sign in sign up
<<
Home , Paromomycin

, AND IN EXPERIMENTAL CHOLERA By

N.K. DUTTA AND M.V. PANSE1 From the Haffkine Institute, Bombay tReceioed October 20, 19(3)

Paromomycin, polymyxin B and furazolidone were tested for their ability to inhibit the growth of V. Cholerae and their effectiveness in curing rabbit cholera, AIi the three drugs possess powerful vibriostatic and vibriocidal action. Paromomycin and furazol- done could cure about 50 per cent of the animals suffering from cholera if the treatment was instituted ear Iy. Polymyxin B on the other hand possesses only preventive but no curative value. 'I he usefulness of these drugs in clinical cases is discussed. '

Cholera may exhibit a range of signs and symptoms from milddiarrhoea to its severe form, the collapse. But the one which attracts the physician' attention follows a characteristic pattern. After an incubation period of twos to five days, the patient generally starts with severe diarrhoea, accompanied or more commonly, followed by vomiting. The stools become watery (rice water) and are excreted ill bulk. Acidosis developes unless the water and electrolytes are restored and the case may terminate fatally within a few hrs or days. Such a condition calls for immediate treatment. While quick attention in replacing fluid loss and electrolyte ,imbalance will benefit a large number of patients. yet there is no drug today which claims to have cured the disease. Experimentally, it has been demonstrated that , , etc. may be of' value if administered at the early stage of the disease (Dutta and Habbu, 1935; Dutta and Colah, 1958, and Dutta, Colah and Vaidya, 1958),but not so when the disease has advanced. The clinical observations also indicate so. A search for a more effective remedy against cholera has, therefore, been continued. Paromomycin, polymyxin B and furazolidone have been chosen for the present study.

Paromomycin is an derived from a strain of rimosus forma Paramomycinus, Chemically it is a glycoside of d-glucosamine. It is water soluble and possesses marked antibacterial activity against a number of gram negative and gram positive bacteria.

Present address. Research Division, Hindusthan Anti 'otic Ltd . Pimpri, Near Poona, for- merly Assistant Research Officer, I. C.M.R. N. K. DUTTi\ AND M. V. PANSE 81

Polymyxin B is obtained from B. polymyxa; polyrnvxin B sulphate is freely soluble in water. It is useful against many gram negative organisms. It is ND FURAZOLIDONE poorly absorbed from and its use has been recognized in OLERA the treatment of intestinal disorders such as pseudomonas enteritis, shigellosis and other enteric diseases. It may also be given parenterally,

Furazolidone (3-(5-nitro furfurylideneamino)-2-oxazolidinone; -5-nitro- 2.furfurylidene)-3-amino-2-oxazolidone) occurs as a yellow crystalline powder, sparingly soluble in water. It displays a wide spectrum of acti-

tested for their ability to inhibit vity. It has inhibiting activity against E. histolytica and Trichomonas foetus. It .ng rabbit cholera. AIi the three is used in Salmonellosis . on. Paromomycin and furazol- ing from cholera if the treatmen t METHODS possesses only preventive but no Bacteriostatic or bactericidal properties.- These tests were carried out by the I cases is discussed. serial dil utiou method in which the total volume of the casein hydrolysate medium in each tube was kept constant (10 ml) but the concentrations of the rrmptoms from mild diarrhoea compounds to be tested were varied. All dilutions were made in a protein hich attracts the physician> free casein hydrolysate medium (Sokhey et al., 1950). Rabbit passaged strain an incubation period of twos of V. Cholerae, (Inaba, 569 B), was used as the test organism. The potency of vere diarrhoea, accompanied each drug was tested against 104 vibrios per ml. stools become watery (rice " lopes unless the water and Test on rabbit cholera.- The method of Dutta and Ha bbu (1955) was inate fatally within a few hrs followed. The rabbit passaged strain of.F, Cholera (Inaba, 569 B) was pre· ate treatment. While quick served in a lyophilised state. Before each experiment, the vibrios were rege- imbalance will benefi t a large nerated, suitably cultured and finally administered to J O-day old rabbits hich claims to have cured intraintestinally in a dose of 104 vibrios per 100g of body weight. The nstrated that chloramphenicol treatment was begun either before or after inoculation of vibrios but the if administered at the early drugs were given every 2 to 6 hrs. A total of 6 or 9 doses were administered utta and Colah, 1958, and orally depending on the condition of the animals. With each set of experi- n the disease has advanced. ments, control experiments were carried out in which the infected animals search for a more effective were given no treatment. J n order to confirm that the death was due continued. Paromomycin, to cholera, post mortem examination was done wherever required. n for the present study.

RESULTS a strain of Streptomyces rimosus The vibriostatic and vibriocidal properties of paromomycin, polymyxin ide of d.glucosamine. I t is B and furazolidone are illustrated in the Table I. All were powerful vibrio- I activity against a number of cides against V. Cholerae. They were effective in concentrations of 5 to 25 p.gfml. biotic Ltd., Pimpri, Near Poona, for- In Table 11 it will be seen that when the treatment with paromomycin was begun 1 hr before or 8 hr after , it prevented all the signs and 82 EXPERIMENTAL CHOLERA symptoms of the dsease from appearing. All the 4 animals remained normal although the control rabbit died at the end of 30 hr When the treatment was started 16 hr after infection using a dose of 25 mg per 100 g of body weight, about 44 per cent of the animals survived. In 7 animals there were no symptoms except that they appeared to be ill for sometime. When the dose of the drug was raised beyond 25 mg and up to 100 mg and the inter- vals of administration were reduced from 4 hr to 2 hr, no additional benefit was seen. On the contrary, all the animals so treated, with the exception of one never recovered.

In the case of polymyxin B (Table III), all animals survived when the administration of the drug began 8 hr after infection or earlier with a dose of 100 mg/kg. With -a higher dose of 250 mg/kg, none of the, anima remained alive when the treatment began 16 hr after vibrio inoculation.

Furazolidone (Table IV) in a dose of 100 mg/kg protected all the animals when the drug was given 1 hr before or 8 hr after infection. This dose, however, was ineffective when the administration of the drug first began at a later period, i.e., 16 hr after infection. When the dose was raised still further to 500 mg/kg, furazolidone afforded protection to 50 per cent of the animals. Further increase of the dose to 1 mg/kg had no benefit.

The animals which had received no treatment but were infected with V. cholerae died without exception with typical signs and symtoms of cholera.

TABLE I

Paromomycin, polymyxin B and furazolidone against 10' V. choleraejml

Vibriostatic Vibriccidal Drugs cone. conc.

Paromomycin 5 P.g/ml 10 p.gfml

Polymyxin B 10 p.gfml 25 p.gfml

Furazolidone 10 P.g/ml 25 P.gfml _. ::l (!) ..• 0:> 0 ~ aq ~ '"C c..:> ;;. en aq ... ::I ;::;> ::I 0 ol>- ::'. :;;:. ~ 0 :;;:. o· ::; 0 )0 ~ s (!) ::l (!) ="":r- o ...., '" , ~ aq (!) o· =:r- ... •.... ~ •.... ::I 0- ::I ... :3 '?Cl Er ~ ~..• - 0' e- ::l ~ ... ::I P- =:r- ~ (!) -e o ~ ;; <.Jl ..• ri ~ ~ ... v;- ::I 0 en "" ~ en S» o S» .•.. p.. 0 -...J P- ~ (!) P- o· ...., eT 0 0 3' "< ::l 0 ;::> aq ..• 0 8 :-=::: -::;: (!) ~ en aq ~ S» --.J en ::l a, S» El 8 ~ en 8 (!) ::l (!) ~ ... o· (!) ~ ::l e- e-, (!) '"C ..• ~. P- aq '"C ;::,- 0 ..• 0 0 ..• ~. &. (!) ..• Cl> ::;: -e ;:r (!) Cl> 0- 0- S' 0 ..• S» ::l -< ~ 8 <.J1 S» ..• S' ~ 0 .....• e: 8 s· ..• "" 0 tn (!) n <' ..• ... e ... S "" "" ..,e;: .'".. ::l 0 CA ~ iii' Cl> Cl> :::r 0- ~ ~ •....• =:r- S» "" "1: S' aq n ~ ~. 0- Cl> o· en "1: "1: o a. 0.....• Cl> "0 n 0 Cl> e c. '" iii' ::n ..• ... ;; Cl &- ;:r ::l 0 s· ~ ~ o o '" o o Cl> ::n 0- ..• ... ..• Cl> ~ ..• ..• ..• o· ('D Cl> ~ :::r :::r ..• e. !i. :::r ~. CA ~ aq 0- e. '0: Cl> CA S» ..• e. - •• o· >< ('D ~ C'D 8.. 0. C'l C'D ..• ? ::l :::r C'l :::r C'D ~ C'D 0. 0. C'D 0- ..• 0 ::l / 0- ~ S» CD C'D CD .....• S» .0.• .' ::l 0 ::l "0 CD •... ;:l ..• 0 n .., Cl> ;:l ;:l 0- ..• S» .... Cl> ..• ~. aq - 2. CD ... ~ S ... :::r ..• ..• CD ... 0 ..• •..... III :::r o· Cl> :::r Cl> 0. Cl> :::r 0.....• •..... ::l S 0 :::r ::n ...... ::l sPo> rn' Cl> S» .....• ::l Cl> ('D CD ..• I -e ..• -

TABLE n Action of paromomycin (oral) on infant rabbits infected with V. cholerae (Inaba 569 B) 10',100 g body weight

Treated infected rabbits Treatment started Untreated infected rabbits (control) (hr before or after I Mean time of Dose/lOOg ,onset of diarrhoea I Mean survival I Mean time of I Mean survival inf'.) time hr) Mortality onset of diarrhoea I Mortality (hr) (hr) time (hr)

hr before 10 mg. 6 hrly, 0/2 8 doses 22 30 :z: 8 hr after -<10- } 1/1 0/2 ?'= e 16 hr after ~ 25 mg, 18 (9) 23 9/16 18 20 >i 4 hrly, 1/1 )- 8 doses )- Z t:J 16 hr after 30 rng, 25 (2) 32 19 4 hrly, 2/2 23 1/1 ~ 7 doses :<: 'Il 16 hr after > 50 mg, 25 (4) 36 4/4 20 z 4 hrly, 25 1/1 '"~ 7 doses

16 hr after 100 mg, 26 (3) 30 3/4 18 4 hrly, 28 1/1 6 doses

16 hr after 100 rng, 20 (4) 31 4/4 20 28 2 hrly, 1/1 9 doses

0:> Figures in the parenthesis indicate the number of animals suffered from diarrhoea ~ (Ill --.J

~

C:) ~

TABLE III

Action of polymyxin B (oral) on infant rabbits injected with V. cholerae (Inaba, 569 B) 10'/100 g body weight

Treated infected rabbits. I Untreated infected rabbits (control) Treatment started Mean time of Mean survival Mean time of Ionset of diarrhoea I Mortality onset of diarrhoea I Mean survival I Mortality (hr before or after Dose/lOOg lime (hr) time (hr) inf.) (hr) )I (hr) I M X '1:l ••• 1 hr before 10 mg, -- -- 0/4 22 32 1/1 ::0 6 hrly, -:.:: 8 doses \ M .Z., )- 8 hr after IQ mg, -- -- 0/4 20 30 1/1 t" hrly, 6 0 8 doses \ :I: 0 t" M 16 hr after 25 mg, 20 31 (6) 6/6 I 18 (2) 28 2/2 4 hrly, I '")- 5 doses

Figures in parenthesis indicate the number of animals suffered from dia'rrhoca mg, I (6) 6/6 18 (2) 28 2/2 4 hrly, , 5 doses

Figures in parenthesis indicate the number of animals suffered from diarrhoea

/

TABLE IV

Action of furazolidone (oral) on infant rabbits infected with V. Cholerae (Inaba 569 B) 101/100 g body weight

Treated infected ra bbits I Untreated infected rabbits '<0",<01) Treatment started -- \ Mean time of Mean survival Mean time of \ Mean survival (hr before or after Dose/lOO g onset of diarrhoea I Mortalit onset of diarrhoea Mortality time (hr) time (hr) inf.) (hr) I (hr) I br before 10 mg, 0/2 22 30 III 6 hrly, 8 doses :-: ~ 8 hr after 10 mg, 0/4 18 20 III Cl 6 hrly, c:: 8 doses ~ > > 16 hr after 10 mg, 23 (4) 33 4/4 19 24 2/2 Z 6 hrly, Cl 8 doses re :<: 16 hr after 50 mg, 24 (2) 34 2/4 22 32 III ~ 6 hrly, )0 7 doses Z Cl> t'l 16 hr after 100 rng, 28 (4) 32 4/4 18 34 1/1 6 hrly, 5 doses

Figures in parenthesis indicate the number of animals suffered from diarrhoea 86 EXPERIMENTAL CHOLERA

DISCUSSION

Before attempting to interpret the results it would be necessary to exa- mine the infant rabbit model in relation to other methods commonly used in the laboratory for evaluation of drugs against cholera. In this respect mice have been used extensively. Rao and Ganapathi (1941). Griffitts (1942). Bharnagar 119~8) and others (reported by Pollitzer, 1960) have demonstrated the usefulness of sulphonamides in protecting mice against cholera. Felsen· feld and Soman (1952) observed that poorly absorbed such as , neomycin and bacitracin were of no use in mice infected with V. cholerae, but chloramphenicol and gave better results and , the best, with 98 per cent survival. In mouse cholera where vibrios are injected intraperitoneally with mucin, one deals with a condition of bacteraemia. Any drug which possesses vibriostatic or vibriocidal action should be useful provided it is absorbed in the system. It is natural that substances like streptomycin, neomycin and bacitracin, which are not easily absorbed, would but be ineffective in the mouse infection.

Felsenfeld and Soman (1952) have used monkeys to demonstrate the effect of drugs in cholera. They showed that Macaca rhesus heavily infected with V. cholerae intragastrically could be saved when treated with oxytetracy- cline or neomycin either given orally or parenterally.

Collier, Hall and Waterhouse (1949) had developed the "mouse faecal suspeusion test" for evaluation of remedies for cholera. but the method had no relation to clinical cholera.

Burrows (1953) used gumeapigs to demonstrate the efficacy of sulphona- mides, giving each drug to groups of cholera infected animals. He found that sulfadiazine and sulphaguanidine reduced the total number of bacteria and percentage of cholera vibrios in the stools.

In clinical cases, the vibrios remain localized within the gut and the individual suffers from massive loss of fluid and extreme dehydration which are the primary reasons for death. Neither the mice or the guineapigs nor the monkeys exhibit such symptoms. A parallel condition, however, is seen in infant rabbits infected intraintestinally with V. cholerae (Dutta and Habbu, 1955). In the rabbit, one observes the fatal diarrhoea with characteristic rice water stools (Jenkin and Rowley, 1959) and other morphologic changes in the gut (Norris, Dutta, Finkelstein, Formol and Sprinz, 1963) akin to human biopsies. The vibrios too remain within the intestines (Dutta and Habbu, 1955). N.K.DUTTA AND M. V.PANSE 87

Now turning towards the therapeutic tests as a criterion in judging the experimental model for its closeness to the human disease, one would observe that both the sulphonamides and the antibiotics were effective in mice, mon- uld be necessary to ex a- keysand guineapigs, but no such actions of the drugs have been seen in well ethods commonly used developed clinical cases. In rabbit cholera too, these drugs had little value cholera. In this respect once the diarrhoea had set in. In short, both from the point of symptomato- hi (1941). Griffitts (1942). logy and the results of drug treatment in clinical cases and in the rabbit cho- 1960) have demonstrated lera, one would like to conclude that there is no behavioural differences against cholera. Felsen- between the two models. rbed antibiotics such as ase in mice infected with In the rabbit infected with V. cholerae, diarrhoea makes its appearance, ne gave better results and on an average, at the end of 24 hr and the animal lives for another 10 or 12 . In mouse cholera where hr. In this study, as well as in earlier investigations, the schedule of treatment ne deals with a condition has been arbitrarily divided into four groups. To the first group, the treat- tic or vibriocidal action ment began I hr before infection, to the second 8 hr after infection, to' the stem. It is natural that third 16 hr after and in the last, the therapy begun only after the onset of cin, which are not easily diarrhoea By drawing an analogy from human cases where the incubation fection. period is greater and the physician gets more time to treat, it may be pre- keys to demonstrate the sumel that "one hr before infecti JIl" treatment schedule would correspond aca rhesus heavily infected to the prophylactic value of a drug. If there be any benefit from treatment treated with oxytetracy- which begins 8 or 16 hr after infection then one may perhaps presume, it has y. been due to treating an early case of cholera. Drug therapy in a rabbit where diarrhoea has set in would indicate, as if, treating a well established eloped the "mouse faecal case of cholera. None of the drugs used here, i e., paromomycin, polymyxin era, but the method had Band furazolidone have proved their usefulness once the diarrhoea had be- gun. evertheless. they could be used in the prevention of disease in contact cases Some benefit was noted when the treatment had started 8 or 16 hr e the efficacy of sulphona- after infection, with paromomycin Or furazolidone. Or in other words they ected animals. He found would be of some value if given at the early stage of clinical cholera. total number of bacteria Generally speaking by the time the patient offers himself for treatment as a cholera case, it is probably too late for any vibriostatic or vibriocidal within the gut and the drug to have any real benefit. By that time there has been a massive multip-Ii reme dehydration which cation of vibrios followed by lysis with the release of cholerogenic material (s). e or the guineapigs nor the Wheather the latter is secreted or leached out of the cells is yet to be ition, however, is seen in decided. The manifestatioan of the disease is due to the local and systemic crae (Dutta and Habbu, actions of the released m aterialrs). Under the circumstances, bacterios- oea with characteristic rice tatic and bactericidal actions of the drug will have a limited value in arresting orphologic changes in the the further growth of the bacteria and freeing the stools of vibrios earlier, z,1963) akin to human provided the subject survives. Work of Dutta and Panse (1963) that berberine !e intestines (Dutta and which has no vibriostatic or vibriocidal actions could as well be effective as 88 EXPERIMENTAL CHOLERA sulphonamides or antibiotics in the treatment of experimental cholera con' firms such presumption. Therefore, the real treatment of cholera still lies in fluid and electrolytes replacement. Paromomycin and polymyxin B may at least be used as supportive treatment.

We thank Dr. R.J. Modi, Parke, Davis (India). Ltd. for a gift of paromomycin andto Dr. C. B. Andrade, Burrough's Welcome and Co, (India) Private Ltd, for a gift of poly. myxin B. The assistance Shri G. B. Borkar is acknowledgedwith thanks.

REFERENCES

Bhatnagar,S.S.,deS.J.Fernandes, F, and Divekar P. V. (1948). Brit. Med. J., 1,719. Burrows, W. (1953). J. Infect. Dis., 92,152. ColIier, H.O.J.,HaIl I. F. andWaterhouse,P.D. (1949). Ann. Trop. u«. Parasa., 43,155. Dutta,N.K. and Colah, R.B.M. (1958). Jour. Postgrad. Med., 4, I. Dutta, N.K., Colah, R.B.M. and"Vaidya, S.D. (1958). Proceedings of the Symposiumon Chemotherapy,pp. 60, Central Drugs Laboratory, Lucknow. Dutta, N.K. and Habbu, M.K. (1955). Brit. Jour. Pharmacol., 10,153. Dut+a, N.K. and Panse, M.V. (1962). Ind. J. Med. R~., 50,732. Dutta,N.K. Panse,M.V. and Kulkarni,D.R. (1959). J. Bact.,78,594. Felsenfeld, 0., and Soman,D.W. (1952). Ann. N.r. Acad. Sei., 55,1059. Griffitts,J.J. (1942). Publ. Hlth. Rep. (Wash),57,814. Jenkin, C.R., and Rowley, D. (1959). Jr. Gen. Microbiol., 21,191. Norris,H.T.,Dutta,N.K., Finkelstcin, R.A., Formal, S.B. and Sprinz, H. (1963). Fed. Proceedings,22, No. 2, 512. Pollitzer, R. (1959). Cholera,World Health Organization, Geneva. Rao, R.S. and Ganapathi, K. (1941). Ind. Med. Gaz., 76,78. Sokhey, S.S., Habbu, M.K. and Bharucha, K.H. (1950). Bul. Wrd. Hlth... Ovg., 3,25.