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Studies on chemotherapy of parasitic helminths: Effects of avermectin Bla on Angiostrongylus Cantonensis in rats
lnternatlonai Journal for Parasitology Vol. 13, No, 5, pp. 49 I--498, 1983. Prtnted in Greal Britain.
0020-7519/83 23.00 + 0.00
Pergamon Pres~ Ltd.
1983 Al~trallon $ociety for l~r~z~llology
S T U D I E S O N C H E M O T H E R A P Y OF P A R A S I T I C H E L M I N T H S : EFFECTS OF A V E R M E C T I N B 1a O N ANGLOS TR ONG YL US C A N T O N E N S I S IN RATS A . I. I s a n , M . TERADA, H . IONo, M . HAYASHI a n d M. SAr¢O Department of Parasitology, Hamamatsu University School of Medicine, Hamamatsu, Japan (Received 4 October 1982) Abstract~Ismt A. 1., T~rtAt~AM., IONOH., HAYAStnM. and SANoM. 1983. Studies on chemotherapy of parasitic helminths: effects of avermectin Bla on Angioszrong.vlus cantonensis in ra~s. International Journal for Parasiloiogy 13: 491--498. The in viva effects of avermectin Bin against Angiostrong),lus eantonen$is in rata were examined. Two distinguishable effects of this drug were suggested; a paralyzing effect against adult worms and a direct vcrmicidal effect against larval worms. When the drug was given intraperitoneallyat O.0l or 0-1 mr/ks at 6 weeks after infection, a sustained paralysis of worms by this drug was suggested from the following aspects; the change of the first stage larval count in feces, histological observations of lung tissues of the host and the reproductive system of female worms, and the motility of recovered worms i~ vilro. However, no direct vermicidal effect was observed in this treatment, because there was no significant difference in the recovery rate of worms from that of controt rats. On the other hand, when the drug was given orally at a dose of 1-0 rag/ks at 3 days after infection (larval stage treatment), a significant reduction in the recovery rate of worms was observed compared to those in the adult stage treatment (drug administrationat 7 weeks after in fection) and control groups. INDEX KEY WORDS: Angiostrongylus cantonens~ avermeetin Bla; chemotherapy; in viva effects; paralysis; rat. INTRODUCTION AVEItMECTIJ¢ Bin is one of the antiparasitic macrocyclic lactones produced by a ~ described actinomycete, Streptomyces avermitilis (Burg, Miller, Baker, Birnbaum, Currie, Hartman, gong, Monaghan, Olson, Putter, Tunac, Wallick, Stapley, Oiwa & Omura, 1979; Miller, Chalet, Cole, Cole, Flor, Goegelman, Gullo, Joshua, Kempf, Krdtwitz, Monaghan, Ormond, Wilson, Albers-Schonberg & P u t t e r , 1979; E g e r t o n , O s t l i n d , Blair, Eary, Suhayda, Riek& Campbell, 1979). There have been many reports referring to the in viva effects of this c o m p o u n d against gastro-intestinai a n d blooddwelling nematodes and otto-parasitic arthropods o f domestic animals such as sheep, cattle, dogs and chickens (Blair & Campbell, 1978a,b; Campbell & Blair, 1978; Egerton e t a L , 1979; James, Picton & Rick 1980; Roncalli, 1980). Recently the paralyzing effects of avermectin B l a on a rat lungworm, Angiostrongylus cantonensis have been observed at concentrations o f 3"6 × 10-'s M or more, using an in vitro method that has been developed for studying the effects of drugs o n the motility of smaUe.• heiminths (Sano, Terada, Ishii & Kino, 1981; Sano, Terada, Ishii, Kino & Hayashi, 1981). Because of the medical importance of h u m a n angiostrongylosis causing eosinophilic m e n i n g o cephalitis, studies have bccn carried out to determine
the efficacy of certain drugs such as thiabendazole (Cuckler, Egerton & Alicata, 1965; Nishimura, 1965/ 1966), /-tetramisole (Jindrak & Alicata, 1969) and mebendazole (Larnmler & Weidner, 1975) against this parasite in viva. However, no a d e q u a t e chemotherapy has been introduced for this parasitic disease. In the present study, the effects o f avermectin Bla on A. cantonensis in rats were examined to determine the in viva effects and also to define its relationship to the in vitro effects.
491
MATERIALS AND METHODS Materials. The tested compound, avermectin Bin (MK931, L-686, 895-4)0P)was offered from Merck Sharp & Dohme Research Laboratories and was dissolved in propylene glycol for administrationto rats, Animal treatment. Male Wistar albino rats. 4 weeks old. wet.~ used in all experiments as a definitive host. Infective third stage larvae of Angiostrongylus cantonensis were obtained from experimentally infected snails, Biomphalaria glabrata, by artificial digestion using 0.2% pepsin for I hr at 370C. in experiment I, the relationship between the dose of drug and its effects on the adult stage was examined. All animals were inoculated through an esophageal tube with 40 larvae suspended in Tyrode's solution and divided into two treated groups of 10 and a control group of five rats. At 6 weeks a~cr infection, when adult worms were present and fi~t stage larvae were dete~ted in rat's fecet, the two treated groups received avermccfin Bin intraperitoneallyas
A. I. lgHn, M. TERADA,H. Kl~rO,M. I'IAYASHIand M. SANd
492
I.J.P. VOL. 13. 1983
a single dose of 0-01 or 0-1 mg/kg of body weight, respec- difference in the recovery rates between the treated tively. The infected control group received equivalent and control groups. volumes of vehicle only, In addition, a second treatment The first stage larval count per female worm was was performed in the 0.1 mg/kg treated group at 13 weeks shown as an average number from three rats in each after infection at the same dose rate. From 6 weeks after infection, the first stage larval count in the feces was o f treated and control groups. As shown in Fig. l, examined at weekly intervals from three rats in each group, the larval count remarkably decreased in both treated being represented as larvae per gram of feces (LPG) per live groups of 0.01 and 0.1 m g / k g at 2 weeks after treatadult female worm recovered. At 2 days and 4 weeks after ment. At 3 weeks after treatment, the larval count treatment, respectively, one rat from the 0-01 mg/kg slightly increased in the 0.01 m g / k g treated group, treated group was sacrificed and the motility of recovered but no first stage larva could be detected in the 0.1 worms was observed using the isotonic transducer method m g / k g treated group. From 4 weeks after treatment, (Sand, Terada, Ishii, Kind & Hayashi, 19811. As to the 0.l however, larvae reappeared and after 5 weeks the mg/kg treated group, observations of the motility were carried out at 2 and 4 weeks after the first treatment. For number (LPG) increased to the same level as that o f histological observations of rat's lung tissue, one rat from the control group. In the second treatment for the the 0.01 mg/kg treated group was sacrificed at 2 days, !, 2, 0.1 m g / k g treated group, a similar pattern to the first 3 and 4 weeks after the first treatment, respectively, and one treatment was observed. from the control group at 10 weeks after infection. The On histological observations o f lung tissues by rest of the treated and control groups were sacrificed 20 hematoxylin and eosin staining in the 0.1 mg/kg weeks after infection and the number of worms in the heart treated group, some interesting results were obtained and lungs was examined to determine the recovery rate. In (Fig. 2). The sections o f lung tissue at 2 days after experiment 2, the relationship between the worm stages and treatment showed almost the same features as those effects of drug was examined. All animals were inoculated orally with 20 larvae and divided into two treated and two o f the control lung tissue, showing the various stage control groups of 15, 10, 4 and 6 rats, respectively. Aver- eggs and the first stage larvae. At 1 week, however, the early stage eggs remarkably decreased. At 2 mectin Bla in propylene glycol was admit~i~tered orally to weeks, few early stage eggs were seen and the first the treated groups with a single dose of 1.0 mg/kg at 3 days or 7 weeks after infection. One control group was given stage larvae markedly decreased. At 3 weeks very few propylene glycol in volumes equivalent to that administered first stage larvae were observed but unicellular eggs to the treated groups. The other control received no treator early stage eggs reappeared, suggesting that the meat, The number of the first stage larvae in feces was reproduction o f worms was slightly restored. At 4 counted from 7 weeks after infection. All rats were sacriweeks after treatment, the various stage eggs and the ficed at I 1 weeks after infection, and the numbers of worms first stage larvae were observed similar in appearance present was determined. Pharrn~cological t~ts. The whole worm preparation was to the control section. It is intere~ing that the female suspended in Tyrode's solution in a thermostatically con- worms [n all observed sections of the treated group had eggs in their uteri and that the internal structure trolled organ bath (7 ml capacity) at 35"C and gassed o f eggs seemed not to be disorganized as far as could slightly with air. Responses of the preparation to drugs were recorded isotonically on a recorder (Toa, EPR.100A) be seen with a light microscope. with an isotonic transducer (Nihon Koden, TD-112S), Figure 3 shows the tnotility o f recovered worms producing a magnification of 15-30-fold and exerting a recorded by the isotonic transducer method. Retension of 0.g-1.0 g. The following drugs were used: covered worms could respond against mechanical eserine salicylate ISigma], picrotoxin hydrochloride [Tokyokaseil, and s-methyicytisine (r~-MC), an alkaloid stimuli to produce a distinct and transient movement. from Sophoraflavescens, was kindly offered from Dr. T. The motility o f all worms recovered both at 2 days after treatment with 0.01 m g / k g and at 2 weeks after Noro (Shizuoka College of Pharmacy). treatment with 0.01 m g / k g was observed to be remarkably inhibited. The motility o f some o f the RESULTS worms recovered at 4 weeks after treatment with 0.01 or0-1 mg/kg was, however, slightly restored, Though The relationship between the dose o f drug and its eserine (an inhibitor o f eholineslerase) and N-methyleffects on the adult stage (experiment D cyctine (N-MC) (a stimulator o f release o f aeetylAs summarized in Table 1, there was no significant choline) caused a remarkable contraction in control TABL~I--EFrEcTsor AV~Pa~ECTINBla oN ,4. cantonensis tN PArs WHENGIVESar~TR^PEStTONE^LtVAr 6 WEEKS ^~t ~R I~ECTtO~ Dosage avermectin
No. of rats used
B Ia (mg/kg) 0"01 0"I None
8 5 4
No. of larvae
No. of worms
Recover rate
given
recovered (Mean -. S.D.)
(%)
40 40 40
34'9 ± 1.96 34"8 :I:0"45 36-0 ± 2-45
~7.2 87'0 90.0
There is no significant difference in the recovery rates (Student's t test).
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In vivo effects of avermectin Bla on A. cantonertsis
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Fzo. 1. Changes of the mean number of the first stage larvae per gram feces per female worm of A. cantonensis. The mean number was calculated from LPG examined for three rats in each group. Az the point DRUG, avermectin Bla was administered to rats in treated groups. A _ A; control group, • - - O; treated group with a dose of 0.01 mg/kg once at 6 weeks after infection. O - - C); tr¢atccl group with a dose of 0.1 mg/kg twice at 6 and 13 weeks after infection.
worms, these spasmogens had little effect on the worms pretreated with avermectin B l a in rive.
The relationship between the worm stages and effects o f drugs (experiment 2) Table 2 shows results o f the recovery rate in experiment 2. In the larval treatment, avermectin B l a at. 1-0 m g / k g gave a significant reduction o f about 55% in the recovery rate compared to those o f control and adult treatment groups. In addition, it is interesting to note that a few males and females from the adult treatment group were yellowish in colour and were stretched loosely compared to those from the control group. Furthermore, four females T ~ t ~ 2--EF~c'T'S OF AVERMECnN Trealment
f r o m three rats of the adult treatment group apparently showed disorganization of internal structures (Fig. 4). On fecal examination, the first stage larvae could be detected in the larval treatment group, but no larvae were found in the adult treatment group until the rats were sacrificed 4 weeks after treatment. Eggs at various stag¢~ o f development and first stage larvae were observed in histological sections of lung tissues from rats in the larval treatment group. In the adult treatment group, however, very few unicellular eggs and no larvae were seen at 4 weeks after treatment, though many eggs were observed in the uteri (Fig. 5).
B l a ON ,4. o m l o n e r t s i , s IN r~ATS WHEN OWEN OIC~LLY AT 3 DAYS OK '7 WIEF.KS AFTER L~IFECTION
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No. of rats used
No. of larvae given
after infection Avermeczin Bla £.0 mi~/k s
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Fzo. 2. Light micrographs of rat lung tissues in the control and the O.I mg/kg treated groups. Note the apparent changes of the number of eggs and larvae in the lung tissue with the lapse of time after treatment and the presence of eggs in the uteri of A. cantonensis in the 0.1 mg/kg tr~tcd group. 2a; control group: 2b-2f; 0.t mg/kg treated group, 2b; 2 days, 2c; i week, 2d; 2 weeks, 2c; 3 weeks, 21";4 weeks after treatment. Scale bar; 0.5 ram. DISCUSSION it was reported that avermectin B l a may act on the nervous system o f various nematodes as well as other animals including .crustacca and mammals, po~slbly through an effect on y-amino-butyric acid (GABA) (Fritz, Wang & Gorio, 1979; Williams & Yarbrough, 1979; Kass, Wang, Walrond & Strctton, 1980; Paul, Skolniek & Zatz 1980). In our in vitro experiments, it was also su~estod that avcrracctin B l a pa.ralyzcs Angiostrongy(~.s cantonen.~, a tissue
parasite, through a neuropharmacological mcchanEsm including G A B A and acetylcholine (Sano, Terada, lshii, Kino & Hayashi, 1981). It is likely that gastro-intestinal nematodes which were paralyzed by the dI~g are expelled by the pcrislalsis o f the host digestive tracl. In the present in vivo experiments, animals wer© treated at 6 wc~ks (Cxl~rimcnt 1) or 7 weeks (cxpcriraent 2) after infection when the inoculawd larvae are known to become adult~ (Maekerras & Sanders,
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Flo. 3. Effects of some neuropharma~olosi~ qczlr~ on the motility of A. (~ntonenxis; u n ~ t ~ l worms and worms pr~'x~led t~]th l,vtrmew~in B I I In rats. ~ w~rc cum~zd*a~ i~v~z and plor~ox~, N - o ~ (N-MC) mu~ in (B), picrmoxin and ~V-MCin (C) and N-MC, p i t . t o x i n and e~rine in (D) were suc,~.~lvety gtvl~. At tl~ pol~ W, the prepzmflon was washed by " i ' ~ e ' s toludon. Eu~rine and N-MC csttu~ It remarkable c~atraction in control wormL but theze spLtmoscns hgvc little elTtgl on the worn~ pre-trealed L¢vivo,
A. I. IsHn, M. TaP,Av^, H. KINo, M, H^Y^sm and M. S^~o
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Fro. 4. Light micrographs of worms from the rontrol (4a) and the 1.0 mg/kg treated (4b) groups. Note the apparent disorganization of internal structures of the worm from the treated group. Scale bar: 4a; 1 mrn, 4b; 5 ram.
Fio. 5. Light micrograph of rat lung tissue at 4 weeks after treatment in the adult treatment group (I.0 mg/kg of avermeetin B1a). Note very few unicellular eggs and no larvae in the lung tissue, though many eggs were observed in the uterus. Scale bar: 0.$ ram. 1955). In these adult stage treatments, no direct vermicidal effect o f avermc~'tin B l a was suggested from the recovery rates o f living worms (Tables 1, 2). On the other hand, there was a remarkable inhibition in the reproduction and this inhibition was sustained and dose-related. From the results o f the larval counts in feces and observatiom o f host lung tissue sections, the reproduction o f eggs might be inhibited for almost 3 weeks in the. O. 1 m g / k g treated gxoup (experiment 1). Furthermore, from the in vitro obser-
rations of worms pretreated with a v e r m ~ t i n B l a in vivoo it is suggested that this drug may temporarily paralyze adult worms in vivo as well as in vitro and therefore, that this inhibition o f motility probably resulted in the reduction o f egg laying. In the 1.0 mg/kg treated group (experiment 2), this inhibition of motility was severe and the larval output was completely inhibited until the hosts were sacrificed at 4 weeks after treatment. Though this drug has no direct vermicidal effect on adult worms, it is pmsible
I.J.P. VOL. 13. 1983
In vivo effects of avermectin Bla on A. cantonensis
that the severely paralyzed worms might be attacked and killed secondarily by host reactions. Regarding the development o f A . cantonensis, it was reported that eggs o f this worm are hatched about 1 week after oviposition (Alicata & Jindrak, 1970), so no larvicidal effect on the first stage larvae is suggested from the observations mentioned above. Similar effects o f this drug have been reported in dogs infected with Dirofilaria immitis (Campbell & Blair, 1978). Treatment with avermectin B l a at a dose o f 0.1 mg/kg completely suppressed microfilaremia for 6 weeks after treatment. However, microfilariae were again demonstrated in the blood on the 9th week after treatment and living adults were present in the heart on the 6th month after treatment. In addition to the inhibitory effects, a direct verrnicidal effect against the larval stage was confirmed which may be the third stage at 3 days after infection. This effect could not be detected in the in vitro experiments because .the adult worm was used for the material (Sano, Terada, Ishii, Kino & Hayashi, 1981). The recovery rate of worms was significantly lower in the larval treatment group than the adult treatment and control groups (Table 2). Similar effects were also r e p o r t ~ in dogs (Campbell & Blair, 1978b) or in ferrets (Blair & Campbell, 1978) infected with 19. immitis. When animals received avermectin B l a orally at a dosage of 0.02 m g / k g on 5 successive days, this drug was highly effective against 2-month-old (in dogs) or 4-month-old (in ferrets) precardiae larvae. To determine this larvicidal effect of avermectin B l a on the third stage larvae o f A . cantonensis in detail, further studies, such as mechanism o f action and stage specificity will be necessary. No grossly notable toxic reactions were observed in any rats treated with the levels of 0.01-1.0 m g / k g o f this drug in the present experiments. However, more detailed studies on side effects, especially on the central nervous system, should be carried out before avermectin B l a will be introduced clinically as a therapeutic against meningoencephalitis caused by A. cantonensis, because the G A B A system in the central nervous system is also reported to be sensitive to this drug (Williams & Yarbrough, 1979; Paul et ai,, 1980).
REFERENCES ALICATAJ. E. & JINDgAKK. 1970. Life cycle and biology. In: Angioatrongylosis in the PaeOic and Southeast Asia (Edited by A~D~I~soN H. H.), pp. 17-27. Charles C. Thomas, Springfield, Illinois. Bt.Am L. S. & C~i,at~t.L W. C. 1978a. Efficacy of avermectins against Anc.viostoma caninum in dogs, Journal o f Helminthoiogy 52: 305-307, B,.^tit L. S. & CmaPaeLa W, C. 1978b. Trial of avermectin Bla, mebendazole and melarsoprol against pre.cardiac DiroJTluria lmmltt, in the ferret (Mustela putorius faro). Journal o f Parasitotopj 64: 1032-1034.
497
Buao, R. W., MtLLea B. M., BAaaa E. E., BtanaAUM J., CuamES. A., HaI~TV,AN R., Kono Y. L., Mo~,~OH.~N R. L., O~o~ G., PVTT~a I., TU~^C J. B., W,~LL|CKH., STAPLaYE. O., Otw^ R. & OMUaAS. 1979. Avermectins, new family of potent anthelmintic agents: Prod~icing organism and fermentation. ¢4ntimicrobial Agents and Chemotherapy 15: 361-367. CA~tPa~LX. W. C. & BLAre L. S. 1978. Efficacy of avermectlns against Dirofilaria immitis in dogs. Journal o f Helm inthoiogy 52 "-308-310. CUCKLEa A. C., EOERXONJ. R. & ALICATAJ, E, 1965. Therapeutic effect of thiabendazolg on Angiostrongylus cantonensis infections in rats. Journal o f Parasitotogy 51: 392-396. EGERTONJ. R., OSTLINDD. A., BLAIRL. S., EARYC. a., SUP.AYD^ D., Ring R. F. & CAMPaELL W. C. 1979. Avermectins, new family of potent anthelmintie agents: Efficacy of Bla component. Antimicrobial Agents and Chemotherapy IS: 372-378. FatTz L. C,, W^No C. C. & OoRIo A. 1979. Avermectin Bla irreversibly blocks postsynaptic potentials at the lobster neuromuscular junction by reducing muscle membrane resistance. Proceedings o f the National
Academy o f Sciences o f the United States o f America 76: 2062-2066. JAMES P. S., PIc'ror~ J. & RInK R. F, 1980. Insecticidal activity of the avermectins. Veterinary Record 106: 59. J a q D ~ K. & A]LteATAJ, E. 1969. Effect of I-tetramisole on Angiostrongylus cantonensis infection in rats. Chemotherapy 14: 244-252. lO,ss I. S., W^No C. C., WALaOr~D J. P. & SraE'rro~ A. O. W. 1980. Avermectin Bla, a paralyzing anthelmintic that affects interneurons and inhibitory motoneurons in Ascaris. Proceedings o f the National
Academy o f Sciences o f the United States o f America 77:6211-6215. L.g~tuLta G. & WEmNER E. 1975. Zur larviziden Wirkung yon Amhelminthika gegen Angiostrongylus cantonensis.
Berliner und Mginchener Tierarztliche WochensehrO't 88: 152-156. MACtC~RaASM. J. & S^r;DArts D. F. 1955. The life history of the rat lung-worm. Angiostrongylus cantonensis (Chen) (Nematoda : Metastrongylldae). Australian Journal o f Zoology 3: 1-21. MtLktm T. W., CmUET L., CoL~ D. J., CoI.~ L. J., FLoa J. E., GO£OELM~..,*R, T,, GULLOV. P., JosardA H,, KF..MPF A. J., KRELLWVfZW, g,, MONAOHANR. L., O~OND R. E., Wit.soN K. E . AL~m,.s-ScHot~aEgoO. & Ptrrr~a I. 1979. Avermectins, new family of potent anthelmintic agents: Isolation and chromatographic properties. Antimicrobial Agents and Chemotherapy !$: 368-371. NtsmMu~A K. 1965/66..Experimental studies on the chemotherapy of rat lungworm, Angiostrongylus cantonettsis, in rats, Chemotherapia 10:164-175. PAut S. M.. SKOt.NtCK P. & ZArz M. 1980. Avermectin Bin: art irreversible activator of the r-aminobutyric acid-b~nzodiazepine-ehloride-ionophore receptor complex, Biochemical and Biophysical Research Communicatibns 96:632--638. RoNc^tu R. A. 1980. Clinical evaluation of the avermecti~s, a new da.~s of broad-spectrura anthdminfic agents. The Argentinian Veterinary Congress, Buenos Aires, Argentina. S ~ o M., Tagm3A M., IsRu A. I. & Kmo H. 1981. Effects o f avermectin Bla on the motility of various parasitic helminths. J~xperiemia 37: 844.846.
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A, I.Ist~ix,M, TERADA, H. lONO, M. HAYASHI and M. SASO
S^No M., TERAO^ M., ISHIIA. 1., KxbtoH. & HAYASHIM. 1981. Studies on chemotherapy of parasitic hclmiths (l). On the in vitro methods and paralyzing e.ffects of avermectin Bia on Angiostrongylus cantonensis. Japanese Journal of Parasitology 30:305-314,
tJ.p. VOL. 13. 1983
WILLJAM.~M. & YARBgOUOHG. G. 1979. Enhancement of in vitro binding and some of the pharmacological properties of diazepam by a novel anthelmintic agent, avermectin Bla. European Journal of Pharmacology $6: 273-276.
0020-7519/83 23.00 + 0.00
Pergamon Pres~ Ltd.
1983 Al~trallon $ociety for l~r~z~llology
S T U D I E S O N C H E M O T H E R A P Y OF P A R A S I T I C H E L M I N T H S : EFFECTS OF A V E R M E C T I N B 1a O N ANGLOS TR ONG YL US C A N T O N E N S I S IN RATS A . I. I s a n , M . TERADA, H . IONo, M . HAYASHI a n d M. SAr¢O Department of Parasitology, Hamamatsu University School of Medicine, Hamamatsu, Japan (Received 4 October 1982) Abstract~Ismt A. 1., T~rtAt~AM., IONOH., HAYAStnM. and SANoM. 1983. Studies on chemotherapy of parasitic helminths: effects of avermectin Bla on Angioszrong.vlus cantonensis in ra~s. International Journal for Parasiloiogy 13: 491--498. The in viva effects of avermectin Bin against Angiostrong),lus eantonen$is in rata were examined. Two distinguishable effects of this drug were suggested; a paralyzing effect against adult worms and a direct vcrmicidal effect against larval worms. When the drug was given intraperitoneallyat O.0l or 0-1 mr/ks at 6 weeks after infection, a sustained paralysis of worms by this drug was suggested from the following aspects; the change of the first stage larval count in feces, histological observations of lung tissues of the host and the reproductive system of female worms, and the motility of recovered worms i~ vilro. However, no direct vermicidal effect was observed in this treatment, because there was no significant difference in the recovery rate of worms from that of controt rats. On the other hand, when the drug was given orally at a dose of 1-0 rag/ks at 3 days after infection (larval stage treatment), a significant reduction in the recovery rate of worms was observed compared to those in the adult stage treatment (drug administrationat 7 weeks after in fection) and control groups. INDEX KEY WORDS: Angiostrongylus cantonens~ avermeetin Bla; chemotherapy; in viva effects; paralysis; rat. INTRODUCTION AVEItMECTIJ¢ Bin is one of the antiparasitic macrocyclic lactones produced by a ~ described actinomycete, Streptomyces avermitilis (Burg, Miller, Baker, Birnbaum, Currie, Hartman, gong, Monaghan, Olson, Putter, Tunac, Wallick, Stapley, Oiwa & Omura, 1979; Miller, Chalet, Cole, Cole, Flor, Goegelman, Gullo, Joshua, Kempf, Krdtwitz, Monaghan, Ormond, Wilson, Albers-Schonberg & P u t t e r , 1979; E g e r t o n , O s t l i n d , Blair, Eary, Suhayda, Riek& Campbell, 1979). There have been many reports referring to the in viva effects of this c o m p o u n d against gastro-intestinai a n d blooddwelling nematodes and otto-parasitic arthropods o f domestic animals such as sheep, cattle, dogs and chickens (Blair & Campbell, 1978a,b; Campbell & Blair, 1978; Egerton e t a L , 1979; James, Picton & Rick 1980; Roncalli, 1980). Recently the paralyzing effects of avermectin B l a on a rat lungworm, Angiostrongylus cantonensis have been observed at concentrations o f 3"6 × 10-'s M or more, using an in vitro method that has been developed for studying the effects of drugs o n the motility of smaUe.• heiminths (Sano, Terada, Ishii & Kino, 1981; Sano, Terada, Ishii, Kino & Hayashi, 1981). Because of the medical importance of h u m a n angiostrongylosis causing eosinophilic m e n i n g o cephalitis, studies have bccn carried out to determine
the efficacy of certain drugs such as thiabendazole (Cuckler, Egerton & Alicata, 1965; Nishimura, 1965/ 1966), /-tetramisole (Jindrak & Alicata, 1969) and mebendazole (Larnmler & Weidner, 1975) against this parasite in viva. However, no a d e q u a t e chemotherapy has been introduced for this parasitic disease. In the present study, the effects o f avermectin Bla on A. cantonensis in rats were examined to determine the in viva effects and also to define its relationship to the in vitro effects.
491
MATERIALS AND METHODS Materials. The tested compound, avermectin Bin (MK931, L-686, 895-4)0P)was offered from Merck Sharp & Dohme Research Laboratories and was dissolved in propylene glycol for administrationto rats, Animal treatment. Male Wistar albino rats. 4 weeks old. wet.~ used in all experiments as a definitive host. Infective third stage larvae of Angiostrongylus cantonensis were obtained from experimentally infected snails, Biomphalaria glabrata, by artificial digestion using 0.2% pepsin for I hr at 370C. in experiment I, the relationship between the dose of drug and its effects on the adult stage was examined. All animals were inoculated through an esophageal tube with 40 larvae suspended in Tyrode's solution and divided into two treated groups of 10 and a control group of five rats. At 6 weeks a~cr infection, when adult worms were present and fi~t stage larvae were dete~ted in rat's fecet, the two treated groups received avermccfin Bin intraperitoneallyas
A. I. lgHn, M. TERADA,H. Kl~rO,M. I'IAYASHIand M. SANd
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I.J.P. VOL. 13. 1983
a single dose of 0-01 or 0-1 mg/kg of body weight, respec- difference in the recovery rates between the treated tively. The infected control group received equivalent and control groups. volumes of vehicle only, In addition, a second treatment The first stage larval count per female worm was was performed in the 0.1 mg/kg treated group at 13 weeks shown as an average number from three rats in each after infection at the same dose rate. From 6 weeks after infection, the first stage larval count in the feces was o f treated and control groups. As shown in Fig. l, examined at weekly intervals from three rats in each group, the larval count remarkably decreased in both treated being represented as larvae per gram of feces (LPG) per live groups of 0.01 and 0.1 m g / k g at 2 weeks after treatadult female worm recovered. At 2 days and 4 weeks after ment. At 3 weeks after treatment, the larval count treatment, respectively, one rat from the 0-01 mg/kg slightly increased in the 0.01 m g / k g treated group, treated group was sacrificed and the motility of recovered but no first stage larva could be detected in the 0.1 worms was observed using the isotonic transducer method m g / k g treated group. From 4 weeks after treatment, (Sand, Terada, Ishii, Kind & Hayashi, 19811. As to the 0.l however, larvae reappeared and after 5 weeks the mg/kg treated group, observations of the motility were carried out at 2 and 4 weeks after the first treatment. For number (LPG) increased to the same level as that o f histological observations of rat's lung tissue, one rat from the control group. In the second treatment for the the 0.01 mg/kg treated group was sacrificed at 2 days, !, 2, 0.1 m g / k g treated group, a similar pattern to the first 3 and 4 weeks after the first treatment, respectively, and one treatment was observed. from the control group at 10 weeks after infection. The On histological observations o f lung tissues by rest of the treated and control groups were sacrificed 20 hematoxylin and eosin staining in the 0.1 mg/kg weeks after infection and the number of worms in the heart treated group, some interesting results were obtained and lungs was examined to determine the recovery rate. In (Fig. 2). The sections o f lung tissue at 2 days after experiment 2, the relationship between the worm stages and treatment showed almost the same features as those effects of drug was examined. All animals were inoculated orally with 20 larvae and divided into two treated and two o f the control lung tissue, showing the various stage control groups of 15, 10, 4 and 6 rats, respectively. Aver- eggs and the first stage larvae. At 1 week, however, the early stage eggs remarkably decreased. At 2 mectin Bla in propylene glycol was admit~i~tered orally to weeks, few early stage eggs were seen and the first the treated groups with a single dose of 1.0 mg/kg at 3 days or 7 weeks after infection. One control group was given stage larvae markedly decreased. At 3 weeks very few propylene glycol in volumes equivalent to that administered first stage larvae were observed but unicellular eggs to the treated groups. The other control received no treator early stage eggs reappeared, suggesting that the meat, The number of the first stage larvae in feces was reproduction o f worms was slightly restored. At 4 counted from 7 weeks after infection. All rats were sacriweeks after treatment, the various stage eggs and the ficed at I 1 weeks after infection, and the numbers of worms first stage larvae were observed similar in appearance present was determined. Pharrn~cological t~ts. The whole worm preparation was to the control section. It is intere~ing that the female suspended in Tyrode's solution in a thermostatically con- worms [n all observed sections of the treated group had eggs in their uteri and that the internal structure trolled organ bath (7 ml capacity) at 35"C and gassed o f eggs seemed not to be disorganized as far as could slightly with air. Responses of the preparation to drugs were recorded isotonically on a recorder (Toa, EPR.100A) be seen with a light microscope. with an isotonic transducer (Nihon Koden, TD-112S), Figure 3 shows the tnotility o f recovered worms producing a magnification of 15-30-fold and exerting a recorded by the isotonic transducer method. Retension of 0.g-1.0 g. The following drugs were used: covered worms could respond against mechanical eserine salicylate ISigma], picrotoxin hydrochloride [Tokyokaseil, and s-methyicytisine (r~-MC), an alkaloid stimuli to produce a distinct and transient movement. from Sophoraflavescens, was kindly offered from Dr. T. The motility o f all worms recovered both at 2 days after treatment with 0.01 m g / k g and at 2 weeks after Noro (Shizuoka College of Pharmacy). treatment with 0.01 m g / k g was observed to be remarkably inhibited. The motility o f some o f the RESULTS worms recovered at 4 weeks after treatment with 0.01 or0-1 mg/kg was, however, slightly restored, Though The relationship between the dose o f drug and its eserine (an inhibitor o f eholineslerase) and N-methyleffects on the adult stage (experiment D cyctine (N-MC) (a stimulator o f release o f aeetylAs summarized in Table 1, there was no significant choline) caused a remarkable contraction in control TABL~I--EFrEcTsor AV~Pa~ECTINBla oN ,4. cantonensis tN PArs WHENGIVESar~TR^PEStTONE^LtVAr 6 WEEKS ^~t ~R I~ECTtO~ Dosage avermectin
No. of rats used
B Ia (mg/kg) 0"01 0"I None
8 5 4
No. of larvae
No. of worms
Recover rate
given
recovered (Mean -. S.D.)
(%)
40 40 40
34'9 ± 1.96 34"8 :I:0"45 36-0 ± 2-45
~7.2 87'0 90.0
There is no significant difference in the recovery rates (Student's t test).
t . ~ . e , VOL. 13.
In vivo effects of avermectin Bla on A. cantonertsis
1983
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Fzo. 1. Changes of the mean number of the first stage larvae per gram feces per female worm of A. cantonensis. The mean number was calculated from LPG examined for three rats in each group. Az the point DRUG, avermectin Bla was administered to rats in treated groups. A _ A; control group, • - - O; treated group with a dose of 0.01 mg/kg once at 6 weeks after infection. O - - C); tr¢atccl group with a dose of 0.1 mg/kg twice at 6 and 13 weeks after infection.
worms, these spasmogens had little effect on the worms pretreated with avermectin B l a in rive.
The relationship between the worm stages and effects o f drugs (experiment 2) Table 2 shows results o f the recovery rate in experiment 2. In the larval treatment, avermectin B l a at. 1-0 m g / k g gave a significant reduction o f about 55% in the recovery rate compared to those o f control and adult treatment groups. In addition, it is interesting to note that a few males and females from the adult treatment group were yellowish in colour and were stretched loosely compared to those from the control group. Furthermore, four females T ~ t ~ 2--EF~c'T'S OF AVERMECnN Trealment
f r o m three rats of the adult treatment group apparently showed disorganization of internal structures (Fig. 4). On fecal examination, the first stage larvae could be detected in the larval treatment group, but no larvae were found in the adult treatment group until the rats were sacrificed 4 weeks after treatment. Eggs at various stag¢~ o f development and first stage larvae were observed in histological sections of lung tissues from rats in the larval treatment group. In the adult treatment group, however, very few unicellular eggs and no larvae were seen at 4 weeks after treatment, though many eggs were observed in the uteri (Fig. 5).
B l a ON ,4. o m l o n e r t s i , s IN r~ATS WHEN OWEN OIC~LLY AT 3 DAYS OK '7 WIEF.KS AFTER L~IFECTION
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Fzo. 2. Light micrographs of rat lung tissues in the control and the O.I mg/kg treated groups. Note the apparent changes of the number of eggs and larvae in the lung tissue with the lapse of time after treatment and the presence of eggs in the uteri of A. cantonensis in the 0.1 mg/kg tr~tcd group. 2a; control group: 2b-2f; 0.t mg/kg treated group, 2b; 2 days, 2c; i week, 2d; 2 weeks, 2c; 3 weeks, 21";4 weeks after treatment. Scale bar; 0.5 ram. DISCUSSION it was reported that avermectin B l a may act on the nervous system o f various nematodes as well as other animals including .crustacca and mammals, po~slbly through an effect on y-amino-butyric acid (GABA) (Fritz, Wang & Gorio, 1979; Williams & Yarbrough, 1979; Kass, Wang, Walrond & Strctton, 1980; Paul, Skolniek & Zatz 1980). In our in vitro experiments, it was also su~estod that avcrracctin B l a pa.ralyzcs Angiostrongy(~.s cantonen.~, a tissue
parasite, through a neuropharmacological mcchanEsm including G A B A and acetylcholine (Sano, Terada, lshii, Kino & Hayashi, 1981). It is likely that gastro-intestinal nematodes which were paralyzed by the dI~g are expelled by the pcrislalsis o f the host digestive tracl. In the present in vivo experiments, animals wer© treated at 6 wc~ks (Cxl~rimcnt 1) or 7 weeks (cxpcriraent 2) after infection when the inoculawd larvae are known to become adult~ (Maekerras & Sanders,
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Flo. 3. Effects of some neuropharma~olosi~ qczlr~ on the motility of A. (~ntonenxis; u n ~ t ~ l worms and worms pr~'x~led t~]th l,vtrmew~in B I I In rats. ~ w~rc cum~zd*a~ i~v~z and plor~ox~, N - o ~ (N-MC) mu~ in (B), picrmoxin and ~V-MCin (C) and N-MC, p i t . t o x i n and e~rine in (D) were suc,~.~lvety gtvl~. At tl~ pol~ W, the prepzmflon was washed by " i ' ~ e ' s toludon. Eu~rine and N-MC csttu~ It remarkable c~atraction in control wormL but theze spLtmoscns hgvc little elTtgl on the worn~ pre-trealed L¢vivo,
A. I. IsHn, M. TaP,Av^, H. KINo, M, H^Y^sm and M. S^~o
496
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Fro. 4. Light micrographs of worms from the rontrol (4a) and the 1.0 mg/kg treated (4b) groups. Note the apparent disorganization of internal structures of the worm from the treated group. Scale bar: 4a; 1 mrn, 4b; 5 ram.
Fio. 5. Light micrograph of rat lung tissue at 4 weeks after treatment in the adult treatment group (I.0 mg/kg of avermeetin B1a). Note very few unicellular eggs and no larvae in the lung tissue, though many eggs were observed in the uterus. Scale bar: 0.$ ram. 1955). In these adult stage treatments, no direct vermicidal effect o f avermc~'tin B l a was suggested from the recovery rates o f living worms (Tables 1, 2). On the other hand, there was a remarkable inhibition in the reproduction and this inhibition was sustained and dose-related. From the results o f the larval counts in feces and observatiom o f host lung tissue sections, the reproduction o f eggs might be inhibited for almost 3 weeks in the. O. 1 m g / k g treated gxoup (experiment 1). Furthermore, from the in vitro obser-
rations of worms pretreated with a v e r m ~ t i n B l a in vivoo it is suggested that this drug may temporarily paralyze adult worms in vivo as well as in vitro and therefore, that this inhibition o f motility probably resulted in the reduction o f egg laying. In the 1.0 mg/kg treated group (experiment 2), this inhibition of motility was severe and the larval output was completely inhibited until the hosts were sacrificed at 4 weeks after treatment. Though this drug has no direct vermicidal effect on adult worms, it is pmsible
I.J.P. VOL. 13. 1983
In vivo effects of avermectin Bla on A. cantonensis
that the severely paralyzed worms might be attacked and killed secondarily by host reactions. Regarding the development o f A . cantonensis, it was reported that eggs o f this worm are hatched about 1 week after oviposition (Alicata & Jindrak, 1970), so no larvicidal effect on the first stage larvae is suggested from the observations mentioned above. Similar effects o f this drug have been reported in dogs infected with Dirofilaria immitis (Campbell & Blair, 1978). Treatment with avermectin B l a at a dose o f 0.1 mg/kg completely suppressed microfilaremia for 6 weeks after treatment. However, microfilariae were again demonstrated in the blood on the 9th week after treatment and living adults were present in the heart on the 6th month after treatment. In addition to the inhibitory effects, a direct verrnicidal effect against the larval stage was confirmed which may be the third stage at 3 days after infection. This effect could not be detected in the in vitro experiments because .the adult worm was used for the material (Sano, Terada, Ishii, Kino & Hayashi, 1981). The recovery rate of worms was significantly lower in the larval treatment group than the adult treatment and control groups (Table 2). Similar effects were also r e p o r t ~ in dogs (Campbell & Blair, 1978b) or in ferrets (Blair & Campbell, 1978) infected with 19. immitis. When animals received avermectin B l a orally at a dosage of 0.02 m g / k g on 5 successive days, this drug was highly effective against 2-month-old (in dogs) or 4-month-old (in ferrets) precardiae larvae. To determine this larvicidal effect of avermectin B l a on the third stage larvae o f A . cantonensis in detail, further studies, such as mechanism o f action and stage specificity will be necessary. No grossly notable toxic reactions were observed in any rats treated with the levels of 0.01-1.0 m g / k g o f this drug in the present experiments. However, more detailed studies on side effects, especially on the central nervous system, should be carried out before avermectin B l a will be introduced clinically as a therapeutic against meningoencephalitis caused by A. cantonensis, because the G A B A system in the central nervous system is also reported to be sensitive to this drug (Williams & Yarbrough, 1979; Paul et ai,, 1980).
REFERENCES ALICATAJ. E. & JINDgAKK. 1970. Life cycle and biology. In: Angioatrongylosis in the PaeOic and Southeast Asia (Edited by A~D~I~soN H. H.), pp. 17-27. Charles C. Thomas, Springfield, Illinois. Bt.Am L. S. & C~i,at~t.L W. C. 1978a. Efficacy of avermectins against Anc.viostoma caninum in dogs, Journal o f Helminthoiogy 52: 305-307, B,.^tit L. S. & CmaPaeLa W, C. 1978b. Trial of avermectin Bla, mebendazole and melarsoprol against pre.cardiac DiroJTluria lmmltt, in the ferret (Mustela putorius faro). Journal o f Parasitotopj 64: 1032-1034.
497
Buao, R. W., MtLLea B. M., BAaaa E. E., BtanaAUM J., CuamES. A., HaI~TV,AN R., Kono Y. L., Mo~,~OH.~N R. L., O~o~ G., PVTT~a I., TU~^C J. B., W,~LL|CKH., STAPLaYE. O., Otw^ R. & OMUaAS. 1979. Avermectins, new family of potent anthelmintic agents: Prod~icing organism and fermentation. ¢4ntimicrobial Agents and Chemotherapy 15: 361-367. CA~tPa~LX. W. C. & BLAre L. S. 1978. Efficacy of avermectlns against Dirofilaria immitis in dogs. Journal o f Helm inthoiogy 52 "-308-310. CUCKLEa A. C., EOERXONJ. R. & ALICATAJ, E, 1965. Therapeutic effect of thiabendazolg on Angiostrongylus cantonensis infections in rats. Journal o f Parasitotogy 51: 392-396. EGERTONJ. R., OSTLINDD. A., BLAIRL. S., EARYC. a., SUP.AYD^ D., Ring R. F. & CAMPaELL W. C. 1979. Avermectins, new family of potent anthelmintie agents: Efficacy of Bla component. Antimicrobial Agents and Chemotherapy IS: 372-378. FatTz L. C,, W^No C. C. & OoRIo A. 1979. Avermectin Bla irreversibly blocks postsynaptic potentials at the lobster neuromuscular junction by reducing muscle membrane resistance. Proceedings o f the National
Academy o f Sciences o f the United States o f America 76: 2062-2066. JAMES P. S., PIc'ror~ J. & RInK R. F, 1980. Insecticidal activity of the avermectins. Veterinary Record 106: 59. J a q D ~ K. & A]LteATAJ, E. 1969. Effect of I-tetramisole on Angiostrongylus cantonensis infection in rats. Chemotherapy 14: 244-252. lO,ss I. S., W^No C. C., WALaOr~D J. P. & SraE'rro~ A. O. W. 1980. Avermectin Bla, a paralyzing anthelmintic that affects interneurons and inhibitory motoneurons in Ascaris. Proceedings o f the National
Academy o f Sciences o f the United States o f America 77:6211-6215. L.g~tuLta G. & WEmNER E. 1975. Zur larviziden Wirkung yon Amhelminthika gegen Angiostrongylus cantonensis.
Berliner und Mginchener Tierarztliche WochensehrO't 88: 152-156. MACtC~RaASM. J. & S^r;DArts D. F. 1955. The life history of the rat lung-worm. Angiostrongylus cantonensis (Chen) (Nematoda : Metastrongylldae). Australian Journal o f Zoology 3: 1-21. MtLktm T. W., CmUET L., CoL~ D. J., CoI.~ L. J., FLoa J. E., GO£OELM~..,*R, T,, GULLOV. P., JosardA H,, KF..MPF A. J., KRELLWVfZW, g,, MONAOHANR. L., O~OND R. E., Wit.soN K. E . AL~m,.s-ScHot~aEgoO. & Ptrrr~a I. 1979. Avermectins, new family of potent anthelmintic agents: Isolation and chromatographic properties. Antimicrobial Agents and Chemotherapy !$: 368-371. NtsmMu~A K. 1965/66..Experimental studies on the chemotherapy of rat lungworm, Angiostrongylus cantonettsis, in rats, Chemotherapia 10:164-175. PAut S. M.. SKOt.NtCK P. & ZArz M. 1980. Avermectin Bin: art irreversible activator of the r-aminobutyric acid-b~nzodiazepine-ehloride-ionophore receptor complex, Biochemical and Biophysical Research Communicatibns 96:632--638. RoNc^tu R. A. 1980. Clinical evaluation of the avermecti~s, a new da.~s of broad-spectrura anthdminfic agents. The Argentinian Veterinary Congress, Buenos Aires, Argentina. S ~ o M., Tagm3A M., IsRu A. I. & Kmo H. 1981. Effects o f avermectin Bla on the motility of various parasitic helminths. J~xperiemia 37: 844.846.
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A, I.Ist~ix,M, TERADA, H. lONO, M. HAYASHI and M. SASO
S^No M., TERAO^ M., ISHIIA. 1., KxbtoH. & HAYASHIM. 1981. Studies on chemotherapy of parasitic hclmiths (l). On the in vitro methods and paralyzing e.ffects of avermectin Bia on Angiostrongylus cantonensis. Japanese Journal of Parasitology 30:305-314,
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WILLJAM.~M. & YARBgOUOHG. G. 1979. Enhancement of in vitro binding and some of the pharmacological properties of diazepam by a novel anthelmintic agent, avermectin Bla. European Journal of Pharmacology $6: 273-276.