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CLINICAL AND PHARMACOLOGICAL ASPECTS OF THE TOXICITY OF STREPTOMYCIN
9
death more often than do surgeons engaged in special branches of our art. Even so, let him who thinketh he standeth take heed lest he fall from unpreparedness. To prick the ventricle within three-quarters of a minute of ascertaining that the heart has ceased to beat, and, in the likely event of this simple procedure being ineffective, forthwith to massage the heart, will, I am sure, recall some of those whom Charon had started to ferrv across the. waters of the Styx. REFERENCES
Adams, H. D., Hand, L. V. (1942) J. Amer. med. Ass. 118, 133. Bailey, H. (1927) Practitioner, 118, 368. (1941) Brit. med. J. ii, 84. (1946a) Ibid, i, 29. (1946b) Ibid, p. 661. Bodon, C. (1923) Lancet, i, 586. Dodd, H., Prescott, F. (1943) Surg. Gynec. Obstet. 77, 645. Easton, A. M. (1942) Brit. med. J. i, 651. Elam, J. (1944) Brit. J. Anœsth. 19, 31. Grimshaw, C. (1942) Brit. med. J. i, 387. —
—
—
MacLeod, N., Schnipelsky, L. A. (1942) Ibid, p. 610. Morley, J. (1946) Ibid, i, 177. Nicholson, J. C. (1942) Ibid, i, 385. Primrose, W. B. (1935) Ibid, ii, 540. Vernon, H. K. (1943) Lancet, i, 6. Waters, R. M., Gillespie, N. A. (1944) Anesthesiology, 5, 113.
CLINICAL AND PHARMACOLOGICAL ASPECTS OF THE TOXICITY OF STREPTOMYCIN MADIGAN D. G. P. N. SWIFT M.B. N.U.I. M.R.C.S. TUBERCULOSIS
CHEMOTHERAPY UNIT,
COUNTY
HOSPITAL,
FARNBOROUGH, KENT
GEORGE BROWNLEE B.Sc.
Glasg., Ph.D. Lond.
OF THE WELLCOME RESEARCH
_
LABORATORIES, BECKENHAM,
KENT
As pharmacological and clinical experience with streptomycin (Schatz et al. 1944) accumulates, the potential and actual toxicity of the antibiotic and its concentrates becomes apparent. Already it is possible to indicate those intoxications due to the pure drug and those associated with known and unknown impurities. Thus, the fatty metamorphoses in the parenchymal cells of the liver, and to a lesser extent in the tubular epithelium of the kidneys, described by Molitor et al. (1946), are reversible changes produced in susceptible laboratory animals after the parenteral injection of pure streptomycin. We have observed these changes and the probably associated albuminuria in man. On the other hand, the reactions reported by Hettig and Adcock (1946)-facial flushing, headache, and fall of blood-pressure, appearing promptly and lasting 10-60 min.-are attributed to histamine-like impurities Molitor et al. (1946), who also reported the presence of an antidiuretic impurity. Most workers associate skin eruptions, rashes, and to some extent local pain with the presence of impurities. Whether the fever,
by
invariably accompanied by myalgias and arthralgias in the temporomandibular joints and suboccipital region (Hettig and Adcock 1946), and the clinical evidence of toxic effects on the eighth nerve seen in 3 of 34 patients by Hinshaw and Feldman (1945), are due to streptomycin or its impurities is a subject for further inquiry. We report here the results obtained from the examination in animals and man of the pharmacological effects of some seventeen batches of streptomycin. This material, though of varying purity, was free from histamine-like and other toxic substances, thus allowing large doses to be given intrathecally without incident. In well-marked contrast were the findings of Cairns et al. (1946), who recorded fatal reactions following injection of streptomycin of low dosage (Merck and Co. Inc., lots 200 and 213) in two out of seven infections. It is evident that streptomycin concentrates, though of high purity, are persistently contaminated by histamine-like substances (Molitor et al. 1946) ; hence it is
desirable that streptomycin biological control.
should be subjected to
PHARMACOLOGY
potencies of the batches of streptomycin from one (w.F.) are described in terms of weight of pure streptomycin base C21H37N7O12 (M.w.579) and have been derived from assay with a working standard of streptomycin sulphate, 11/1 H2SO4 (M.w.726), of known potency. It follows that streptomycin sulphate contains 79-8% equivalence of base ; in the accompanying graph this figure has been rounded off to 80%. Assay.-Assessment in terms of the working standard by a dilution test, using a constant inoculum of a susceptible The
source
strain of Bact. coli, C.N.1360, where the interval of each increment is
20%, yields an answer of potency ± S.D. 20%. Chemotherapy.—The potency and therapeutic activity of the batches studied were controlled by assay, in terms of a working standard, in which three doses of test were compared with three doses of standard in groups of six
mice infected with a number of lethal doses of a virulent strain of Bact. coli, c.N.348. Acute Toxicity in Mice.-The acute toxicity of streptomycin concentrates was studied after the intravenous injection of two doses into two groups each of ten animals weighing 18-20 g. The onset of symptoms was immediate in the case of toxic doses, and took the form of irregular breathing, dyspnoea, and apnoea. Arrest of the heart and death followed in three or four minutes and was due to asphyxia. There were no late deaths. The slopes of the line relating the probits corresponding to the per cent. mortalities and the logarithms of the two doses are all of the same order and reveal no artefact. The average lethal doses (L.D.50), derived graphically, range from 1-05 g. to 0.27 g. per kg. and are remarkable for the lack of toxicity which they reflect. This fact is illustrated on the accompanying graph, which shows the simple relation between acute toxicity and potency for the seventeen batches (w.F.) we have examined. The least potent batch (w.F.ll), of some 30% purity, is a fifth as toxic as pure streptomycin base, and the most potent batch, of some 80% purity, is two-thirds as toxic as pure streptomycin base. For comparison the acute toxicities and potencies of twenty-eight lots of streptomycin from one source (Merck Research Laboratories) derived from the paper of Molitor et al. (1946) are included in the graph. Most of the batches, though varying in potency between a quarter and a third of the potency of pure streptomycin, are seen to be twice to four times as toxic. Molitor et al. attribute this toxicity mainly to the presence of a histamine-like impurity ; an antidiuretic factor is also present. Included in the graph are the data on one sample of streptomycin sulphate from another sourcePfizer, lot 459. Circuda.tor2t Effects.—When tested in cats anæsthetised with pentobarbitone sodium (30 mg. per kg. intraperitoneally), no significant effects on blood-pressure or respiration were recorded in any of the seventeen batches of streptomycin concentrates. Antidiuretic Effects.-The acute effect of streptomycin concentrates on water diuresis was studied in groups of four rats. Food, but not water, was withdrawn on the previous night, and in the morning 5 ml. of tap water per 100 g. of body-weight was administered with a stomachtube, and a dose of 250 mg. per kg. of streptomycin concentrate was given subcutaneously. Urine was measured at hourly intervals for six hours. With two exceptions, batches 20 and 15, the shape of the normal diuresis curve remained unaltered, except that the two-hour peak was a little flattened, but the total urinary excretion was not significantly different. In the case of batch 15 the shape of the curve was slightly modified, but the total excretion was not significantly different ; with batch 20 there was retardation of diuresis for two hours, but thereafter it
10 was
normal, and the total urinary excretion
was
not
significantly altered. Intrathecal Injection
in Rabbits.-Rabbits anaesthetised with pentobarbitone sodium (40 mg. per kg. i.p.) tolerate without detectable reaction 10 nig. per kg. equivalence of streptomycin base injected cistern ally in 0-5 ml. of sterile distilled water following withdrawal of 0-5 ml. of cerebrospinal fluid.’ Under the same conditions two out of three rabbits did not survive the injection of 20 mg. per kg. equivalence of streptomycin base. Death followed in 10-15 min. from respiratory failure and was smooth and free from any other symptom. STUDIES IN MAN
In the
of the evaluation of antituberculous chemotherapeutic drugs in this hospital the opportunity to evaluate the status of streptomycin in a side-by-side comparison with established sulphone chemotherapy presented itself in June, 1946. A limited supply was available from one source (w.F.) in the form of seventeen batches which had been subject to pharmacological control. The material was available, in sterile rubbercapped containers, in the form of a freeze-dried powder and contained the equivalent of 0.2 g. of pure streptomycin base, together with a supply of sterile pyrogenfree distilled water. The biological control of the material before it reached us included tests for sterility and freedom from pyrogens, freedom from histaminelike impurities, and, with one exception, from antidiuretic substances. The average lethal doses in an acute experiment for mice of these batches were less than that of pure streptomycin. On the occasion of intrathecal injection an additional tonicity test was made on the batches used for this purpose. However, the figures obtained subsequently proved to be average values for all batches, an isotonic solution being obtained when one container (0-2 g.) was dissolved in 18 ml. of distilled water. In practice one container was mixed with 2 ml. of distilled water, but even with such a hypertonic solution no acute toxic effects were encountered. The usual precautions were taken of withdrawing at least an equal volume of cerebrospinal fluid, the use of a narrow-bore lumbarpuncture needle, very slow injection, and subsequently raising the foot of the bed. In the course of this trial every attempt was made to evaluate the toxic manifestations of streptomycin both clinically and biochemically. Fourteen patients, aged 2-60 years, served as subjects. The total doses of streptomycin varied from 0-4 g. given in 9 hours to 108 g. given in 90 days. All of the fourteen patients received streptomycin by intermittent intramuscular injection at four-hour intervals ; in addition four of the fourteen patients received a daily dose of 0-2 g. intrathecally by the lumbar route, or into the cisterna magna. Preliminary intramuscular injection in one of us (G. B.) proved the streptomycin to be painful ; hence at the start of the trial, and subsequently in those patients who complained of pain, streptomycin was administered in the presence of 0-5% procaine hydrochloride. Animal protein enzymic hydrolysate and glucose, of each 1 g. per kg. of body-weight, were given by mouth to all patients on streptomycin to reduce liability to renal and hepatic damage. Most patients were so seriously ill as to preclude systematic pathological investigations, but in most cases complete blood-counts (including differential leucocyte counts), haemoglobin estimations, and urine analysis were done at short intervals before, during, and after the experimental trial. The quantity of streptomycin circulating in the blood at four hours after the intramuscular injection of 0-2 g. of streptomycin was estimated from time to time throughout the trial- by the slide technique (Fleming 1943) with a strain of Bact. coli, c.N.1360. Mean blood concentrations of 8 µg. per ml. {minimum 4, maximum 16) were found. In cerebrocourse
Relation between potency and toxicity of batches of streptomycin from different sources. Wellcome Foundation batches fall on two curves, A and B.
spinal
fluid twenty-four hours after intrathecal injection of 0-2 g. (two specimens), the concentration was 3-2 and 6-4 µg. per ml. ; after intramuscular injection alone no streptomycin could be detected in the cerebrospinal fluid. No evidence of toxic effects on the hæmatopoietic system was observed, and the total and differential blood-counts revealed no fluctuations outside the normal limits. The urines of five subjects showed the presence of leucocytes and epithelial cells during the experimental period ; haematurias were not seen, but red corpuscles were found in two cases. Albuminuria was recorded in one case. Casts were not found on any occasion. No facial flushing or headache or any other evidence of histamine-like reaction, such as described by Hettig and Adcock (1946), was seen. One observation was made which seemed to us to be associated with the toxicity of pure streptomycin. This took the form of an increase of the normal diurnal excursion of the temperature range relative to the pyrexia. It was clearly demonstrated in three of the fourteen cases and showed no obvious relationship to any particular batch. It was not associated with myalgia or arthralgia nor did any symptom appear with it. In one case of advanced miliary tuberculosis with peritonitis and meningitis which came to necropsy, a diffuse fatty degeneration of the liver was found. This may have been due to the tuberculosis or to the streptomycin. No dermatoses were encountered, except a transient papulo-squamous lesion on the dorsum of the hands of a Thailand railway worker ex-prisoner-of-war. It is doubtful if this condition was due to streptomycin. No toxic effects on the auditory nerve appeared. On two occasions swelling and pain round the site of injection, with regional adenitis, developed after intramuscular injection but settled down with conservative, treatment in three or four days. DISCUSSION
The pharmacological and clinical study of seventeen batches of streptomycin from one source reveals several interesting differences between this material and that described elsewhere. The major differences are freedom from acute toxicity of an order greater than that of pure streptomycin, an effect associated to some extent with freedom from histamine-like and antidiuretic impurities. Clinically, ten patients, all seriously ill with tuberculosis, were treated with 1-2 g. daily for ninety days by intermittent intramuscular injectiona total of 108 g. of streptomycin drawn from several batches. Four patients were treated for shorter periods with 1.0 g. intramuscularly and 0-2 g. intrathecally daily. Our experiences were free from the hazards and perils
11
which have characterised previously reported trials, and have convinced us that streptomycin, of the purity which we used, can be administered in safety at the dose levels which we adopted. Differences of this kind have come to be expected from concentrates of antibiotics of natural origin and are inherent in the method of preparation. The nature of the medium and the extraction procedure are only two of the many variables. Since it is clear that streptomycin of a purity closely approaching chemical purity may vary in its intravenous acute toxicity between one and four times (Molitor et al. 1946), it is evident that the control of individual batches by biological standardisation is a necessity. It is desirable that streptomycin for parenteral use should be free from histamine-like substances and antidiuretic factors. Ilowever, it seems that streptomycin may be contaminated by an additional hitherto unidentified substance of greater toxicity but whose toxic effects are similar to the effect of toxic doses of streptomycin itself. The additional safeguards required of streptomycin for intrathecal use is that it should be free from pyrogens and have an acute toxicity for mice not greater than the pure streptomycin of L.D.50, 220 mg. per kg. In this connexion it is of interest to refer to the graph relating potency and toxicity of the batches we have examined. Six batches may be considered to fall on a smooth curve {curve A) with a point of origin at the L.D.50 of pure streptomycin (220 mg. per kg.). Eleven batches may be considered to fall on a smooth curve (curve B) with a point of origin corresponding to L.D.50, 100 mg. Should this be so, these batches may be considered to be mixtures consisting of streptomycin of L.D.50, 200 mg. per kg., and a compound of toxicity greater than L.D.50, 100 mg. per kg. Alternatively the batches may be considered to be impure concentrates of a second pure streptomycin having an L.D.50 of some 100 mg. per kg. SUMMARY
-
Seventeen batches of streptomycin from one source (w.F.) have been examined pharmacologically and found to be free from histamine-like impurities and, with one exception, antidiuretic substances ; moreover, a simple relation has been found between potency and toxicity. This is in contrast with the observations previously published, which difference may be attributed to the biological variations which exist in the preparation of natural antibiotics. Important among these are the choice of medium and the method of extraction. A graph relating toxicity and potency reveals the presence of a hitherto uncharacterised substance more toxic than pure streptomycin, of acute toxicity to mice corresponding to L.D.50, 220 mg. per kg. This may be a very toxic material present in low concentration or a second of acute
toxicity to mice, corresponding to or thereby. Fourteen patients have been treated systematically with these batches. Eleven received 1.2 g. daily by intermittent intramuscular injection for ninety days, a total of 108 g. Four patients received 1-0 g. daily intramuscularly and 0.2 g. daily intrathecally for shorter periods. The toxic manifestations were trivial.
streptomycin
L.D.50, 100 mg. per kg.
We are indebted to the Wellcome Foundation Ltd. for the supply of seventeen batches of streptomycin. We wish to thank Mr. P. A. Young, Mr. M. Woodbine, and Mr. M. Cheeseman, of the Wellcome Research Laboratories, for the estimates of potency and toxicity, and Dr. J. Fine, of the County Hospital, Farnborough, Kent, for invaluable help with the biochemical and pathological aspect. The animal experiments were made by Dr. G. Brownlee and Mr. M. Woodbine. NOTE
After this paper was prepared for publication we had access to the report of the National Research Council (1946), whose committee on chemotherapeutics attribute hypersensitive reactions, such as skin eruptions and fever,
and
neurological disturbances, such as vertigo, tinnitus, deafness, and parsesthesia, to the active substances and not to impurities. They further found that the incidence of reactions increased with the total daily dose. When the average daily dose was more than 1 g. there was a striking increase in reactions. Among patients receiving 3 g. a day 46% had reactions ; and, when the daily dose was 4 g. or more, 60% had reactions. In our series of cases the average dose was 1’2 g. daily for ninety days, and such reactions, though diligently searched for, were not seen. REFERENCES
Cairns, H., Duthie, E. S., Smith, H. V. (1946) Lancet, ii, 153. Fleming, A. (1943) Ibid. ii, 434. Hettig, R. A., Adcock, J. D. (1946) Science, 103, 355. Hinshaw, H. C., Feldman, W. H. (1945) Proc. Mayo Clin. 20, 313. Molitor, H., Graessle, O. E., Kuna, S., Mushett, C. W., Silber, R. H. (1946) J. Pharmacol. 86, 151. National Research Council (1946) J. Amer. med. Ass. 132, 4, 70. Schatz, A., Bugie, E., Waksman, S. A. (1944) Proc. Soc. exp. Biol., N.Y. 55, 66.
CEREBRAL BERIBERI (WERNICKE’S ENCEPHALOPATHY) REVIEW OF 52 CASES IN A SINGAPORE PRISONER-OF-WAR HOSPITAL
H. E. DE WARDENER M.B.E., M.B. Lond., M.R.C.P.
B. LENNOX
CAPTAIN R.A.M.C.
LATE CAPTAIN R.A.M.C.
M.D. Durh.
IN 1881 Wernicke described three fatal cases of ataxia, ophthalmoplegia, and clouding of consciousness, which he called acute superior hsemorrhagic polioencephalitis and believed to be inflammatory. Subsequent descriptions emphasised the association with alcohol. The of vascular necropsy findings symmetrical change and haemorrhages, with glial proliferation and neuron degeneration in the paraventricular grey matter, usually best seen in the mammillary bodies, have often been described. From 1933 onwards a deficiency origin began to be suspected, and more recent clinical and experimental work has pointed to vitamin-B, deficiency as the principal factor. The disease in the past has been regarded as rare and chiefly diagnosed post mortem, and descriptions of the signs have been largely limited to those of the terminal stages with gross ocular and mental changes. An opportunity for placing a large number of healthy adults simultaneously on a standardised deficient diet and observing the results over a period of years is one which the many workers on the vitamin-B complex must have coveted. To some extent the capitulation of Singapore supplied these conditions : some 32,000 troops (British alone are included) were within a very few days of Feb. 15, 1942, changed suddenly from normal British rations to a grossly unbalanced diet consisting mainly of polished rice. Largely owing to the efforts of Colonel J. Bennet, A.M.s., consulting physician, Malaya Command, the medical services were from the first alive to the opportunities of study likely to be presented. All severe cases were admitted to the Roberts Hospital, Changi. In spite of considerable departures from the strict standards of a formal deprivation experiment-e.g., the diet was not, especially at first, completely standardised, and a severe epidemic of bacillary dysentery complicated and intensified the picture-and in spite of manifold difficulties encountered-e.g., laboratory facilities were primitive, drugs for curative experiments in short supply or non-existent, paper for records almost unobtainable, and the medical officers concerned, who never were themselves subjects of the experiment, completely fit-many of the observations made may be worth recording. A general account of the deficiency symptoms encountered in these men, with the biochemical findings, has already appeared (Burgess 1946, Cruickshank 1946) ; we present here a detailed description of 52 cases A2
death more often than do surgeons engaged in special branches of our art. Even so, let him who thinketh he standeth take heed lest he fall from unpreparedness. To prick the ventricle within three-quarters of a minute of ascertaining that the heart has ceased to beat, and, in the likely event of this simple procedure being ineffective, forthwith to massage the heart, will, I am sure, recall some of those whom Charon had started to ferrv across the. waters of the Styx. REFERENCES
Adams, H. D., Hand, L. V. (1942) J. Amer. med. Ass. 118, 133. Bailey, H. (1927) Practitioner, 118, 368. (1941) Brit. med. J. ii, 84. (1946a) Ibid, i, 29. (1946b) Ibid, p. 661. Bodon, C. (1923) Lancet, i, 586. Dodd, H., Prescott, F. (1943) Surg. Gynec. Obstet. 77, 645. Easton, A. M. (1942) Brit. med. J. i, 651. Elam, J. (1944) Brit. J. Anœsth. 19, 31. Grimshaw, C. (1942) Brit. med. J. i, 387. —
—
—
MacLeod, N., Schnipelsky, L. A. (1942) Ibid, p. 610. Morley, J. (1946) Ibid, i, 177. Nicholson, J. C. (1942) Ibid, i, 385. Primrose, W. B. (1935) Ibid, ii, 540. Vernon, H. K. (1943) Lancet, i, 6. Waters, R. M., Gillespie, N. A. (1944) Anesthesiology, 5, 113.
CLINICAL AND PHARMACOLOGICAL ASPECTS OF THE TOXICITY OF STREPTOMYCIN MADIGAN D. G. P. N. SWIFT M.B. N.U.I. M.R.C.S. TUBERCULOSIS
CHEMOTHERAPY UNIT,
COUNTY
HOSPITAL,
FARNBOROUGH, KENT
GEORGE BROWNLEE B.Sc.
Glasg., Ph.D. Lond.
OF THE WELLCOME RESEARCH
_
LABORATORIES, BECKENHAM,
KENT
As pharmacological and clinical experience with streptomycin (Schatz et al. 1944) accumulates, the potential and actual toxicity of the antibiotic and its concentrates becomes apparent. Already it is possible to indicate those intoxications due to the pure drug and those associated with known and unknown impurities. Thus, the fatty metamorphoses in the parenchymal cells of the liver, and to a lesser extent in the tubular epithelium of the kidneys, described by Molitor et al. (1946), are reversible changes produced in susceptible laboratory animals after the parenteral injection of pure streptomycin. We have observed these changes and the probably associated albuminuria in man. On the other hand, the reactions reported by Hettig and Adcock (1946)-facial flushing, headache, and fall of blood-pressure, appearing promptly and lasting 10-60 min.-are attributed to histamine-like impurities Molitor et al. (1946), who also reported the presence of an antidiuretic impurity. Most workers associate skin eruptions, rashes, and to some extent local pain with the presence of impurities. Whether the fever,
by
invariably accompanied by myalgias and arthralgias in the temporomandibular joints and suboccipital region (Hettig and Adcock 1946), and the clinical evidence of toxic effects on the eighth nerve seen in 3 of 34 patients by Hinshaw and Feldman (1945), are due to streptomycin or its impurities is a subject for further inquiry. We report here the results obtained from the examination in animals and man of the pharmacological effects of some seventeen batches of streptomycin. This material, though of varying purity, was free from histamine-like and other toxic substances, thus allowing large doses to be given intrathecally without incident. In well-marked contrast were the findings of Cairns et al. (1946), who recorded fatal reactions following injection of streptomycin of low dosage (Merck and Co. Inc., lots 200 and 213) in two out of seven infections. It is evident that streptomycin concentrates, though of high purity, are persistently contaminated by histamine-like substances (Molitor et al. 1946) ; hence it is
desirable that streptomycin biological control.
should be subjected to
PHARMACOLOGY
potencies of the batches of streptomycin from one (w.F.) are described in terms of weight of pure streptomycin base C21H37N7O12 (M.w.579) and have been derived from assay with a working standard of streptomycin sulphate, 11/1 H2SO4 (M.w.726), of known potency. It follows that streptomycin sulphate contains 79-8% equivalence of base ; in the accompanying graph this figure has been rounded off to 80%. Assay.-Assessment in terms of the working standard by a dilution test, using a constant inoculum of a susceptible The
source
strain of Bact. coli, C.N.1360, where the interval of each increment is
20%, yields an answer of potency ± S.D. 20%. Chemotherapy.—The potency and therapeutic activity of the batches studied were controlled by assay, in terms of a working standard, in which three doses of test were compared with three doses of standard in groups of six
mice infected with a number of lethal doses of a virulent strain of Bact. coli, c.N.348. Acute Toxicity in Mice.-The acute toxicity of streptomycin concentrates was studied after the intravenous injection of two doses into two groups each of ten animals weighing 18-20 g. The onset of symptoms was immediate in the case of toxic doses, and took the form of irregular breathing, dyspnoea, and apnoea. Arrest of the heart and death followed in three or four minutes and was due to asphyxia. There were no late deaths. The slopes of the line relating the probits corresponding to the per cent. mortalities and the logarithms of the two doses are all of the same order and reveal no artefact. The average lethal doses (L.D.50), derived graphically, range from 1-05 g. to 0.27 g. per kg. and are remarkable for the lack of toxicity which they reflect. This fact is illustrated on the accompanying graph, which shows the simple relation between acute toxicity and potency for the seventeen batches (w.F.) we have examined. The least potent batch (w.F.ll), of some 30% purity, is a fifth as toxic as pure streptomycin base, and the most potent batch, of some 80% purity, is two-thirds as toxic as pure streptomycin base. For comparison the acute toxicities and potencies of twenty-eight lots of streptomycin from one source (Merck Research Laboratories) derived from the paper of Molitor et al. (1946) are included in the graph. Most of the batches, though varying in potency between a quarter and a third of the potency of pure streptomycin, are seen to be twice to four times as toxic. Molitor et al. attribute this toxicity mainly to the presence of a histamine-like impurity ; an antidiuretic factor is also present. Included in the graph are the data on one sample of streptomycin sulphate from another sourcePfizer, lot 459. Circuda.tor2t Effects.—When tested in cats anæsthetised with pentobarbitone sodium (30 mg. per kg. intraperitoneally), no significant effects on blood-pressure or respiration were recorded in any of the seventeen batches of streptomycin concentrates. Antidiuretic Effects.-The acute effect of streptomycin concentrates on water diuresis was studied in groups of four rats. Food, but not water, was withdrawn on the previous night, and in the morning 5 ml. of tap water per 100 g. of body-weight was administered with a stomachtube, and a dose of 250 mg. per kg. of streptomycin concentrate was given subcutaneously. Urine was measured at hourly intervals for six hours. With two exceptions, batches 20 and 15, the shape of the normal diuresis curve remained unaltered, except that the two-hour peak was a little flattened, but the total urinary excretion was not significantly different. In the case of batch 15 the shape of the curve was slightly modified, but the total excretion was not significantly different ; with batch 20 there was retardation of diuresis for two hours, but thereafter it
10 was
normal, and the total urinary excretion
was
not
significantly altered. Intrathecal Injection
in Rabbits.-Rabbits anaesthetised with pentobarbitone sodium (40 mg. per kg. i.p.) tolerate without detectable reaction 10 nig. per kg. equivalence of streptomycin base injected cistern ally in 0-5 ml. of sterile distilled water following withdrawal of 0-5 ml. of cerebrospinal fluid.’ Under the same conditions two out of three rabbits did not survive the injection of 20 mg. per kg. equivalence of streptomycin base. Death followed in 10-15 min. from respiratory failure and was smooth and free from any other symptom. STUDIES IN MAN
In the
of the evaluation of antituberculous chemotherapeutic drugs in this hospital the opportunity to evaluate the status of streptomycin in a side-by-side comparison with established sulphone chemotherapy presented itself in June, 1946. A limited supply was available from one source (w.F.) in the form of seventeen batches which had been subject to pharmacological control. The material was available, in sterile rubbercapped containers, in the form of a freeze-dried powder and contained the equivalent of 0.2 g. of pure streptomycin base, together with a supply of sterile pyrogenfree distilled water. The biological control of the material before it reached us included tests for sterility and freedom from pyrogens, freedom from histaminelike impurities, and, with one exception, from antidiuretic substances. The average lethal doses in an acute experiment for mice of these batches were less than that of pure streptomycin. On the occasion of intrathecal injection an additional tonicity test was made on the batches used for this purpose. However, the figures obtained subsequently proved to be average values for all batches, an isotonic solution being obtained when one container (0-2 g.) was dissolved in 18 ml. of distilled water. In practice one container was mixed with 2 ml. of distilled water, but even with such a hypertonic solution no acute toxic effects were encountered. The usual precautions were taken of withdrawing at least an equal volume of cerebrospinal fluid, the use of a narrow-bore lumbarpuncture needle, very slow injection, and subsequently raising the foot of the bed. In the course of this trial every attempt was made to evaluate the toxic manifestations of streptomycin both clinically and biochemically. Fourteen patients, aged 2-60 years, served as subjects. The total doses of streptomycin varied from 0-4 g. given in 9 hours to 108 g. given in 90 days. All of the fourteen patients received streptomycin by intermittent intramuscular injection at four-hour intervals ; in addition four of the fourteen patients received a daily dose of 0-2 g. intrathecally by the lumbar route, or into the cisterna magna. Preliminary intramuscular injection in one of us (G. B.) proved the streptomycin to be painful ; hence at the start of the trial, and subsequently in those patients who complained of pain, streptomycin was administered in the presence of 0-5% procaine hydrochloride. Animal protein enzymic hydrolysate and glucose, of each 1 g. per kg. of body-weight, were given by mouth to all patients on streptomycin to reduce liability to renal and hepatic damage. Most patients were so seriously ill as to preclude systematic pathological investigations, but in most cases complete blood-counts (including differential leucocyte counts), haemoglobin estimations, and urine analysis were done at short intervals before, during, and after the experimental trial. The quantity of streptomycin circulating in the blood at four hours after the intramuscular injection of 0-2 g. of streptomycin was estimated from time to time throughout the trial- by the slide technique (Fleming 1943) with a strain of Bact. coli, c.N.1360. Mean blood concentrations of 8 µg. per ml. {minimum 4, maximum 16) were found. In cerebrocourse
Relation between potency and toxicity of batches of streptomycin from different sources. Wellcome Foundation batches fall on two curves, A and B.
spinal
fluid twenty-four hours after intrathecal injection of 0-2 g. (two specimens), the concentration was 3-2 and 6-4 µg. per ml. ; after intramuscular injection alone no streptomycin could be detected in the cerebrospinal fluid. No evidence of toxic effects on the hæmatopoietic system was observed, and the total and differential blood-counts revealed no fluctuations outside the normal limits. The urines of five subjects showed the presence of leucocytes and epithelial cells during the experimental period ; haematurias were not seen, but red corpuscles were found in two cases. Albuminuria was recorded in one case. Casts were not found on any occasion. No facial flushing or headache or any other evidence of histamine-like reaction, such as described by Hettig and Adcock (1946), was seen. One observation was made which seemed to us to be associated with the toxicity of pure streptomycin. This took the form of an increase of the normal diurnal excursion of the temperature range relative to the pyrexia. It was clearly demonstrated in three of the fourteen cases and showed no obvious relationship to any particular batch. It was not associated with myalgia or arthralgia nor did any symptom appear with it. In one case of advanced miliary tuberculosis with peritonitis and meningitis which came to necropsy, a diffuse fatty degeneration of the liver was found. This may have been due to the tuberculosis or to the streptomycin. No dermatoses were encountered, except a transient papulo-squamous lesion on the dorsum of the hands of a Thailand railway worker ex-prisoner-of-war. It is doubtful if this condition was due to streptomycin. No toxic effects on the auditory nerve appeared. On two occasions swelling and pain round the site of injection, with regional adenitis, developed after intramuscular injection but settled down with conservative, treatment in three or four days. DISCUSSION
The pharmacological and clinical study of seventeen batches of streptomycin from one source reveals several interesting differences between this material and that described elsewhere. The major differences are freedom from acute toxicity of an order greater than that of pure streptomycin, an effect associated to some extent with freedom from histamine-like and antidiuretic impurities. Clinically, ten patients, all seriously ill with tuberculosis, were treated with 1-2 g. daily for ninety days by intermittent intramuscular injectiona total of 108 g. of streptomycin drawn from several batches. Four patients were treated for shorter periods with 1.0 g. intramuscularly and 0-2 g. intrathecally daily. Our experiences were free from the hazards and perils
11
which have characterised previously reported trials, and have convinced us that streptomycin, of the purity which we used, can be administered in safety at the dose levels which we adopted. Differences of this kind have come to be expected from concentrates of antibiotics of natural origin and are inherent in the method of preparation. The nature of the medium and the extraction procedure are only two of the many variables. Since it is clear that streptomycin of a purity closely approaching chemical purity may vary in its intravenous acute toxicity between one and four times (Molitor et al. 1946), it is evident that the control of individual batches by biological standardisation is a necessity. It is desirable that streptomycin for parenteral use should be free from histamine-like substances and antidiuretic factors. Ilowever, it seems that streptomycin may be contaminated by an additional hitherto unidentified substance of greater toxicity but whose toxic effects are similar to the effect of toxic doses of streptomycin itself. The additional safeguards required of streptomycin for intrathecal use is that it should be free from pyrogens and have an acute toxicity for mice not greater than the pure streptomycin of L.D.50, 220 mg. per kg. In this connexion it is of interest to refer to the graph relating potency and toxicity of the batches we have examined. Six batches may be considered to fall on a smooth curve {curve A) with a point of origin at the L.D.50 of pure streptomycin (220 mg. per kg.). Eleven batches may be considered to fall on a smooth curve (curve B) with a point of origin corresponding to L.D.50, 100 mg. Should this be so, these batches may be considered to be mixtures consisting of streptomycin of L.D.50, 200 mg. per kg., and a compound of toxicity greater than L.D.50, 100 mg. per kg. Alternatively the batches may be considered to be impure concentrates of a second pure streptomycin having an L.D.50 of some 100 mg. per kg. SUMMARY
-
Seventeen batches of streptomycin from one source (w.F.) have been examined pharmacologically and found to be free from histamine-like impurities and, with one exception, antidiuretic substances ; moreover, a simple relation has been found between potency and toxicity. This is in contrast with the observations previously published, which difference may be attributed to the biological variations which exist in the preparation of natural antibiotics. Important among these are the choice of medium and the method of extraction. A graph relating toxicity and potency reveals the presence of a hitherto uncharacterised substance more toxic than pure streptomycin, of acute toxicity to mice corresponding to L.D.50, 220 mg. per kg. This may be a very toxic material present in low concentration or a second of acute
toxicity to mice, corresponding to or thereby. Fourteen patients have been treated systematically with these batches. Eleven received 1.2 g. daily by intermittent intramuscular injection for ninety days, a total of 108 g. Four patients received 1-0 g. daily intramuscularly and 0.2 g. daily intrathecally for shorter periods. The toxic manifestations were trivial.
streptomycin
L.D.50, 100 mg. per kg.
We are indebted to the Wellcome Foundation Ltd. for the supply of seventeen batches of streptomycin. We wish to thank Mr. P. A. Young, Mr. M. Woodbine, and Mr. M. Cheeseman, of the Wellcome Research Laboratories, for the estimates of potency and toxicity, and Dr. J. Fine, of the County Hospital, Farnborough, Kent, for invaluable help with the biochemical and pathological aspect. The animal experiments were made by Dr. G. Brownlee and Mr. M. Woodbine. NOTE
After this paper was prepared for publication we had access to the report of the National Research Council (1946), whose committee on chemotherapeutics attribute hypersensitive reactions, such as skin eruptions and fever,
and
neurological disturbances, such as vertigo, tinnitus, deafness, and parsesthesia, to the active substances and not to impurities. They further found that the incidence of reactions increased with the total daily dose. When the average daily dose was more than 1 g. there was a striking increase in reactions. Among patients receiving 3 g. a day 46% had reactions ; and, when the daily dose was 4 g. or more, 60% had reactions. In our series of cases the average dose was 1’2 g. daily for ninety days, and such reactions, though diligently searched for, were not seen. REFERENCES
Cairns, H., Duthie, E. S., Smith, H. V. (1946) Lancet, ii, 153. Fleming, A. (1943) Ibid. ii, 434. Hettig, R. A., Adcock, J. D. (1946) Science, 103, 355. Hinshaw, H. C., Feldman, W. H. (1945) Proc. Mayo Clin. 20, 313. Molitor, H., Graessle, O. E., Kuna, S., Mushett, C. W., Silber, R. H. (1946) J. Pharmacol. 86, 151. National Research Council (1946) J. Amer. med. Ass. 132, 4, 70. Schatz, A., Bugie, E., Waksman, S. A. (1944) Proc. Soc. exp. Biol., N.Y. 55, 66.
CEREBRAL BERIBERI (WERNICKE’S ENCEPHALOPATHY) REVIEW OF 52 CASES IN A SINGAPORE PRISONER-OF-WAR HOSPITAL
H. E. DE WARDENER M.B.E., M.B. Lond., M.R.C.P.
B. LENNOX
CAPTAIN R.A.M.C.
LATE CAPTAIN R.A.M.C.
M.D. Durh.
IN 1881 Wernicke described three fatal cases of ataxia, ophthalmoplegia, and clouding of consciousness, which he called acute superior hsemorrhagic polioencephalitis and believed to be inflammatory. Subsequent descriptions emphasised the association with alcohol. The of vascular necropsy findings symmetrical change and haemorrhages, with glial proliferation and neuron degeneration in the paraventricular grey matter, usually best seen in the mammillary bodies, have often been described. From 1933 onwards a deficiency origin began to be suspected, and more recent clinical and experimental work has pointed to vitamin-B, deficiency as the principal factor. The disease in the past has been regarded as rare and chiefly diagnosed post mortem, and descriptions of the signs have been largely limited to those of the terminal stages with gross ocular and mental changes. An opportunity for placing a large number of healthy adults simultaneously on a standardised deficient diet and observing the results over a period of years is one which the many workers on the vitamin-B complex must have coveted. To some extent the capitulation of Singapore supplied these conditions : some 32,000 troops (British alone are included) were within a very few days of Feb. 15, 1942, changed suddenly from normal British rations to a grossly unbalanced diet consisting mainly of polished rice. Largely owing to the efforts of Colonel J. Bennet, A.M.s., consulting physician, Malaya Command, the medical services were from the first alive to the opportunities of study likely to be presented. All severe cases were admitted to the Roberts Hospital, Changi. In spite of considerable departures from the strict standards of a formal deprivation experiment-e.g., the diet was not, especially at first, completely standardised, and a severe epidemic of bacillary dysentery complicated and intensified the picture-and in spite of manifold difficulties encountered-e.g., laboratory facilities were primitive, drugs for curative experiments in short supply or non-existent, paper for records almost unobtainable, and the medical officers concerned, who never were themselves subjects of the experiment, completely fit-many of the observations made may be worth recording. A general account of the deficiency symptoms encountered in these men, with the biochemical findings, has already appeared (Burgess 1946, Cruickshank 1946) ; we present here a detailed description of 52 cases A2