

One cannot overemphasize the need for study of biotrans-formation of drugs mediated by P450 enzymes in the brain,particularly, the human brain and understand the downstreamimpact in terms of drug action. As a next step, the characteri-zation of the unique P450 enzymes generated throughalternate splicing in the brain needs to be discerned. TheCNS expression of distinctive P450 enzymes that are notpresent in significant amounts in the liver also needs to belooked at. Further, the differential distribution of P450enzymes in brain and liver also emphasizes the need for charac-terizing the composition and distribution of brain P450enzymes. A detailed study of the above aspects would helpprovide a more comprehensive picture of the drug metabolismin brain and pave the way for effective pharmacologicalintervention, which is the mainstay of several neuropsychiatricillnesses requiring prolonged pharmacotherapy.

The characterization of brain P450 enzymes is importantnot only in terms of metabolism of drugs that act on the brainbut also to understand the metabolism of endogenous com-pounds, such as the eicosanoids which have substantial impacton inflammatory response. This is very important consideringthat inflammatory response in the brain has been shown toplay a role not only in infections of the central nervous systembut also in neurodegenerative disorders and more recently inpsychiatric illnesses.

What are the limitations for studying P450 in the humanbrain? The availability of postmortem human brain tissue is diffi-cult, however brain banks that provide autopsy tissue for researchhave helped ease this difficulty. The low levels of brainP450 enzymes is limiting in some sense and drug metabolismassays are challenging to performusing humanbrainmicrosomes.

Table 4. Distribution of CYP4F forms in brain (percent

total 4Fs measured).

Form Rat (%) Form Mouse (%) Form Human (%)

4F1 36.9 4F13 29.3 4F2 < 14F4 8.1 4F14 20.9 4F3A 84F5 5.6 4F15 44.8 4F3B 14F6 49.4 4F16 3.8 4F8

4F18 1.2 4F11 884F12 34F22

Adapted from Kalsotra and Strobel [7], Sehgal et al. [49] and Bell and Strobel

(manuscript submitted).

Table 5. Examples of brain-active therapeutic drugs

metabolized by human CYP4F11.

Drug CYP4F11

Amitriptyline 28.46 ± 3.53Benzphetamine 253.3 ± 25.4Chlorpromazine 58.0 ± 6.30Imipramine 274.2 ± 8.16Pirenzepine 16.55 ± 2.33Theophylline 260.7 ± 8.69Verapamil 110.8 ± 6.92

Activities are shown in µmol/min/mg protein.

Adapted from Kalsotra et al. [51].



Exp

ert O

pin.

Dru

g M

etab

. Tox

icol

. Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f L

imer

ick

on 0

4/16

/13

For

pers

onal

use

onl

y.

However, cloning and overexpression, in vitro can helpovercome this constraint and further the understanding of thepharmacological consequences of biotransformation of drugsmediated by cytochrome P450 enzymes in the human brain.

Declaration of interest

The authors are supported by the NIH and Department ofScience & Technology, India.

BibliographyPapers of special note have been highlighted as

either of interest (�) or of considerable interest(��) to readers.

1. Halpert JR, Domanski TL, Adali O,

et al. Structure-function of cytochromes

P450 and flavin-containing

monooxygenases: implications for drug

metabolism. Drug Metab Dispos

1998;26(12):1223-31

2. Nelson DR, Kamataki T, Waxman DJ,

et al. The P450 superfamily: update on

new sequences, gene mapping, accession

numbers, early trivial names of enzymes,

and nomenclature. DNA Cell Biol

1993;12(1):1-51. This paper provides a comprehensive

update on the P450 nomenclature.

3. Ortiz de Montellano PR. Cytochrome

P450: structure, mechanism, and

biochemistry. 3rd edition. Kluwer

Academic/Plenum Press, New York; 2005

4. Eichelbaum M, Burk O. CYP3A genetics

in drug metabolism. Nat Med

2001;7(3):285-7

5. Chinta SJ, Kommaddi RP, Turman CM,

et al. Constitutive expression and

localization of cytochrome P-450 1A1 in

rat and human brain: presence of a splice

variant form in human brain.

J Neurochem 2005;93(3):724-36. Provides evidence for the presence of

splice variant of P4501A1 in

human brain.

6. Imig JD, Simpkins AN, Renic M,

Harder DR. Cytochrome

P450 eicosanoids and cerebral vascular

function. Expert Rev Mol Med

2011;13:e7

7. Kalsotra A, Strobel HW. Cytochrome

P450 4F subfamily: at the crossroads of

eicosanoid and drug metabolism.

Pharmacol Ther 2006;112(3):589-611

8. Machala M, Soucek P, Neca J, et al.

Inter-species comparisons of hepatic

cytochrome P450 enzyme levels in male

ruminants. Arch Toxicol

2003;77(10):555-60

9. Dong D, Wu B. Substrate selectivity of

drug-metabolizing cytochrome P450s

predicted from crystal structures and

in silico modeling. Drug Metab Rev

2012;44(1):1-17

10. Otyepka M, Berka K, Anzenbacher P. Is

there a relationship between the substrate

preferences and structural flexibility of

cytochromes P450? Curr Drug Metab

2012;13(2):130-42

11. Pavek P, Dvorak Z. Xenobiotic-induced

transcriptional regulation of xenobiotic

metabolizing enzymes of the cytochrome

P450 superfamily in human extrahepatic

tissues. Curr Drug Metab 2008;9(2):129-43

12. Agarwal V, Kommaddi RP, Valli K,

et al. Drug metabolism in human brain:

high levels of cytochrome P4503A43 in

brain and metabolism of anti-anxiety

drug alprazolam to its active metabolite.

PLoS One 2008;3(6):e2337.. The paper demonstrates how

differential expression of

P450 enzymes impacts on drug

metabolism, in situ in the

human brain.

13. Pai HV, Kommaddi RP, Chinta SJ,

et al. A frameshift mutation and alternate

splicing in human brain generate a

functional form of the pseudogene

cytochrome P4502D7 that demethylates

codeine to morphine. J Biol Chem

2004;279(26):27383-9.. Provides vital evidence for alternate

splicing of P450 enzyme in

human brain.

14. Boyd MR. Evidence for the Clara cell as

a site of cytochrome P450-dependent

mixed-function oxidase activity in lung.

Nature 1977;269(5630):713-15

15. Dutheil F, Jacob A, Dauchy S, et al.

ABC transporters and cytochromes

P450 in the human central nervous

system: influence on brain

pharmacokinetics and contribution to

neurodegenerative disorders. Expert Opin

Drug Metab Toxicol 2010;6(10):1161-74

16. Ghersi-Egea JF, Monkkonen KS,

Schmitt C, et al. Blood-brain interfaces

and cerebral drug bioavailability.

Rev Neurol (Paris)

2009;165(12):1029-38

17. Sasame HA, Ames MM, Nelson SD.

Cytochrome P-450 and NADPH

cytochrome c reductase in rat brain:

formation of catechols and reactive

catechol metabolites. Biochem Biophys

Res Commun 1977;78(3):919-26. The first evidence for presence of

cytochrome P450 in brain.

18. Anandatheerthavarada HK, Shankar SK,

Ravindranath V. Rat brain cytochromes

P-450: catalytic, immunochemical

properties and inducibility of multiple

forms. Brain Res 1990;536(1-2):339-43

19. Kawashima H, Strobel HW.

cDNA cloning of three new forms of rat

brain cytochrome P450 belonging to the

CYP4F subfamily. Biochem Biophys

Res Commun 1995;217(3):1137-44

20. Anandatheerthavarada HK, Shankar SK,

Bhamre S, et al. Induction of brain

cytochrome P-450IIE1 by chronic

ethanol treatment. Brain Res

1993;601(1-2):279-85

21. Ravindranath V,

Anandatheerthavarada HK, Shankar SK.

NADPH cytochrome P-450 reductase in

rat, mouse and human brain.

Biochem Pharmacol 1990;39(6):1013-18

22. Ravindranath V,

Anandatheerthavarada HK, Shankar SK.

Xenobiotic metabolism in human

brain--presence of cytochrome P-450 and

associated mono-oxygenases. Brain Res

1989;496(1-2):331-5.. First evidence of P450-mediated

xenobiotic metabolism in

human brain.

23. Chinta SJ, Pai HV, Upadhya SC, et al.

Constitutive expression and localization

of the major drug metabolizing enzyme,

cytochrome P4502D in human brain.

Brain Res Mol Brain Res

2002;103(1-2):49-61

24. Upadhya SC, Tirumalai PS, Boyd MR,

et al. Cytochrome P4502E (CYP2E) in

brain: constitutive expression, induction

by ethanol and localization by

fluorescence in situ hybridization.

Arch Biochem Biophys

2000;373(1):23-34

25. Pai HV, Upadhya SC, Chinta SJ, et al.

Differential metabolism of alprazolam by

liver and brain cytochrome (P4503A) to



Exp

ert O

pin.

Dru

g M

etab

. Tox

icol

. Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f L

imer

ick

on 0

4/16

/13

For

pers

onal

use

onl

y.

pharmacologically active metabolite.

Pharmacogenomics J 2002;2(4):243-58

26. Bhamre S, Anandatheerathavarada HK,

Shankar SK, et al. Purification of

multiple forms of cytochrome P450 from

a human brain and reconstitution of

catalytic activities. Arch Biochem Biophys

1993;301(2):251-5

27. Bhamre S, Anandatheerthavarada HK,

Shankar SK, Ravindranath V.

Microsomal cytochrome P450 in human

brain regions. Biochem Pharmacol

1992;44(6):1223-5

28. Ferguson CS, Tyndale RF. Cytochrome

P450 enzymes in the brain: emerging

evidence of biological significance.

Trends Pharmacol Sci

2011;32(12):708-14

29. Anandatheerthavarada HK, Boyd MR,

Ravindranath V. Characterization of a

phenobarbital-inducible cytochrome

P-450, NADPH-cytochrome P-

450 reductase and reconstituted

cytochrome P-450 mono-oxygenase

system from rat brain. Evidence for

constitutive presence in rat and human

brain. Biochem J 1992;288(Pt 2):483-8

30. Kommaddi RP, Turman CM,

Moorthy B, et al. An alternatively spliced

cytochrome P4501A1 in human brain

fails to bioactivate polycyclic aromatic

hydrocarbons to DNA-reactive

metabolites. J Neurochem

2007;102(3):867-77

31. Bhagwat SV, Leelavathi BC, Shankar SK,

et al. Cytochrome P450 and associated

monooxygenase activities in the rat and

human spinal cord: induction,

immunological characterization and

immunocytochemical localization.

Neuroscience 1995;68(2):593-601

32. McFayden MC, Melvin WT,

Murray GI. Regional distribution of

individual forms of cytochrome

P450 mRNA in normal adult human

brain. Biochem Pharmacol

1998;55(6):825-30

33. Anzenbacher P, Anzenbacherova E.

Cytochromes P450 and metabolism of

xenobiotics. Cell Mol Life Sci

2001;58(5-6):737-47

34. Miksys S, Rao Y, Hoffmann E, et al.

Regional and cellular expression of

CYP2D6 in human brain: higher levels

in alcoholics. J Neurochem

2002;82(6):1376-87

35. Hipolito L, Sanchez MJ, Polache A,

Granero L. Brain metabolism of ethanol

and alcoholism: an update.

Curr Drug Metab 2007;8(7):716-27

36. Song BJ. Ethanol-inducible cytochrome

P450 (CYP2E1): biochemistry, molecular

biology and clinical relevance:

1996 update. Alcohol Clin Exp Res

1996;20(8 Suppl):138A-46A

37. Watts PM, Riedl AG, Douek DC, et al.

Co-localization of P450 enzymes in the

rat substantia nigra with tyrosine

hydroxylase. Neuroscience

1998;86(2):511-19

38. Grabowski PJ, Black DL. Alternative

RNA splicing in the nervous system.

Prog Neurobiol 2001;65(3):289-308

39. Turman CM, Hatley JM, Ryder DJ,

et al. Alternative splicing within the

human cytochrome P450 superfamily

with an emphasis on the brain: the

convolution continues. Expert Opin

Drug Metab Toxicol 2006;2(3):399-418

40. Cojocaru V, Winn PJ, Wade RC.

Multiple, ligand-dependent routes from

the active site of cytochrome P450 2C9.

Curr Drug Metab 2012;13(2):143-54

41. Kalsotra A, Anakk S, Boehme CL,

Strobel HW. Sexual dimorphism and

tissue specificity in the expression of

CYP4F forms in Sprague Dawley rats.

Drug Metab Dispos 2002;30(9):1022-8

42. Kikuta Y, Kusunose E, Kusunose M.

Characterization of human liver

leukotriene B(4) omega-hydroxylase P450

(CYP4F2). J Biochem

2000;127(6):1047-52

43. Stark K, Torma H, Cristea M, Oliw EH.

Expression of CYP4F8 (prostaglandin H

19-hydroxylase) in human epithelia and

prominent induction in epidermis of

psoriatic lesions. Arch Biochem Biophys

2003;409(1):188-96

44. Cui X, Nelson DR, Strobel HW.

A novel human cytochrome P450 4F

isoform (CYP4F11): cDNA cloning,

expression, and genomic structural

characterization. Genomics

2000;68(2):161-6

45. Bylund J, Bylund M, Oliw EH. cDna

cloning and expression of CYP4F12, a

novel human cytochrome P450.

Biochem Biophys Res Commun

2001;280(3):892-7

46. Hashizume T, Imaoka S, Hiroi T, et al.

cDNA cloning and expression of a novel

cytochrome p450 (cyp4f12) from human

small intestine. Biochem Biophys

Res Commun 2001;280(4):1135-41

47. Stark K, Schauer L, Sahlen GE, et al.

Expression of CYP4F12 in

gastrointestinal and urogenital epithelia.

Basic Clin Pharmacol Toxicol

2004;94(4):177-83

48. Lefevre C, Bouadjar B, Ferrand V, et al.

Mutations in a new cytochrome

P450 gene in lamellar ichthyosis type 3.

Hum Mol Genet 2006;15(5):767-76

49. Sehgal N, Agarwal V, Valli RK, et al.

Cytochrome P4504f, a potential

therapeutic target limiting

neuroinflammation. Biochem Pharmacol

2011;82(1):53-64. Role of brain P4504f enzymes in

reducing neuroinflammation.

50. Kikuta Y, Kusunose E, Endo K, et al.

A novel form of cytochrome

P-450 family 4 in human

polymorphonuclear leukocytes.

cDNA cloning and expression of

leukotriene B4 omega-hydroxylase.

J Biol Chem 1993;268(13):9376-80

51. Kalsotra A, Turman CM, Kikuta Y,

Strobel HW. Expression and

characterization of human cytochrome

P450 4F11: putative role in the

metabolism of therapeutic drugs and

eicosanoids. Toxicol Appl Pharmacol

2004;199(3):295-304.. Demonstrates that P450 enzymes that

are known to metabolize endogenous

compounds, such as eicasanoids can

also metabolize psychoactive drugs.

52. Sehgal N, Kumawat KL, Basu A,

Ravindranath V. Fenofibrate reduces

mortality and precludes neurological

deficits in survivors in murine model of

Japanese encephalitis viral infection.

PloS One 2012;7(4):e35427

AffiliationVijayalakshmi Ravindranath†1 &

Henry W Strobel2

†Author for correspondence1Indian Institute of Science,

Centre for Neuroscience,

C.V. Raman Avenue, Bangalore 560012, India

Tel: +91 80 2293 3433;

Fax: +91 80 2360 3323;

E-mail: [email protected] University of Texas Health Science Center

at Houston-Medical School,

Department of Biochemistry &

Molecular Biology,

Houston, TX 77030, USA



Exp

ert O

pin.

Dru

g M

etab

. Tox

icol

. Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f L

imer

ick

on 0

4/16

/13

For

pers

onal

use

onl

y.

Documents

Cytochrome P450-mediated metabolism in brain: functional roles and their implications