Vaccine-induced alterations in hepatic drug metabolism

Vaccine-induced alterations in hepatic drug metabolism Sherry Ansher*, Walter Thompson and William Habig A dministration of Diphtheria and Tetanus Toxoids and Pertussis Vaccine Adsorbed (D TP) vaccine to mice causes dose- and time-dependent alterations in hepatic drug metabolism as determined by hexobarbital-induced sleep time and several direct measurements of soluble and microsomal enzyme activities. Vaccines containing only tetanus and/or diphtheria toxoids did not alter hepatic drug metabolism, implicating the pertussis component as the cause of the observed changes. Other pyrogenic whole cell vaccines such as typhoid and cholera also had no effect on drug metabolizing capacity. However, polyriboinosinic polyribocytidylic acid (poly I.C), a compound thought to exert its effects through the induction of interferon, induced changes comparable to those seen with DTP vaccine. The similarities in the effects following administration of DTP vaccine and poly L C suggest that vaccine-induced alterations of drug metabolism may be mediated by immunomodulatory agents such as interferons and interleukins. Studies with purified cytokines are planned to address this question. Keywords:drug metabolism; diphtheria; pertussis; tetanus; sleep time

INTRODUCTION There have been several reports of inhibition of drug metabolism following the administration of vaccines and interferon-inducing agents in animals and man ~-7. The relationship of these observations to the reported adverse reactions following immunization of man is not available, nor is the mechanism for the inhibition of drug metabolism known. There is, however, the suggestion that the immune system is involved. Administration of vaccines has been associated with an increase in serum interferon levels in rodents and rabbits, and administration of interferon-inducing agents depresses hepatic drug metabolism 6,s. Most reports are limited to inhibition of cytochrome P-450-dependent mixed function oxidase activities as demonstrated either directly by measuring enzyme activity or indirectly by measuring barbiturateinduced sleep time 6's. Inhibition of the capacity for metabolizing drugs is an important concern. Many clinically important xenobiotics are metabolized in reactions catalysed by cytochrome P-450 to the pharmacologically active form or are inactivated by P-450-dependent reactions prior to excretion. Severe clinical manifestations can result from the inhibition of these enzymes. Theophylline, for example, has been reported to reach dangerously high levels when administered to asthmatic children, following influenza virus immunization if drug metabolism is Division of Bacterial Products, Center for Biologics Evaluation and Research, FDA, Bethesda, MD 20892, USA. *To whom correspondence should be addressed at: Food and Drug Administration, Building 29, Room 103, 8800 Rockville Pike, Bethesda, MD 20892, USA. (Received 2 May 1990; revised 22 August 1990; accepted 17 September 1990)

0264-410X/91/040277-07 © 1991 US Government

depressed3'5.. For chemotherapeutic agents that are metabolized to their active form, such inhibition might prevent an effective therapeutic concentration from being reached. Cancer chemotherapy regimens now often combine traditional drugs with cytokines such as interferons and interleukins. We and others have shown that cytokines are able to inhibit drug metabolism 9-11 Inhibition of drug metabolism would alter the pharmacokinetics determined with single agents. Nor are cytochrome P-450-dependent reactions the only important enzymes in the metabolism of foreign compounds. Effects of vaccines on enzymes in the pathways of glucuronidation, sulphation, and mercapturic acid formation must also be considered, although they have been less well studied. Agents which inhibit drug metabolism are widely reported. Treatment of animals with polyriboinosinic polyribocytidylic acid (poly I:C), a double stranded RNA polymer, is thought to inhibit microsomal drug metabolism through the induction of interferon 12,1a. The effects of administration of poly I:C on a variety of drug metabolizing enzymes have been well documented. Serum interferon levels have been reported to be increased within a few hours of poly I:C administration, while the inhibition of hepatic enzyme activities seems to take at least a day la. Because the effects of poly I:C administration have been characterized in several strains of mice, we compared the effects of vaccine administration to those of poly I:C administration in C57B/16 mice. The studies reported in this paper address three main questions: Which enzyme systems are affected by vaccine administration and to what extent? What is the time course of the effect? Which of the vaccine components are responsible for the observed effects?

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MATERIALS

AND

METHODS

Polyriboinosinic polyribocytidylic acid (Poly I:Ct and coli endotoxin (serotypc 055:B5) were purchased from Sigma Chemical Co. (St Louis, MO). Bordetella pertussis endotoxin was purchased from List Biological Labs (Campbell, CA 1. Vaccines were samples submitted to Center for Biologics Evaluation and Research, F D A , for control testing. Acellular pertussis vaccine and purified pertussis toxin were supplied by the Laboratory of Pertussis, FDA, Bethesda, M D. Endotoxin content was determined by the Division of Product Quality Control, FDA, using a standard protocol ~ Escherichia

Animals C57/B16 female mice (6 10 weeks of age, 10 15g) were purchased from NCI or Charles River (Small Animal Section, NIH). This strain of mice was chosen because of their sensitivity to vaccine-induced alterations of drug metabolizing capacity. They were given free access to food and water and were maintained on a 12 hour light and dark cycle. Animals were acclimatized several days prior to the start of an experiment. All injections were administered intraperitoneally (i.p.)in a volume of 0.5 ml. Poly I:C or endotoxin was dissolved in sterile, pyrogenfree saline prior to injection.

Sleep time Hexobarbital sodium (Sigma Chemical Co.. St Louis, MO), 50rag, was dissolved in 0.3ml 1M N a O H and diluted to 10ml in water immediately before use. Mice were weighed and injected i.p. with a dose of 100 mg kg Sleep time was measured as the time elapsed from injection of hexobarbital to the return of the redressment reflex.

formaldehyde. Benzopyrene monooxygenasc activity was determmed by the method of DePicrrc e¢ al. ~". Benzpyrenc was obtained from Aldrich and [3H]benzpyrene (40Ci mmol 1) from Amersham. [3H]Benzpyrene was purified before use as described in the literature 1°. Activity was expressed as nmol h L mg 1 microsomal protein. Uridine diphosphate (UDP) glucuronyl transferase was assayed radiochemically with [~aC] p-nitrophenol ( 7 9 m C i m m o l 1 Amersham)eO and epoxide hydrase was assayed radiochemically with [3H]styrene oxide (99 mCi mmol ~, Amersham) ~9. Activity was expressed as nmol h 1 mg ~ microsomal protein. The cytoso!ic glutathione lransferases were assayed with l-chloro-2,4-dinitrobenzene ( C D N B ) a n d 1,2-dichloro-4-nitrobenzene (DCNB) as substrates e~. Dicoumarol-inhibitable quinone reductase was assayed according to Ernster ee with the modifications described by Benson et al. e3 using 2,6-dichloroindophenol as the electron acceptor in a final volume of 1.0 ml. All cytosolic activities are expressed as nmol min-~ mg ~ cytosolic protein. Protein was determined with bicinchoninic acid ::4 with bovine serum albumin (BSA) as a standard. Data were analysed by Student's t test or analysis of variance using Statview (Macintosh) on a PC.

Plasma Blood was collected into heparinized tubes by retroorbital bleeding immediately before killing. Plasma was prepared by centrifugation in a microfuge at high speed for 10min. The plasma samples were used for the determination of glucose levels and blood enzymes by MetPath (Rockville, MD). Interferon assays were performed by the Division of Cytokine Biology, CBER, FDA, using L929 cells with mouse g a m m a interferon as a standard 2s.

Enzyme assays hnmediately after killing the mice by cervical dislocation, the livers were removed, weighed and homogenized in 3 volumes ice-cold isotonic KC1. Cytosolic and microsomal fractions were prepared according to Hodgeboom 15. Microsomes were resuspended in 2ml 0.1M potassium phosphate at pH 7.4, containing 20% glycerol, frozen immediately in liquid nitrogen, stored at 70, and used within 1 week of preparation. No loss of activity was seen upon storage when samples were handled in this manner. Cytochrome P-450 levels were measured according to the method of O m u r a and Sato ~6 using a Model A4 50A Hewlett Packard spectrophotometer. Values reported are in nmol mg-~ microsomal protein. Benzphetamine demethylase activity' was assayed spectrophotometrically according to the method of Astrom et al. 17. Enzyme activity is expressed as nmol min 1 mg ~ microsomal protein. Ethylmorphine was obtained from Alltech Applied Science(Deerfield, IL). Ethylmorphine demethylase activity was assayed by the method of Astrom et al.17 with the following modifications: a total volume of 0.25ml was used to which an equal volume of trichloroacetic acid (TCA) was added to stop the reaction. Formaldehyde production was determined according to the Nash procedure 1s. Activity was expressed as ~lg formaldehyde formcd h - I mg microsomal protein based on a standard curve with

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RESULTS

Hexobarbital-induced sleep time following the administration of vaccines and poly I:C The ability of different vaccines to alter hexobarbitalinduced sleep times was compared to the effect of poly I:C. Vaccines or poly I:C were administered 24h before (for one dose evaluation) or 48 h and 24 h (for two dose evaluation)prior to hexobarbital injection. Vaccines were administered at a dose of 0.5 ml (one single human dose) i.p. and poly I:C was administered i.p. at a dose of 1 0 m g k g 1 Of the vaccines listed in Table 1, only diphtheria and tetanus toxoids and pertussis vaccine adsorbed (DTP) vaccine increased sleep time over that of controls. Both D T P vaccine and poly l: C significantly increased hexobarbital-induced sleep time after one or two doses. Tetanus Toxoid Fluid, Tetanus Toxoid Adsorbed and Diphtheria and Tetanus Toxoids Adsorbed (DT) had no effect on sleep time. Because D T P vaccine markedly altered sleep time, it was studied in more detail. The increase in hexobarbital-induced sleep observed with D T P vaccine was dose dependent (Figure 1). Mice were injected with doses corresponding to 1/10 single human dose to two single human doses. However, significant increases in sleep time 1 week following injection were observed only at a minimurn of one single human dose (0.5 ml). Larger quantities of vaccine led to

Vaccines and metabolism: S. Ansher et al. Table 1 Comparisons of one or two doses of vaccine or poly I:C on sleep times in mice Number of doses

Sleep time index ~

Tetanus Toxoid Fluid

1 2

1.0 1.0

Tetanus Toxoid Adsorbed

1 2

1.0 1.0

Diphtheria and Tetanus Toxoids-Adsorbed

1

1.0

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1 2

1.4 ~ 2.3 ~

1 2

1.8 ~ 2.7 b

Treatment

Poly I:C

single injection. For cytochrome P-450, a spectral assay detected a 10% decrease 2h after poly I:C injection, which declined to 70% of control level by 8 h. Interferon levels were increased in the plasma by 80- to 125-fold for up to 8-h after poly I:C. Microsomal cytochrome P-450 and other enzyme activities were measured in a parallel experiment with different animals (Figure 2b). Spectrally assayed cytochrome P-450 levels were decreased by 50% 7 days after a single dose of vaccine. They returned slowly toward normal but had not exceeded 80% of control values by 28 days. The microsomal mixed function oxidase activities showed a similar pattern of activity for each of the enzymes assayed (Figure 3a). Inhibition was greatest 7-10

"Sleep time index represents the mean sleep time of treated animals (four per group) divided by the mean sleep time of control animals bp < 0.05

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significantly differentfrom control (p <0.05) mortality of some mice when the hexobarbital was given. All further studies employed one single human dose (Figure I) and sleep times were measured 1 week following the single injection of vaccine, Time course of vaccine effects

In order to evaluate whether the effects of the D T P vaccine were direct or were mediated by an immune or other secondary pathway, mice were injected with a single dose of vaccine and several enyzme activities were measured from 1 to 28 days postinjection. Hexobarbitalinduced sleep times were increased within 24h and reached a 2.2- to 2.4-fold maximum above controls at 7-10 days (Figure 2a). The effect declined rapidly to levels (1.2-fold) not significantly different from controls by day 14. Separate studies found that increases in sleep time (l.5-fold) can be observed at 12h, but not 6h, after a

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Time a f t e r injection (days) Hexobarbital-induced sleep time and cytochrome P-450 levels Figure 2 following administration of DTP vaccine show a similar pattern of response. (a), The time course of the increase in sleep time relative to control is shown from 1 to 28 days following a single injection of vaccine. The data are depicted as % control which is calculated as the mean sleep time for treated animals divided by the mean sleep time for control animals (four animals per group) at each time point. Sleep times were significantly increased (p < 0.05) from days 4 to 10. (b), Spectrally assayed cytochrome P-450 levels in liver microsomes from control and DTP treated mice were compared from days 1 to 28 as described in (a). Each point represents the mean of three treated animals divided by the mean of three control animals. Values marked * were significantly different from controls (p<0.05). Control values were ~0.9nmol mg ~ protein

Vaccine, Vol. 9, April 1991

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Vaccines and metabolism. S. Ansher et al. 120

somal epoxidc hydrasc activity remained at this level for 21 days, then returned to 80% of control levels (Figure 3c). U D P glucuronyl transfcrasc activity returned to 80% of control levels at day 4 and to 100% by day ]!4 (Ficjure 3c). The alterations in enzyme activities were accompanied by marked increases m the weights of liver {twofold) and spleen (four- to sixfold) compared I:o saline-injected controls. Histologic examination of the liver 7 days after vaccine administration showed significant multifocal random inflammatory infiltrates consisting mostly of macrophages and lymphocytes (Dr P. Snoy, personal communication).

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Time a f t e r injection ( d a y s ) Figure 3 Microsomal and cytosolic enzyme activities after injection of mice with DTP vaccine. (a), The microsomal mixed function oxidase activities benzphetamine demethylase (BZ) ( I ) , ethylmorphine demethylase (EM) ( ~ ) and benzo(a)pyrene monooxygenase (BP) ( 0 ) are shown for the ratio of treated divided by control. Each value represents the mean of three livers divided by three controls. Each sample is the pool of two livers. Control enzyme activity units ~ 8.0 nmol min ~mg for BZ, 4 y g formaldehyde formed h ~ mg ~for EM and 5nmol h ~ mg ' for BP. (b), The cytosolic enzyme activities quinone reductase (QR) ( I ) and glutathione transferases with the substrates CDNB(GST) (1~) and DCNB(GST) ( 0 ) are shown. Mean control values were 90 nmol min m g ' for QR, 2000nmol min ' m g 1 for CDNB(GST) and 60nmol min mg ' for DCNB(GST). (c), The microsomal enzyme activities UDPglucuronyl transferases (UDPGT) ( I ) and epoxide hydrolase (EH) (O) are shown as described in the text. Mean control values for UDPGT w e r e 4 0 0 n m o l h ' mg ~and for EH w e r e 3 0 n m o l h ' mg '

days after vaccine administration and then returned to ~ 8 0 % of control levels by day 28. A different pattern of activity was observed for the cytosolic enzymes (Fiqure 3h). After an initial decrease in the glutathione (GSH) transferase activities lasting for up to 4 days, GSH transferase increased twofold at 14 days and then slowly returned to basal levels. Quinonc reductase activity, in contrast, was elevated to 150% of control activity by day 4 persisting through to day 28 of the experiment. Both microsomal epoxide hydrase and U D P glucuronyl transferases were inhibited by ~ 35 40% within 24 h. Micro-

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The effects described above are not limited to D T P wmcine: however, they do seem to be associated with the pertussis component of the vaccine. Whole cell pertussis vaccines (fluid or a.dsorbed) caused increased hexobarbitalinduced sleep time similar to those seen following administration of D T P vaccine, whereas an acelluhlr vaccine composed of inactivated pertussis toxin and filamentous haemagglutinin (FHA) did not alter sleep time (Figure 4). These studies were conducted 7 days after a single injection based on the results of the time course for the D T P wiccine {Figure 2a). Purified active pcrtussis toxin increased sleep time slightly (20 30%) when administered tit 1 Itg per mouse, a dose much higher than would normally be present in commercial whole cell pertussis vaccines. Commercial vaccine preparations contain I0 20ng perlussis toxin per dose. The increased sleep time was not caused by a component present in all whole cell vaccines, since cholera, plague and typhoid vaccines did not affect sleep time. One component of concern in all vaccines is endotoxin. Purified endotoxin from E. colt or B. pertussis caused dose dependent increases in sleep times 24 h after a single injection. The endotoxin content and the sleep time index for each of the purified vaccines and components tire shown in Fioure 5. For the pertussis components there 100

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Figure 4 Hexobarbital-induced sleep time after vaccine administration. Sleep time (min) are compared for control, Diphtheria and Tetanus Toxoids Adsorbed (DT), DTP, whole cell pertussis vaccine (P), acellutar pertussis vaccine (APT) and purified pertussis toxin (PT) given at a dose of 0.5 l~g, 1 week following administration. Each bar represents the mean of five animals per group

Vaccines and metabolism: S. Ansher et al.

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Endotoxin content (O) and sleep time index (m) for vaccines and purified components. Endotoxin content was measured by the Limulus Amoebacyte Lysis test for each of the compounds listed: Diphtheria and Tetanus Toxoids (DT), accellular pertussis vaccine (AP), influenza virus vaccine (Flu), purified pertussis toxin, 1 ,ug (PT), plague vaccine (Plague), DTP vaccine (DTP), purified Bordetella pertussis lipopolysaccharide, 100#g (LPS) and cholera vaccine (Cholera) and compared to sleep times in control mice 1 week following administration. Sleep time index represents the mean sleep time of treated mice divided by the mean sleep time of control mice

Table 2 Microsomal enzyme activities after vaccine or poly I:C administration

P-450

Benzpyrene monooxygenase

Benzphetamine demethylase

9.8 _+0.72 8.36_+0.67 (85) 5.57_+0.90 ~ (57) 8.68-+1.0 (89) 8.26-+0.55 (84) 7.56-+0.73 (77) 4.16_+0.8 (43)

Treatment a

(%)

(%)

Control DT adsorbed

0.90_+0.03 0.76_+0.03 (84) 0.44_+0.18 ~ (49) 0.59_+0.04 ~ (66) 0.78_+0.01 (86) 0.73_+0.01 (81) 0.45_+0.1 (50)

4.49+0.32 3.77_+0.03 (84) 2.01 +0.48 ~ (45) 3.15_+0.46 ~ (70) 4.96_+0.06 (111) 4.11 _ + 0 . 1 8 (92) 3.14_+0.8 (70)

DTP Pertussis whole cell Acellular pertussis Pertussis toxin Poly I:C

(%)

aVaccines were administered seven days and poly I:C one day prior to killing of the animals. Each value represents the mean of three animals per group. Numbers in parentheses are the mean treated values divided by the mean control values for each treatment expressed as a percentage bp < 0.05

is a correlation between B. pertussis endotoxin content and sleep time index, but this is not evident for the other vaccine preparations containing endotoxin from other sources. Inhibition of spectral cytochrome P-450 and microsoreal enzyme activities were observed in mice treated with whole cell pertussis vaccines (DTP and whole cell pertussis vaccine) and poly I:C, but not D T or acellular pertussis (Table 2). These results further demonstrate that sleep time measurements correlate well with changes in microsomal enzyme activities. Plasma e n z y m e measurements

Plasma enzyme profiles were compared for untreated,

Table 3

Serum parameters after DTP vaccine and poly I:C

Days after injection

Glucose

Alkaline phosphatase

SCOT

SGPT

1 4 7 10 14 21 28 Poly I:C

0.54 a 0.74 0.68 0.57 0.65 0.83 0.80 0.87

0.50 0.33 0.36 0.49 0.60 0.84 0.93 0.54

1.30 0.79 0.77 1.29 0.89 1.00 0.76 1.02

0.78 1.13 2.03 6.15 2.62 1.52 0.79 2.20

"Numbers represent the ratio of treated/control serum. Samples were a pool of serum from two mice. In each case three separate samples were analysed and the results averaged. Poly I:C was administered 24 h before samples were taken

poly I:C and D T P treated mice. There were significant decreases in alkaline phosphatase and serum glucose after D T P vaccine or poly I:C (Table 3). There were also increases in serum glutamic-pyruvic transaminase (SGPT) activity (1.5 to 6-fold) and some changes in serum glutamic-oxaloacetic transaminase (SGOT) activity. However, only alkaline phosphatase appeared to correlate well with the alterations in sleep times or drug metabolism. Although alkaline phosphatase seemed to follow the time course of the sleep time data, it is the least specific of the serum parameters for liver disease. Interferon levels were increased within 8h of poly I:C administration but this was not observed following administration of D T P vaccine. By 24 h, interferon levels were not significantly different from controls. DISCUSSION These studies demonstrate that administration of D T P vaccine and other agents can cause alterations in a number of hepatic drug metabolizing enzymes. Our

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Vaccines and metabolism: S. Ansher et al.

results with tetanus toxoid adsorbed vaccine {Table 1) were inconsistent with reports by Descotes et al. "'7 which reported an increase in pentobarbital-induced sleep time in mice and rabbits following injection of adsorbed, but not fluid, tetanus toxoid vaccine. An increase in serum interferon levels 10h after vaccine administration was also reported 67. We were unable to demonstrate an increase in hexobarbital-induced sleep time with any of the vaccines tested containing only tetanus and/or diphtheria toxoids. However, D T P vaccines from four different manufacturers all produced comparable increases in hexobarbital-induced sleep times. Different lots from several manufacturers were tested for both endotoxin content and sleep time effects. We have evaluated three different strains of mice with one or two doses of either fluid or adsorbed vaccine preparations, including the strain of mice used in Descotes' et al. studies <~'. The route of administration of vaccine does not alter hexobarbital-induced sleep time. Comparisons of sleep time in mice injected with the same lot of D T P vaccine by i.m., s.c. and i.p. routes all produced comparable increases 7 days after injection. Our results demonstrating alterations in hepatic drug metabolism in mice following D T P vaccine administration, but not DT vaccine administration, may be consistent with the increased adverse reactions reported in children immunized with D T P vaccine over those immunized with DT vaccine. Although the majority of infants and children experience only minor reactions to immunization, a small proportion of recipients experience more severe side effects such as convulsions and collapse 26. Children immunized with only D T exhibit fewer reactions, consistent with the assumption that the pertussis component is generally responsible for most adverse reactions 26 28. While hepatotoxicity in animals and adverse reactions in man may appear to parallel each other for D T P and DT vaccines, no causal relationship between these effects has been established. The dose of vaccine administered to animals is about 400-fold greater on a weight basis than that given to an infant. The use of high doses is important in toxicological studies with small numbers of animals to produce side effects which otherwise might be observed at very low frequency. I f D T P vaccine is capable of suppressing hepatic drug metabolism in infants, additional adverse reactions may occur in infants taking other drugs concomitantly with vaccine administration. The ability to monitor infants and children for potential adverse reactions would be an important concern. To address this issue, serum parameters were monitored following injection of D T P vaccine in mice. Unfortunately, attempts to correlate a measurable serum parameter with the increased sleep times or inhibition of enzyme activities were not successful. The increases in transaminases were not consistent with the other parameters measured in the liver, nor were there any consistent changes in any of the other serum enzymes. Studies to correlate a serum parameter with the measured enzyme inhibition will be continued since this might lead to a way for predicting or monitoring liver damage. We have observed similar responses to D T P vaccine and to poly I:C in mice. Both D T P vaccine and poly I:C caused significant inhibition of microsomal enzyme activities and cytochrome P-450 levels. The D T P vaccine caused dose-dependent and time-dependent alterations in enzyme activities. In addition to D T P vaccine, other

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Vaccine, Vol. 9, April 1991

vaccines with pertussis components caused alterations in drug metabolizing capacity. This suggests that the toxic component may be associated with permssis or is onl~ activated in the prcsencc of pertussis vaccine or components. One common component of most vaccines is endotoxin, and its presence could enhance or be responsible for the observed effects on hepatic drug metabolism. As shown in Fiqm'e 5, the endotoxin content of a single vaccine dose wtried widely from < 10 units to > 1 million units per dose. For products containing B. pertussis endotoxin, endotoxin content seemed to correlate with the increase in sleep time. Cholera w~ccinc had the highest endotoxin content but hexobarbital-induced sleep time was not changed from control values. Thus, although endotoxin may contribute to the inhibition of drug metabolizing enzyme activities, it is not the only cause of the inhibition. With purified cndotoxin, our results were consistent with those of others s with regard to the inhibition of cytochrome P-450 dependent enzyme activities. Similar effects on drug metabolizing capacity were seen following the administration of poly I:(', although no detectable endotoxin was present in the preparations of poly I:C injected into mice. Because of the similarity in the effects of administration of poly I:C and of D T P vaccine, it is possible that the inhibition could be mediated by immunomodulatory agents such as interferons or interlcukins. The ability of poly i:C to both induce interferon and inhibit drug metabolism supports the idea. Administration of interferon-~ alone does not alter drug metabolism, although interleukin-2 alone or in combination with interferon-:< does so significantly ~'. Studies are in progress with purified cytokines to assess their contribution to the inhibition of hepatic drug metabolism. Because cytokines are now emerging as possibilities for the treatment of cancer and other diseases, their effect on the metabolism of endogenous compounds and those given in conjunction with other drugs is of importance. Further studies may allow differentiation of those vaccine component(st responsible for toxicity from those necessary for immunity. With a readily available animal model for such potential side effects, it may be possible to screen potential vaccine components and combinations prior to testing in man. ACKNOWLEDGEMENTS The authors are grateful to Ms Joan Enterline of the Division of Cytokine Biology for the Interferon assays. Evelyn Rivera and [)on Hochstein of the Division of Product Quality Control for the endotoxin assays, and Dr Phil Snoy for the pathology reports. REFERENCES 1

2

3

4 5

Williams, J. and Szenivanyi, A. Depression of hepatic drugmetabolizing enzyme activity of B. pertussis vaccination. Eur. J. Pharmacol. 1977, 43, 281 284 Meredith, C., Christian, C., Johnson, R., Troxell, R., Davis, G. and Schenker, S. Effects of influenza virus vaccine on hepatic drug metabolism. Clin. Pharmacol. Ther. 1985, 37, 39£:~401 Hannan, S., May, J., Pratt, D., Richtsmeier, W. and Bertino, J. The effect of whole virus influenza vaccination on theophylline pharmacokinetics. Am. Rev. Respir. Dis. 1988, 137, 903-906 Kramer, P. and McClain, C. Depression of aminopyrine metabolism by influenza vaccination. N. Engl. J. Med. 1981, 385, 1262 1264 Renton, K., Gray, J. and Hall, R. Decreased elimination of

Vaccines a n d m e t a b o l i s m : S. A n s h e r et al.

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