Nippostrongylus brasiliensis: Effect of dexamethasone upon anthelmintic efficacy

EXPERIMENTAL

33, lo-16

PARASITOLOGY

Nippostrongylus

(1973)

brasiliensis: Effect of Dexamethasone upon Anthelmintic Efficacy D.

Health

and Nutrition Research

HMS

KENDALL

Department, Shell Development Company, Center, P.O. Box 4248, Modesto, California (Submitted

for publication,

3 January

Biological 95352

Sciences

1972)

HASS, D. KENDALL 1973. Nippostrongylus bra&lien& Effect of dexamethasone upon anthelmintic efficacy. Experimental Parasitology 33, 10-16. A study has been made to determine the chemotherapeutic susceptibility of natural and “steroid sustained” Nippostrongylus brasiliensis infections in rats. Dexamethasone in the drinking water (2.5 X lo* mgjml) aided establishment of greater parasite populations but restricted growth of young rats. Comparison trials with thiabendazole, cambendazole, and parbendazole, as well as 10 candidate drugs, showed that the parasiticidal activity of these materials was not significantly altered between the lo-day-old naturally maintained and the I7-day-old steroid-treated parasitic infections. As expected, 17day-old naturally maintained parasite populations were highly susceptible to the anthelmintic action of these materials. In one instance, the toxic response elicited by a candidate drug was apparently potentiated by dexamethasone. INDEX

therapy; Rats.

DESCRIPTOFB:

Anthehnintics;

Nippostrongylus brasiliensis; Thiabendazole; Technique;

infection is limited. Therefore, it is common practice that 7- to l&day-old infections are utilized so that the investigator can work with a stabilized population. Because of the population variations induced by the immune response, evaluation of the anthelmintic efficiency of candidate materials is not reliable with older parasitic infections. The immune response can be inhibited, however, by such glucocorticosteroids as cortisone or its potent analog, betamethasone (Ogilvie 1965). To date, however, no reports are available in the literature concerning parasitic infections in the glucocorticosteroid-treated rat for chemotherapeutic evaluation of potential anthel-

Nippostrongylus brdk& infections in the rat are easily accomplished and have been studied extensively: (1) to further our understanding of parasitism (Ogilvie and Jones 1971); and (2) as a test model for screening candidate anthelmintic drugs. The utility of this trichostrongylid parasite as a test organism for the screening of anthelmintics was first proposed by Whitlock ( 1943) and Whitlock and Bliss ( 1943). The methods and procedures described by these authors were further refined and reduced to practice (Standen 1963). Today we find that this host-parasite combination is an essential part of the drug-screening program of nearly all of the major laboratories with interests in parasite chemotherapy. Because of the immune reaction initiated in the host by this parasite, the duration of

mintics.

Inc. resewed.

This

communication

presents

an

evaluation of “steroid sustained” infections of Nippostrongylus brasiliensis in the rat as a test model for anthelmintic screening. 10

Co yri ht 0 1973 by Academic Press, ~$rfg%ts of reproduction in any form

Dexamethasone; Steroid; ChemoCambendazole; Parbendazole;

N. bUZSih43?l8k: DEXAMETHASONE EFFECT TABLE

11

I

Zncrease of Body Weight of Growing Rats Relative to Nippostrongylus Injection and Dexamethasone Treatment

brasiliensis

Mean f SD percent gain of body weight6 Dexamethasonea (mg X lo-‘/ml)

Parasitized

20.0 10.0 5.0 2.5 2.5 None None

Yes Yes Yes Yes No Yes No

10 Day

59 f 64 f 134 f 135 f

9 9 12 16

17 Day

(30) (10) (30) (20)

- 4f 21 f 44 f 93 f 106 f 197 f 208 f

0.7 7 10 18 11 26 24

(10)" (10) (10) (40) (10) (40) (20)

a Concentration of dexamethasone in the drinking water. b Based upon mean body weight at start (range = 42-61 g) and 10 or 17 days of treatment. Numbers in parentheses indicate the number of animals weighed at that interval. c Only three animals survived. MATERIALS AND METHODS

The strain of N. bra.dknsis used in this laboratory originated from the University of Maryland and was supplied by Dr. R. B. Wescott of the University of Wisconsin. The culture methods were essentially those of Standen ( 1963), and a routine exposure to 200 infective larvae was made with a single subcutaneous injection. Commercially obtained male Sprague-Dawley rats, weighing 51 f 7 g, were housed in 25 x 45 x 15-cm polycarbonate cages in air-conditioned quarters. Food (commercial rat chow) and water were available ad lib. Groups of 10 rats each were used for the dexamethasone effect and benzimidazole ( thiabendazole, cambendazole, and parbendazole) anthelmintic studies, while five rats were used for each of the 10 candidate anthelmintic materia1s.l A total of 465 rats was utilized in these studies. Single doses of the anthehnintics were administered by intragastric intubation using a blunt 1%gauge needle. The animals 1 These compounds represent anthelmintically active organophosphate and heterocyclic materials selected to confirm the parasiticidal response observed with the benzimidazole drugs.

were then maintained 24 hr without feed, killed with carbon dioxide, eviscerated, and the small intestine examined for parasites. Counting of the nematodes was facilitated by pressing the intestines between glass plates. An injectable solution of dexamethasone ( Azium, Schering Corporation, Bloomfield, NJ ) containing 2 mg of drug per ml was added to the drinking water of the test animals at concentration of 2.5 x 10-4-20.0 x 1O-4 mg/ml. Statistical evaluation of the body-weight data was made by an analysis of variance. RESULTS

Rats receiving 20 x 1O-4 mg/ml of dexamethasone in the drinking water for 17 days became listless, there was evidence of inflamed eyelids, a moderate to severe diarrhea, and coughing. Seven of the 10 test animals died. At autopsy, these animals had a marked intestinal inflammation and the N. bm.siZiems-ispopulations had moved from the proximal to the distal portions of the small intestine. None of the survivors demonstrated a gain of body weight (Table I) which is characteristic of steroid therapy. The remaining signs were more probably

12

HASS

those of a secondary microbial infection. Reduction of the steroid concentration diminished severity of the drug effect and the depression of weight gain, At 2.5 x 1tY4 mg/ml, only the diminished body weight gain was noted. Ten days after initiating the studies, steroid-treated (2.5 x 1O-4mg/ ml) rats increased their body weights about 60%. In contrast, the nonsteroid-treated rats gained up to 135% of their initial body weight. The inhibition of growth induced by dexamethasone was further emphasized at the 17-day interval. Also, at 17 days, a 10% reduction in growth could be attributed to parasitic infection, The mean body weights determined for these groups are graphically presented in Fig. 1. An analysis of variance with these data disclosed that all of the steroid-treated rat body weights were significantly reduced (P 2 0.01) relative to both parasitized and control groups. Further, the differences of weight gains between the parasitized and nonparasitized rats were also significant (P 5 0.01) but only at the 17-day interval. The mean number of N. bra..sdi&s recovered from the dexamethasone-treated and control rats at 10 or 17 days postexpo-

l4Oti

IO

0 DURATION

I?

(DAYS)

FIG. 1. Effect of Nippostrongylus brasiliensis infection and/or dexamethasone upon rat growth.

TABLE

II

Numbers of Nippostrongylus brasihensis Recoveredat 10 and 17 Days Postexposure from Dexamethasone-Treated and Control Rats

Dexamethasonea (mg X 10-4/ml) 20.0 10.0 5.0 2.5 0

Mean number worms f SD (no. animals) b 10 Day

128 f 25 (10) 85 f 18 (15)

17 Day 80 + 83 f 63 f 81 f 45 i

14 7 15 26 25

( 3)c (10) (10) (50) (60)

5 Amount of dexamethasone mixed in the drinking water. b Each animal received a standard exposure of 200 infective larvae. c Only 3 of 10 rats survived.

sure are shown in Table II. Rats given 2.5 X 1O-4 mg/ml of dexamethasone in the drinking water had approximately 50% more worms present at 10 days postexposure versus those found in the control animals. One week later ( 17 days), approximately 80% more worms were counted from the steroid-treated rats. However, the parasite populations were also reduced by 45% (control) or 37% (steroid treated) at this same 17-day observation. No significant differences were noted in the numbers of worms recovered at 17 days relative to the concentration of steroid in the drinking water. Three known anthehnintic drugs, thiabendazole, cambendazole, and parbendazole, were tested for their anthehnintic effimciency against 17-day-old N. bra.&eti infections in rats given dexamethasone at 2.5 x 1C4 mg/ml in the drinking water continuously from the day of parasite exposure. Similarly, nonsteroid-treated rats received the same dosages of the three benzimidazole anthelmintics. These data, along with the established lo-day routine test data, are summarized in Table III. Ex-

N. bra.dien.sb:

DFXAMETHASONE

TABLE E$cucy

EFFECT

13

III

of Three Benzimidazole Anthelmintics in Nippostrongylus brasiliensisInjected Rata Treated with Dexamethasone in the Drinking Water

Mean i SD percent reduction of parasite populations at 17 days postexpsoure” lo-Day routine

17-Day steroida

17-Day routine

250 125 62

84 f 22 56 f 28 23 i 16

47 f 24 37 f 14

99*

Cambendazole

250 125 62

82 f 22 78 f 20 67 f 25

87i 88f 78f

500 250 125

22 f 15 18 f 14 15 * 12

19 i 12

Parbendazole

Drug

Thiabendazole

Dose (wdkg)

8 9 11

2

100

99f 98f

6 4

85 &221

0

a Based upon the mean number of worms recovered from representative controls versus those worms from the treated animals. Each value represents at least 10 or more rats tested. b Received dexamethasone at 2.5 X 1W4 mg/ml in the drinking water from the date of parasite exposure to end of study (17 days).

pressed as the mean percentage reduction of parasite populations, it was found that thiabendazole at 62 mg/kg effectively removed one-fourth of the lo-day nonsteroid treated, three-eighths of the 17-day dexamethasone-treated, and all of the 17-day nonsteroid treated parasite populations. Similarly, cambendazole removed from two-thirds to nearly all of the three types of parasite populations. In contrast, a single 500 mg/kg dosage of parbendazole had little effect upon the 17-day steroid-treated or lo-day natural parasite populations. Increased dosages of thiabendazole or cambendazole did not appreciably change the anthelmintic efficiency between the lo-day routine and the 17-day dexamethasonetreated anthelmintic test systems, although each did respond to the greater amounts of drug used. Data for an additional 10 candidate materials also subjected to a differential comparison of anthelmintic efficacy are summa-

rized in Table IV. With the exception of the first and last compounds (candidate materials A and J, Table IV), there was a definite decrease of anthelmintic efficacy associated with the steroid treatment. The first material (candidate material A, Table IV) did not demonstrate a difference of response associated with either the control or steroid-treated group. The last material (candidate material J, Table IV) proved to be toxic for all of the steroid-treated rats, while only one of the control animals (receiving the same dosage) ‘died of drug intoxication. To determine whether the dexamethasone treatment was influencing the anthelmimic efficacy of the above-mentioned materials, a special test was included and is summarized in Table V. Using a standard 12.5 mg/ml suspension of thiabendazole, two groups of rats with 17-day-old infections (control and steroid treated) were dosed at 125 mg/kg on the basis of their

14

HAS

mean group body weights and the dose volumes noted. Two additional groups of infected rats then received the reciprocal dosage volume which, because of body weight differential accorded to steroid therapy, resulted in doses of 192 and 81 mg/kg. The steroid-treated rat parasitic populations were reduced by ca 75% while both of the control groups were totally cleared.

That dexamethasone can adversely affect the growth of young rats is exempli6ed by the percentage weight gains recorded in Table I. The N. bra.siliensis infections also inhibited growth as reflected by the weight gains, although this response was not as marked as with the steroid therapy. An IV

in NippoEficacy of 10 Candidate Anthelmintics strongylus brasiliensis-Infected Rats Treated with Dexamethasone

in the Drinking

water

Mean f SD percent reduction of parasite populations at 17 days postexposure” Candidate material

Dose (mg/kg)

Treated b

Controls

A B C D E F G H I J

500 500 500 500 250 125 125 62 62 31

26 f 12 69f 9 76 f 20 95f 4 69 f 32 51 f 16 47 f 15 73 f 17 45 f 16 Toxicc

11 f 10 100 100 100 99 32 1.5 93 f 12 100 99 f 1.5 69 f 19 Od

DBased upon mean numbers of worms recovered from representative controls versus those worms from the treated animals (five rats per treatment group). *Received 2.5 X 10e4 mg/ml of dexamethasone in the drinking water from the date of parasite exposure to end of study (17 days). c Five of five rate died from drug intoxication. d One of five rats died.

V

Injluence of Dexamethasone and Dosage Volume upon Anthelmintic Ejicacy of Thiabendazole in

Nippostrongylus

brasiliensis-

Infected Rats

Groupa

DISCUSSION

TABLE

TABLE

1 2 3 4

Mean f SD percent reduction of parasite populations at 17 days postexposurec

Mean body weight cd 153 i 100 f 99 f 154 f

Dose (w/k) * 6 8 7 5

125 125 192” 81f

Treatedd

Control 100

75 f 28 77 f 35 100

a Ten rata per group. *Prepared as a 12.5 mg/ml suspension of thiabendaaole in 1y0 methocel solution. c Based upon mean numbers of worms recovered from representative controls vs those worms from the treated animals. dReceived dexamethasone at 2.5 X 10-j mg/ml in the drinking water from day of parasite exposure to end of study (17 days). e Group 3 received the same dosage volume as did Group 1. f Group 4 received the same dosage volume as did Group 2.

analysis of variance of the body weights (Fig. 1) showed that the parasitic infections inhibited growth significantly (P < 0.01) at the 17-day but not the lo-day level. Dexamethasone, at a concentration of 2.5 x lo-” mg/ml in the drinking water, caused a more marked inhibition of growth which was significant (P < 0.01) at both the lo- and 17-day intervals. The parasitic infection combined with steroid therapy further compounded the reduced body weight gains and the effect was also significant (P < 0.01) at both the lo- and 17day observations. The reduced weight gains experienced with the steroid-treated rats are not necessarily an adverse effect from the therapeutic point of view. Decreased body weights require lesser amounts of chemical to accomplish the same effect for

N.

brasiliensis:

DEXAMETHASONE

a given dosage. This can be especially valuable when the investigator is working with limited quantities of a new candidate material. During the course of these studies, several control and steroid-treated groups of rats were observed for the effect of dexamethasone upon the parasitic infections. From these data (summarized in Table II), it was found that at 10 days postexposure, approximately 85 of the 200 infective larvae had matured to adult worms in the nonsteroid-treated rats. One week later, these populations had been reduced in number to approximately 45 adult worms. The addition of dexamethasone to the drinking water has two apparent effects upon the populations. First, the numN. bm.silk& ber of larvae maturing to adult worms is increased by ca 50% ,at the lo-day interval. Second, the numbers of worms recovered 1 week later are more uniform and of greater number than those found with the nonsteroid-treated animals. Thus, dexamethasone apparently promotes greater numbers of worms to establish residence initially and reduces variability of individual parasite populations at a time when they would be grossly affected by immune reactions from the host. Preliminary investigation found that dexamethasone at a concentration of 2.5 x lo-’ mg/ml in the drinking water aided the establishment of a greater and more uniform infection at the 17-day postexposure interval. This phase of the study was followed by the evaluation of three benzimidazole anthelmintics, thiabendazole, parbendazole, and a relatively new product, cambendazole, as the drug standards. These materials were chosen on the basis that a single oral dosage could be selected so as to yield a partial response. Thus, an increase of efficiency could be matched with a change of dosage or parasite susceptibility. The anthelmintic efficacy of these benzimidazole parasiticides, expressed

EFFECT

15

as percentage reduction of the parasite populations (Table III), did not improve when tested against the dexamethasonetreated N. bms-iliensis infections. Indeed, the anthelmintic response with the “steroid sustained” groups was considerably less than that observed with the control groups. Further, when anthelmintic efficacy data taken from our lo-day routine screening program were compared with these observations, no significant differences could be detected between the lo-day routine and the 17-day “steroid sustained” methods of study. These observations were further exemplified when a variety of candidate materials was also included in the comparison studies of anthelmintic activity (Table IV). With one of the candidate materials, there was no change in anthelmintic efficiency regardless of whether the steroid was administered. In those cases in which activity was well demonstrated with the control 17-dayold parasitic infection, the anthelmintic efficiency was generally marginal with the ~corresponding “steroid sustained” infections. Also to be noted was that one of the chemicals (candidate material J, Table IV) was not active as an anthelmintic with the 17-day-old parasitism and one animal from the control group died from drug toxicity. When the dexamethasone-treated animals were given this chemical, the entire group succumbed to drug intoxication. Thus, one must speculate that the steroid, dexamethasone, also has the capacity to potentiate drug toxicity. An additional factor investigated was the relationship of dosage volume relative to thiabendazole efllcacy and the dexamethasone-treated parasitic infections. Steroidtreated and control groups of N. brasilienA-infected animals were subdivided, weighed, and one set dosed with a standard volume (based upon body weight) of thiabendazole (Table V). Owing to body weight differences as reflected bv the ster-

16

HAS

oid therapy, reciprocal administration of the same 125 mg/kg dosage volumes given the first group resulted in doses of 192 and 81 mg/kg for the second subgrouping of parasitized rats. However, regardless of the dosage, only three-fourths of the dexamethasone-treated parasitic infections were expelled by dose levels equal to or 50% greater than the basic 125 mg/kg. Conversely, a one-third reduction of the basic dose to 81 mg/kg resulted in total elimination of the parasitic populations. These data, then, confirmed that dexamethasone is the major factor influencing the anthelmintic activity rather than amount or volume of the dose. The hypothesis associated with this study was that an aged N. bra&lien& infection should be more susceptible to chemotherapeutic intervention. It has been well described ( Ogilvie and Jones 1971) that the immune response effected by the host will irreversibly damage worms 10 days or more of age. In all probability, therefore, 17-dayold worms should be much more susceptible to effective anthelmintic therapy. Indeed, this has now been shown to be an The dexamethasoneestablished fact. treated infections, however, continued to respond in a fashion similar to the standard lo-day-old infection when treated with known anthelmintics. This response indicates that the age of the infection does not

change the overall parasiticidal efficacy. Further, these data confirm the significant damage inflicted upon the parasite by the rat’s immune response mechanisms. ACKNOWLEDGMENTS The author expresses his sincere appreciation to Mrs. J. J. Boudreau, Mrs. C. J. Signorelli, and Mr. D. B. Holtzclaw for their very able technical assistance. Statistical analyses of the data were conducted by Dr. L. J. Brown. Thanks are also extended to Dr. W. C. Campbell, Merck Institute for Therapeutic Research, Rahway, NJ, for samples of thiabendazole and cambendazole, and to Dr. V. Theodorides, Smith, Kline and French Laboratories, West Chester, PA, for the sample of parbendazole.

REFERENCES OGILVIE, B. M. 1965. Use of cortisone derivatives to inhibit resistance to Nippostringylus brasilien& and to study the fate of parasites in resistant hosts. Parasitology 55, 723-730. OGILVIE, B. M., AND JONES, V. E. 1971. Nippostrongylfts brasiliensis: A review of immunity and the host-parasite relationship in the rat. Experimental Parasitology 29, 138-177. STANDEN, 0. D. 1963. Chemotherapy of helminthic infections. In “Experimental Chemotherapy” eds. ) VoI. (R. J. Schnitzer and F. Hawking, I, Academic Press, New York. WHITLOCK, J. H. 1943. Characteristics of the population available for bioassay of anthehnintics in Nippostrongylus muris infections in albino rats. Journal of Parasitology 29, 42-47. WHITLOCK, J. H., AND BLISS, C. I. 1943. A bioassay technique for anthehnintics. Journal of Pamsitology 29, 48-58.