EUROPEAN JOURNAL OF DRUG METABOLISM AND PHARMACOKINETICS 1999, Vol. 24, No.2, pp. 169-176

Effects of Plasmodium berghei infection on cytochromes P-450 2El and 3A2 K. UHU t , J.M. GRACE I, D.A. KOCISKOI, B.T. JENNINGS I, A.L. MITCHELL I and T.G. BREWER 2 JDepartment of Pharmacology, Walter Reed Army Institute ofResearch, Washington, DC, USA 2Armed Forces Research Institute ofMedical Science, Bangkok, Thailand "Present affiliation: US Food and Drug Administration, Rockville, MD, USA

Receivedfor publication: January 14, 1999

Keywords: Cytochrome PA50, liver microsomes, Sprague-Dawley rats, malaria, Western blotting, isozymes

SUMMARY Metabolism and disposition of most drugs used to treat malaria are substantially altered in malaria infection. Few data are available that specify effects of malaria infection on drug metabolism pathways in humans or animal model systems. In this report, studies were undertaken to determine the effect of Plasmodium berghei infection on cytochrome P-450 (CYP450) 2EI and 3A2-mediated metabolism and enzyme expression in rat liver microsomes. Malaria infection (MAL) resulted in significant decreases in total cytochrome PASO content (56%, P <0.05) and NADPH cytochrome PASO reductase activity (32%, P <0.05) as compared to control (CON) rats. Chlorzoxazone 4-hydroxylase activity (CYP2EI-mediated) showed no significant difference between CON and MAL microsomes while testosterone 6-~-hydroxylase activity (CYP3A2-mediated) was reduced by 41% (P <0.05) in MAL. Enzyme kinetic studies and immunoblot analysis indicate that the loss of activity for CYP3A2 in malaria infection is due to significantly decreased CYP3A2 protein expression. The altered expression of CYP450s in malaria infection should be taken into account when treating patients with malaria in order to minimize drug-drug interactions or toxicity.

INTRODUCTION Over 2.5 billion people in 103 countries worldwide are at risk of contracting malaria. Between 1 and 3 million deaths due to malaria occur each year, the majority of which are in children less than 5 years of age (1). No effective vaccine exists to date and protective drug Please send reprint requests to : Dr Kathleen Uhl, US Food and Drug Administration. Office of Clinical PharmacologylBiopharmaceutics, 9201 Corporate Blvd. HFD-880, Rockville. MD 20850. USA

therapy is not available or practical for many people. Most patients acutely ill with malaria receive therapy with one or more drugs which may have narrow therapeutic indices, a situation exacerbated by the need to use combination therapy for multiple drug resistant parasites. Some data available show dramatic, albeit poorly characterized, changes in the metabolism of antimalarial drugs during malaria infection (2-4). Better definition of the nature, extent, and especially the specific metabolic targets of disease-related changes in hepatic drug metabolism should diminish the risks of drug therapy.

170

Eur. J. Drug Metab. Pharmacokinet. 1999, No.2

Malaria, as well as other infectious states, alters the hepatic phase I (5-7) and, to a much lesser extent, phase II (8-10) drug biotransformation activity. With few exceptions (e.g. atovaquone), antimalarial drugs undergo extensive phase I and/or phase II metabolism. The majority of known phase I oxidations are by capacity limited systems that are more sensitive to changes in enzymatic activity as compared to the phase II 'conjugation' reactions. The in vivo phase I activity is related, in large part, to the aggregate tissue cytochrome P-450 (CYP450) content and activity. This activity can be quantitatively estimated by measurements of proteins, co-factors and the rates of probe substrate oxidation using standard in vitro techniques. The present studies involve an approach in which the activities of CYP450 2EI and 3A2 isozymes were determined in microsomes prepared from both control (CON) rats and those infected with the rodent parasite, Plasmodium berghei (MAL). CYP450 2El and 3A2 activities were determined using chlorzoxazone (CZ) and testosterone (TEST) as probe substrates, respectively. Immunoblot analysis was subsequently performed to quantitate differences in protein expression of CYP2El and 3A2 isoforms.

MATERIALS AND METHODS Chemicals Chlorzoxazone and 6-hydroxychlorzoxazone were purchased from Research Biochemicals Inc. (Natick, MA, USA). Testosterone, 6-~-hydroxytestosterone, cytochrome c, carbon monoxide, sodium dithionite, Tris acetate,Trizma base, bovine non-fat dried milk, KCl, EDTA, BHT, Dglucose-6-phosphate, ~-nicotinamide adenine dinucleotide phosphate (~-NADP), glucose-6-phosphate dehydrogenase, and glycerol were purchased from Sigma Chemical Co. (St Louis, MO, USA). Potassium pyrophosphate, and magnesium chloride hexahydrate were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Glycine was purchased from Pharmacia Biotech (San Francisco, CA, USA). SDS was purchased from Bio-Rad (Hercules, CA, USA). All solvents (HPLC grade) were purchased from Burdick and Jackson, Incorporated (Muskegon, MI, USA) and were degassed prior to use.

Instrumentation HPLC analyses were performed using a Hewlett-Packard 1050 HPLC system equipped with a diode array detector (Wilmington, DE, USA) and a Waters /lBondapak C I8 5

urn (4.6 x 250 mm) column (Milford, MA, USA). For chlorzoxazone determination, the mobile phase consisted of 70:30 acetonitrile:water with 0.05% phosphoric acid and a flow rate of 1.2 ml/min. For testosterone determination, the mobile phase consisted of a linear gradient of 70:30 to 90:10 MeOH:water (v/v) over 30 min. and a flow rate of 1.0 ml/min. Spectrophotometric analysis was performed with a Beckman DU-640 UV-Vis spectrophotometer (Mountain View, CA, USA). Animals Male Sprague-Dawley rats (6 weeks of age, 120 g) were purchased from Charles River Laboratories (Charles River, NY, USA). Animals were matched for age and weight and housed in pairs (n = 6 pairs). Animals were housed in well ventilated cages at a controlled temperature (24°C) with 12 h lightdark cycling. They were allowed to feed ad libitum on pelleted food (Agway Inc., e.G., Syracuse, NY, USA) and tap water. Malaria infection (MAL) of one animal of each pair was established by inoculation with saline-diluted blood from rats previously infected with the ANKA strain of the rodent parasite, Plasmodium berghei (0.2 rnl i.p.; 106 parasites/rnl) (11). The paired control (CON) rat was injected with 0.2 rnl sterile saline solution. Infection was followed and the level of parasitemia was quantitated by microscopic examination of Giemsa-stained thin blood smears. Infected animals consistently reached peak parasitemia between days 9-12 postinoculation, when animals were sacrificed for microsomal liver preparations.

Preparation of rat liver microsomes Individual rat livers were homogenized and fractionated by differential centrifugation as previously described (12) and the microsomal suspensions were stored in 0.10 M potassium phosphate buffer (pH 7.4) containing 20% glycerol at -80°C until needed. The microsomes prepared from each rat were not combined with microsomes from any other animal used in the study. Protein concentration, cytochrome P-450 content, and NADPH cytochrome c reductase (NADPH CYP450 reductase) activity were determined by the methods of Lowry, (13) Omura and Sato, (14) and Phillips and Langdon, (15) respectively.

Substrate turnover in microsomes Optimal conditions were established for each assay. Rat liver microsomes (0.1 mg microsomal protein) were preincubated for 3 min at 37°C in 0.1 M potassium phosphate

K. Uhl et al., Effects of Plasmodium berghei infection on cytochromes P-450 2El and 3A2

buffer (pH 7.4) containing a NADPH regenerating system (0.5 rom NADP, 10 roM glucose-6-phosphate, 1.0 IU/mi glucose-6-phosphate dehydrogenase, 5 roM magnesium chloride). The final incubation volume was 0.5 mi. Chlorzoxazone 6-hydroxylase activity was determined by the method of Peter et al. (16) Reactions were initiated by the addition of chlorzoxazone (0-500 umol/l) and incubated for 10 min. Reactions were terminated with the addition of 50 III 1:5 phosphoric acid:water. Testosterone 6-~-hydroxylase activity was determined by the method of Sonderfan et al. (17). Reactions were initiated by the addition of testosterone (0-1000 umol/l) and incubated for 30 min. Reactions were terminated with the addition of 50 III acetonitrile. The quantitation of each metabolite was determined by an external calibration curve using authentic standards.

Immunoblot techniques and densitometry SDS-PAGE was performed using a Bio-Rad MiniPROTEAN gel system (Hercules, CA, USA) and pre-cast 12% Tris-HCl gels (Bio-Rad). Rat liver microsomes were diluted to 5 ug/ml in running buffer, boiled for 5 min, and then 10 III loaded into each lane. After the gel was run, proteins were transferred onto a Bio-Rad Trans Blot PVDF membrane using a Hoefer TE 22 Mighty Small Transfer unit (Pharmacia Biotech, San Francisco, CA, USA). The membrane was then blocked by overnight incubation in milk at 4°C. Immunoblotting was done by first incubating with a 1/1000 dilution of primary antibody to the appropriate CYP450 (Gentest, Woburn, MA, USA) for 2 h at room temperature. After rinsing, a 1110,000 dilution of peroxidase-conjugated secondary antibody (Calbiochem-Novabiochem Corp., La Jolla, CA, USA) was added and gently shaken over the membrane for 1 h. Finally, after rinsing, Chemiluminescence Reagent (NEN Life Sciences Products, Boston, MA, USA) was added for 1 min and then the membrane was exposed to X-ray film. CYP450 standards used on the gels were purchased from Gentest (Woburn, MA, USA) and diluted with running buffer to 0.1, 0.5 and 1.0 ng of CYP450 protein. Protein bands on X-ray film were quantitated using a GS- 700 imaging densitometer with ChemAnalyst software from Bio-Rad.

Data analysis K m and V max were calculated using the method of HanesWoolf. Data were analyzed with the software program EnzymeKinetics (Trinity Software) installed on a Power Macintosh 8100/80 computer. Data were initially

171

analyzed with descriptive statistics. Pairwise comparison was done using the Wilcoxon Rank Sum Test with StatS 3 software (Spreadware, Palm Desert, CA, USA). Statistical significance was accepted at P :50.05.

RESULTS Mean peak parasitemia of 28% (9-65%) infected red blood cells was achieved consistently between 9-12 days. Total body weight was less for the malaria infected animals (CON, 212 ± 50 g; MAL, 190 ± 55 g, P <0.05). In contrast, total liver weight (CON, 8.7 ± 1.7 g; MAL, 10.2 ± 2.6 g; P <0.05) as well as liver weight expressed as percent total body weight (CON 4.2 ± 0.5%; MAL, 5.5 ± 0.9%; P <0.05) was greater in the malaria infected animals. While total protein was unchanged between CON and MAL microsomes (10.32 ± 3.98 versus 9.57 ± 3.72 mg protein/g liver, P =NS), NADPH CYP450 reductase was reduced by 32% in MAL, with 238.5 ± 106.6 versus 162.1 ± 69.4 nmol reduced cytochrome c/min/mg protein for CON and MAL, respectively (P <0.05). CYP450 content was reduced by greater than 50% in MAL with 0.439 ± 0.161 nmol CYP450/mg protein compared to 0.996 ± 0.165 nmol CYP450/mg protein in CON, P <0.05 (Table I). Single point determinations of activity at V max demonstrate that chlorzoxazone hydroxylation (200 11M chlorzoxazone) was unchanged in malaria infection with 1523.3 ± 457.5 versus 1277.3 ± 380.0 pmol 6-hydroxychlorzoxazone formed/min/mg protein in CON versus MAL, respectively (Table I). In contrast, testosterone 6~-hydroxylation (500 11M testosterone) was impaired with 3019.4 ± 742.3 versus 1789.9 ± 801.9 pmol 6-~­ hydroxytestosterone formed/min/mg protein in CON versus MAL, respectively (P <0.05). This reflects an over 40% reduction in the activity of CYP3A2. Representative incubations for both chlorzoxazone and testosterone are demonstrated in Figure 1; both display Michaelis-Menten kinetics. The formation of 6-hydroxychlorzoxazone in malaria-infected micro somes was not significantly different from controls (top panel). However, testosterone 6-~-hydroxylation was significantly reduced in malaria infection (P <0.05; bottom panel). Kinetic parameters for both CYP2E1 and CYP3A2 are displayed in Table II. The mean K m for chlorzoxazone 6-hydroxylation was 85.2 11M and 69.0 11M for CON and MAL microsomes, (P <0.05) respectively. Mean Vmax was 6345 and 4818 pmol 6-hydroxychlorzoxazone formed/min/mg protein for CON and MAL microsomes (P >0.05),

172

Eur. J. Drug Metab. Pharmacokinet. 1999, No.2

Table I: Assay and probe substrate activities in rat liver microsomes Assay/substrate

Control

Malaria infected

Lowry (mg protein/g liver)

10.32 ± 3.98

9.57 ± 3.72

CYP450 content (nmol P450/mg protein)

0.996±0.165

0.439 ± 0.161 *

-56.0*

NAOPH cytochrome P450 reductase (nmol reduced cyt c/min/mg protein)

238.5 ± 106.6

162.1 ±69.4*

-32.0*

Chlorzoxazone (CZ) (pmol 6-hydroxy CZ formed/min/mg protein)

1523.3 ± 457.5

1277.3 ± 380.0

-16.1

Testosterone (TEST) (pmol 6-~-hydroxy TEST formed/min/mg protein)

3019.4±742.3

1789.9 ± 801.9*

--40.7*

Mean ± SO, n = 6 pairs; substrate concentrations: 200

~M

for CZ, 500

~M

'o" c

~.~ 4000 ~o e a.

-

=5e ,

Control ~--::~::::::=====El"-=

Infected

~.!:

e.§ '0 '0 ~

E 2000

' " l(5..2

E a.

200

400

Chlorzoxazone concentration (J.lM)

'c"

Control

e t: 4000 B "Qj

.....

0

s.. e a. ~E"

--

~c E

l- . -

'0 >-..c::'O "",'"

2000

Infected

, E

'"

0

l-

ao£ E t:

200

400

Testosterone concentration (J.lM)

Fig. I Activity in rat liver microsomes. Representative microsomal incubations for control and infected microsomes are presented. Chlorozoxazone (top) and testosterone (bottom) were incubated separately with rat liver mictrosomes for 10 and 30 minutes, respectively. The formation of 6-hydroxy-chlorozoxazone in malaria-infected microsomes was not significantly different from controls (P = NS). The formation of 6-~-hydroxytestosterone was reduced in microsomes prepared from malaria-infected rats (P <0.05).

Change (%) -7.2

for TEST;*P < 0.05.

respectively. The mean Km for testosterone 6-P-hydroxylation was 745 11M and 467 11M for CON and MAL microsomes (P <0.05), respectively, whereas mean Vmax was 3476 and 2024 nmol 6-P-OH testosterone formed! min/mg protein for CON and MAL microsomes (P <0.05), respectively. Despite the fact that all animals were carefully matched for sex, weight, and age, interindividual differences in activities of probe substrates were notable. Interindividual variability in activity is depicted in Figure 2. The top panel of Figure 2 demonstrates the interindividual differences in chlorzoxazone (200 11M), a probe substrate for 2EI. Even though chlorzoxazone activity in MAL was only decreased by 16% (P > 0.05), there was a 2-fold difference in activity in CON while there was 2.5-fold difference in MAL. The bottom panel of Figure 2 depicts activities for testosterone 6-P-hydroxylase (500 11M) for the individual rat pairs. An overall 41% lower activity was demonstrated in MAL (P <0.05), with 2.5- and 3.5-fold differences in the activities of CON and MAL, respectively. Immunoblot analysis with subsequent densitometric measurements reveal quantitative differences in protein expression of CYP3A2 in malaria infection when compared with matched controls (Fig. 3). Mean densitometry readings for CYP3A2 were 0.48 versus 0.33 00 for CON and MAL (P < 0.05), respectively. Using densitometry, no difference in CYP2EI expression was seen (P > 0.05).

DISCUSSION Hepatic drug metabolism is crucial for the elimination of up to 80% of current therapeutic agents. Among the most important drug metabolizing enzymes are the CYP450

K. Uhl et al., Effects of Plasmodium berghei infection on cytochromes P-450 2El and 3A2

Chlorozoxazone activity

2500

•

Control

o

Infected

173

I

CIl

:5

2000

:;l .5 ~

s2

N

2

c..

~E

1500 -

.:c

~

~

'f

l..

_

1000

-0-0 >..CIl

E

..sr -0 0

0"'" E

500

c..

-4- '-

o

3

4000

4 Rat pair

Testosterone activity

CIl l:

o

l.. l: CIl .... CIl

'" ... B ~ '" c..

~E 0<: l..

.-

E

3000

2000

-0 >.."<:-0 , CIl

"i'-E -0 l.. C5..E E

1000

c..

o '2

4

5

Rat pair Fig. 2. Interindividual differences in chlorozoxazone (top) and testosterone (bottom. Even though chlorozoxazone activity in MAL was only decreased by 16% (P >0.05), there was a two-fold difference in activity in CON while there was 2.5-fold difference in MAL. All incubations performed at 200 11M chlorozoxazone. For testosterone 6-~-hydroxylation, an overall 41 % lower activity was demonstrated in MAL (P <0.05), with 2.5- and 3.5-fold differences in the activities of CON and MAL, respectively. All incubations performed at 500 11M teserosterone.

isozymes, a superfamily of heme-proteins that catalyze the oxidation of a wide variety of substrates. The CYPI-4 families are almost exclusively responsible for xenobiotic (drug) metabolism, with over 50% of drugs being metabolized by CYP3A in humans.

Changes in liver structure and function occur during both the erythrocytic (blood) and exoerythrocytic (liver) stage of malaria infection including changes in hepatic ultrastructure, smooth endoplasmic reticulum and mitochondria (18). This is notable as CYP450s are located in

Table II: Kinetic parameters for CYP2EI and CYP3A2

Vmax Control CYP2EI (CZ) CYP3A2 (TEST)

85.2 ± 32.7 745 ± 223.5

Mean ± SD, n = 6 pairs) *p < 0.05. Units: pmol6-hydroxychlorzoxazone formed/min/mg protein or

Infected

Control

69.0 ± 20.7* 467 ± 130.7

6345 ± 1198 3476 ± 657

nmoI6-~-OH

testosterone formed/min/mg protein.

Infected 4818 ± 1277 2024 ± 538*

174

Eur. J. Drug Metab. Pharmacokinet. 1999, No.2

Fig. 3 Immunoblots. CYPE2EI (top) and CYP3A2 (bottom) were analyzed separately via Western blot techniques. Protein expression ofCYPEl was no different in malaria infection compared with controls (P = NS). Protein expression of CYP3A2 was reduced significantly in microsomes prepared

from malaria-infected rats (P <0.05). C = control; I = infected; S = standards of 0.1, 0.5 and 1.0 ng CYP protein.

greatest variety and content in the liver. Experimental malaria infection has been shown to alter the hepatic phase I and II drug biotransformation enzymes.(5-1O) Plasmodium berghei causes decreases in the microsomal metabolism of CYP450 substrates, metronidazole (19) and caffeine (20), but not antipyrine (19). Microsomes prepared from malaria-infected rats demonstrate intrinsic clearance of phenacetin (CYP1A2-mediated) that was 6-30-fold lower than non-infected (7). A possible isozyme-selective effect of experimental malaria infection has been proposed whereby the clearance of 7ethoxyresorufin (CYPIA2-mediated) was reduced in malaria infection, however, metoprolol (CYP2D1mediated) clearance was unchanged (6). Adedoyin et al. (21) demonstrated selectivity of alterations in the metabolic function ofthe liver whereby CYP2C19 activity was more severely affected by cirrhotic liver disease in humans than CYP2D6. Our data support the selective isozyme effect, because both the activity and expression of CYP3A2 was depressed but CYP2El was relatively unchanged. This apparent selectivity may be due to different time courses of onset and recovery of the effects, which cannot be determined in single time point studies. In

the present study, these changes were detected at peak parasitemia, corresponding with the time when clinical infection is most apparent and therefore when drug treatment will most likely be initiated. Alterations in metabolism and cytochrome P-450 activities have been reported in numerous other infections and disease states. Disease effects upon drug metabolism and clearance, expressed as changes in drug efficacy and toxicity, are reported for many conditions, such as influenza, cancer (22), and malaria (23). Influenza infection (24) or injection of influenza vaccine (25,26) impairs theophylline metabolism via decreased CYP450 mediated metabolism of theophylline. Administration of endotoxin to experimental animals causes decreases in hepatic CYP450 and associated activities (7,27). The precise mediators of the effect and their mechanism(s) remain unclear although there are several candidates for the 'disease-metabolism' effect. The interleukins IL-l, 2, 6, interferons, and tumor necrosis factor have all been correlated with selective suppression of various CYP450 activities in animal studies (28-30). Nonprotein inflammatory mediators, such as eicosanoids, may playa role in CYP450 regulation via increased formation of

K. Uhl et al., Effects of Plasmodium berghei infection on cytochromes P-450 2El and 3A2

arachindonic acid and other related substances (31). Some of the effects of inflammation on CYP450 activity and/or expression may be due to altered secretion of glucocorticoids or growth hormone via activation of the hypothalamopituitary axis (32). Additionally, inflammation results in increased activities of hemo oxygenase (33), xanthine oxidase (34), and nitric oxide synthase (35) resulting in decreased CYP450 activity. Previous attempts to correlate probe substrate clearance with parasitemia have been unsuccessful or have subclassified malaria infection as either low or high parasitemia. By using a multiple regression model, the effect of malaria infection on CYP450 substrate activity appears to be multifactorial and not inversely correlated solely with parasitemia as previously discussed (7). A regression model using parasitemia, total protein, CYP450 content, and NADPH cytochrome P-450 reductase levels collectively, reasonably predicts testosterone 6-~-hydroxylase activity in rat (R2 = 0.634). However, rat liver microsomes have a smaller proportion of CYP450 3A relative to total CYP450 content compared to humans (36). Previous reports on the effect of malaria infection on drug metabolism were based on data from pooled liver microsomes from multiple animals (5-8). Pooled microsomal preparations average out any potential interindividual differences in metabolism that are clinically relevant. Pooled microsomes mayor may not be more appropriate for indicating central tendency, however, since they would miss outliers. Interindividual differences in activities for probe substrates CZ and TEST have been demonstrated in rodents in this study (Fig. 2). Variability in parasitemia alone does not explain the interindividual differences seen with these probe substrate activities (R2 = 0.03, CZ and R2 = 0.002, TEST). A frequent observation from other studies investigating the effect of inflammation and infection on CYP450 is that total CYP450 levels are often less affected than are individual CYP450 proteins, suggesting that some proteins must be unaffected or less affected (37,38). The decline in the activity of hepatic CYP3A2 in this study was accompanied by a decrease in the amount of that enzyme in the microsornes, as supported by immunoblot analysis. Experimental models have suggested that lower CYP450 levels are the result of either inhibition of CYP450 (39) or apoprotein (40) synthesis. However, other studies have demonstrated that decreased protein synthesis is partly explained by decreases in P450 mRNAs, although there is evidence to suggest that RNA translation and protein degradation may also be affected (29,41). The difference in Km for chlorzoxazone does not result from quantitative changes in CYP2El protein expression.

175

Our data show that malaria infection with Plasmodium berghei significantly decreased CYP450 content, NADPH reductase activity, and CYP3A2 activity in rat liver microsomes. This is the first demonstration of impaired protein expression of a CYP450 isozyme during experimental malaria infection. However, malaria infection did not significantly affect total CYP2El protein expression and enzymatic activity. The implication of these findings is that dosage adjustment will depend on which CYP450 enzyme is responsible for the metabolism of the drug of interest and the effect of disease on that specific CYP450. The challenge now lies in predicting clinically significant interactions between malaria infection or other diseases and medications used in treatment.

REFERENCES 1. Oaks S.c., Mitchell V.S., Pearson G.w., Carpenter c.c.]. (eds) (1991) : Malaria: Obstacles and Opportunities, A report of the committee for the study on malaria prevention and control: Status Review and Alternative Strategies. Division of International Health, Institute of Medicine, National Academy Press. 2. Na-Bangchang K., Limpaibul L. (1994) : The pharmacokinetics of chloroquine in healthy Thai subjects and patients with Plasmodium vivax malaria. Br.]. Clin. Pharmacol., 38: 278-281. 3. White N.J.,Looareesuwan S., Warrell D.A.,Warrell M.J., Bunnag D., Harinasuta T (1982) : Quinine pharmacokinetics and toxicity in cerebral and uncomplicated falciparum malaria. Am.]. Med., 73: 564-571. 4. White N.J. (1985) : Clinical pharmacokinetics of antimalarial drugs. Clin. Pharmacokinet., 10: 187-215. 5. Alvares AP., Ueng TH., Scheibel t.w, Hollingdale M.R. (1984) : Impairment of hepatic cytochrome P-450 dependent monooxygenases by the malaria parasite Plasmodium berghei. Mol. Biochem. Parasitol., 13: 277-282. 6. Glazier A.P.,Kokwaro G.O., Edwards G. (1993) : Possible isozyme-specific effects of experimental malaria with Plasmodium berghei on cytochrome P450 activity in rat liver microsomes.]. Pharm. Pharmacol., 46: 352-355. Z Kokwaro G.O., Glazier A.P., Ward S.A., Breckenridge AM., Edwards G. (1993) : Effect of malaria infection and endotoxin-induced fever on phenacetin O-deethylation by rat liver microsomes. Biochem. Pharmacol., 45: 1235-1241. 8. Mihaly GW., Date N.M., Veenendaal I.R, Newman K.T., Smallwood R.A. (1987) : Decreased hepatic elimination of pyrimethamine during malaria infection. Studies in the isolated perfused rat liver. Biochem. Pharmacol., 36: 2827-2829. 9. Mansor S.M.,Edwards G., Roberts P.J., Ward S.A. (1991) : The effect of malaria infection on paracetamol disposition in the rat. Biochem. Pharmacol., 41: 1707-1711. 10. Murdoch R.T., Ghabrial H., Smallwood R.A, Morgan D,J. (1992) : Effect of malaria on phenol conjugation pathways in perfused rat liver. Biochem. Pharmacol., 43: 1229-1234.

176

Eur. J. Drug Metab. Pharmacokinet. 1999, No.2

II. Black R.H., Ray AP. (1977) : Experimental studies of the potentiation of proguanil and pyrimethamine by dapsone using Plasmodium berghei in white mice. Ann. Trop. Med. Parasitol., 71: 131-139. 12. Guengerich EP. (1977) : Separation and purification of multiple forms of microsomal cytochrome P-450: activities of different forms of cytochrome P-450 towards several compounds of environmental interest.]. BioI. Chem., 252: 3970-3979. 13. Lowry O.H., Rosenbrough N}, Farr A.L., Randall R], (1951) : Protein measurement with the Folin phenol reagent.]. BioI. Chem., 193: 265-275. 14. Omura T.,Sato Rj. (1964) : The carbon-monoxide-binding pigment of liver microsomes.]. BioI. Chem., 239: 2370-2378. IS. Phillips AH., Langdon R.G. (1962) : Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization, and kinetic studies. J. BioI. Chem., 237: 2652-2660. 16. Peter R., Boeker R.G., Beaune P.H.,Iwasaki M.,Guengerich EP., Yang CS. (1990) : Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-450 lIE1. Chem. Res. Toxicol.,3: 566-573. I Z Sonderfan A.j., Arlotto M.P., Dutton D.R., McMillen S.K., Parkinson A (1987) : Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450. Arch. Biochem. Biophys., 255: 27-41. 18. Rosen 5., Royeroft DW., Hans ].E., Barry KG. (1967) : The liver in malaria. Arch. Patho!., 83: 271-27Z 19. Kokwaro G.O.,Ismail 5., Glazier AP., Ward SA, Edwards G. (1993) : Effect of malaria infection and endotoxin-induced fever on the metabolism of antipyrine and metronidazole in the rat. Biochem. Pharmacol., 45: 1243-1249. 20. Kokwaro G.O.,Szwandt I.S.E, Glazier A.P., Ward SA, Edwards G. (1993): Metabolism of caffeine and theophylline in rats with malaria and endotoxin-induced fever. Xeniobiotica, 23: 1391-139Z 21. Adedayo A, Arns PA, Richards WO., Wilkinson G.R., Branch RA (1998): Selective effect of liver disease on the activities of specific metabolizing enzymes: Investigation of cytochromes P450 2CI9 and 2D6. Clin. Pharmacol. Ther., 64: 8-IZ 22. Nebert D.W, McKinnon R.A., Puga A (1996)v: Human drugmetabolizing enzyme polymorphisms: effects on risk of toxicity and cancer. DNACell BioI., IS: 273-280. 23. White N.]. (1985) : Clinical pharmacokinetics of antimalarial drugs. Clin. Pharm., 10: 187-215. 24. Chang KC, Lauer B.A, Bell T.D., Chai H. (1978) : Altered theophylline pharmacokinetics during acute respiratory viral illness. Lancet, I: 1132-1133. 25. Renton KW, Gray J.D.,Hall R.1. (1980) : Decreased elirnination of theophylline after influenza vaccination. Can. Med. Assoc. J.. 123: 288-290. 26. Renton KW (1981) : Effects of infection and immune stimulation on theophylline metabolism. Semin. Perinatol., 5: 378-382. 2Z Renton KW, Mannering G.j. (1976) : Depression of hepatic cytochrome P-450-dependent monooxygenase systems with

administered interferon inducing agents. Biochem. Biophys. Res. Commun., 73: 343-348. 28. Chen Y.L., Florentin I., Batt A.M., Ferrari L., Giroud J.P., Chauvelot-Moachon L. (1992): Effect of interleukin-6 on cytochrome P450-dependent mixed-function oxidases in the rat. Biochem. Pharmacol., 44: 137-149. 29. Shedlodsky 5.1., Swim AT., Robinson l.M, Gallicchio V.S., Cohen D.A., McClain cr (1987): Interleukin-I (IL-I) depresses cytochrome P450 levels and activities in mice. Life Sci, 40: 2331-2336. 30. Cribb AE., Renton KW (1993) : Dissociation of xanthine oxidase induction and cytochrome P450 depression during interferon induction in the rat. Biochem. Pharmacol., 46: 2114-211Z 31. Morgan E.T. (1997) : Regulation of cytochromes P450 during inflammation and infection. Drug Metab. Rev., 29: 1129-1188. 32. Chung H., Brown D.R. (1976) : The mechanism of the effect of acute stress on hexobarbital metabolism. Toxicol. Appl. Pharmacol., 37: 313-318. 33. BissellD.M., Hammaker L.E. (1976) : Cytochrome P-450 heme and the regulation of delta-arninolevulinic acid synthetase in the liver. Arch. Biochem. Biophys., 176: 103-112. 34. Koizumi A, Hasegawa L., Rodgers KE., Ellefson D., Walford R., Imamura T. (1986) : Influence of H-2 haplotypes on poly IC induction of xanthine oxidase and poly IC induced decreases in P-450 mediated enzyme activities. Biochem. Biophys. Res. Commun., 138: 246-253..T. (1996) : Endotoxemia in rats is associated with induction of 35. Wink DA, Osawa Y., Darbyshire ].P.,Jones CR., Eshenaur S.C, Nims R.W. (1993) : Inhibition of cytochromes P450 by nitric oxide and a nitric oxide-releasing agent. Arch. Biochem. Biophys., 300: 115-123. 36. Ryan D.E., Thomas P.E., Reik L.M., Levin W (1982) : Purification, characterization and regulation of five rat hepatic microsomal cytochrome P-450 isozymes. Xenobiotica, 12: 727-744. 3Z Sewer M.B., Koop D.R., Morgan E the P4504A subfamily and suppression of several other forms of cytochrome P450. Drug Metab. Dispos., 24: 401-40Z 38. Sakai H., Okamoto T., Kikkawa ]. (1992) : Suppression of hepatic drug metabolism by the interferon inducer, polyriboinosinic acid:polyribocitidylic acid.]. Pharmacol. Exp. Ther., 263: 381-386. 39. Singh G., Renton KW (1984) : Inhibition of the synthesis of hepatic cytochrome P-450 by the interferon-inducing agent poly rl.rCi. Can. J. Physiol. Pharmacol., 69: 379-383. 40. Moochhala A.M., Renton KW. (1989) : The effect ofIFNalpha-Con I on hepatic cytochrome P-450 and protein synthesis and degradation in hepatic microsomes. Int.]. Immunopharmacol., 13: 903-912. 41. Cribb AE., Delaporte E., Kim S.G., Novak R.E, Renton KW. (1994) : Regulation of cytochrome P-4501A and cytochrome P-4502E induction in the rat during the production of interferon alphalbeta.]. Pharmacol. Exp. Ther., 268: 487-494.