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A SLOW-RELEASE MEDIUM FOR ADRENOCORTICOTROPHIC HORMONE
71 A SLOW-RELEASE MEDIUM FOR ADRENOCORTICOTROPHIC HORMONE
H. M. BRUCE B.Sc. Lond.
A. S. PARSES M.A., Sc.D. Camb.,
D.Sc. Lond., F.R.S. From the National Institute for Medical Research, Mill Hill, London
IT is the paradoxical fate of many therapeutic preparations to become less effective as they are made more This is particularly true of extracts of some pure. of the endocrine organs, the steady continuous effects of which are not easily reprbduced by relatively infrequent injections of material rendered rapidly absorbable by purification. A number of techniques, including addition of other substances, esterification, and implantation in the dry state, have been used to delay absorption from the site of administration and thus level up the supply of hormone to the animal (see Deanesly and Parkes 1937). In this way a given total dose can be made as effective by infrequent as by frequent injection. Such problems are not, of course, restricted to steroids or to hormones, and they have been successfully dealt with in the case of insulin and of penicillin, and many other therapeutic substances. Difficulties due to over-rapid absorption after parenteral administration have now arisen in connection with adrenocorticotrophic hormone (A.C.T.H.). Acute effects of A.C.T.H., such as discharge of cortical steroids, depletion of cortical ascorbic acid, and changes in blood morphology, can readily be obtained by the short sharp stimulus given by intravenous injection or by the parenteral administration of a rapidly absorbed preparation. Study of these acute effects shows that A.C.T.H. remains in the circulation for less than 30 minutes, and that adrenal stimulation may be ended within an hour of the A.C.T.H. being absorbed. It follows that once-daily injection of a rapidly absorbed preparation may produce effects for only 1 hour out of the 24. It is therefore not surprising that many workers with A.C.T.H. have reported greater effectiveness when a given total dose is subdivided into frequent injections. The record seems to be held by Clayton and Prunty (1951), who in experiments on wound healing in mice gave no less than 25 injections over 30 hours. In clinical use the administration of A.c.T.H. is probably highly inefficient. Prunty (1951), for instance, reports that dosing every 6 hours gives good results, but that continuous intravenous administration increases effectiveness six to ten times as compared with subcutaneous injection four times daily. Nevertheless, comparatively little attention has been given to adjusting the rate of absorption of A.C.T.H. so as to produce maximal effectiveness. Wolfson et al. (1951) recently reported that A.C.T.H. adsorbed on aluminium and suspended in polyvinylpyrrolidone (P.v.P.) had a clinical effect lasting at least 24 hours, and good results were also obtained with A.C.T.H. tannate. It appears from the report of these workers that the use of aluminium stearate in oil, as introduced for penicillin (Ruckwalter and Dickison 1948), had a similar but somewhat lesser effect in delaying absorption. Prunty et al. (1951) record that the use of P.V.P. as a solvent for A.c.T.H. did little to improve its effectiveness. In experimental animals solid implants containing A.C.T.H. and magnesium stearate diluent have been used with some success (Robson and
to be used for assay purposes, some way would have to’ be found of increasing the effectiveness of modern preparations of A.C.T.H. when given by subcutaneous
was
injection. The first method tried
a once-daily injection of in arachis oil. This gave results little better than those obtained with saline. Twice-daily injection of the oil suspension increased the but not sufficiently to hold out any promise effect of a usable test. The next medium tried, 5% beeswax in arachis oil as used at one time for penicillin (Romansky and Rittman 1944), gave remarkable results in increasing the effectiveness of A.C.T.H., and the present report deals with some of the problems associated with the use of the wax-oil medium.
A.C.T.H.
was
powder suspended
slightly
METHODS
experiments were carried out on nestling days old, used for 3-day experiments in which
The main rats 4-10
distributed between the various group. Subsidiary ones were carried out on adults. The thymuses were dissected out and weighed fresh 24 hours after the last of a series of injections, or as otherwise stated. The normal thymus of the young rat is surprisingly regular in size, and at body weights between 10 g. and 100 g. averages 200-300 mg. per 100 g. body weight. In preparing the wax-oil medium, 5 g. of beeswax was dissolved in 95 g. arachis oil by heating to about 70°C. At room-temperature the mixture is semi-solid but can be reduced to a thick cream by shaking violently. The A.C.T.H. powder was incorporated into the required amount of heated medium by grinding with a small part of it and then with the whole. The suspension was reheated and stirred vigorously before each injection. The concentration was usually 10 mg. per ml. ; Armour’s The Preparation K 29901 was used throughout. cortisone acetate was in the form of Merck’s ’Cortone already suspended in proprietary aqueous medium.
litter-maters
were
treatments,
7-10
Effect of
nature
per
RESULTS
carried out of A.C.T.H.
ofmedium.—Several experiments
were
rats to compare the effectiveness dissolved in saline, suspended in oil, or suspended in wax-oil medium ; cortisone was used at the start as a reference substance. The results of one experiment are shown in the accompanying figure, and of two others in the table, and they can be summarised as follows : 1. Cortisone in daily doses of 0’2 mg. reduced the thymus to diffuse residual tissue, which is very difficult to dissect, on
nestling
phosphate
Sharaf 1951).. In considering possible methods of assaying A.C.T.H. we were led to correlate Moon’s (1940) work on the effect of
the thymus of the nestling rat with Jailor’s (1950) report that rats up to 8 days old did not show a The assay problem will be dealt with stress response. elsewhere. It need only be mentioned here that it verv soon became obvious that if an organ like the with a high threshold and requiring a chronic stimulation,
(
ICM.
A.C.T.H. on
thymus,
Thymus glands of nestling A.C.T.H. in 0 B AN AB
rats 8 aays , after 3 days’ treatment witFI6 various media. Utter-mates in horizontal lines. No treatment. 0-1 ml. 5°o beeswax in oil. A.C.T.H.I mg. in 0-1 mi. oil. A.C.T.H. I mg. in 01 mi. 5%, beeswax in oil.
72
averaged
about but -which in the animals listed in the table This destruction of the thymus may be 5 mg. in weight. regarded as maximal. (A smaller dose of cortisone,. 0-1 mg. daily for 3 days, had much less effect.) 2. A.C.T.H. in saline or arachis oil had no certain effect on
thymus weight.
THE EFFECT OF A.C.T.H..A2iD CORTISONE NESTLING RATS
ON THE THYMUS OF
_
3. Administration of the wax-oil medium alone had no effect on thymus weight, and therefore evoked no stress response detectable by this criterion. 4. A.C.T.H. in the wax-oil medium was highly effective, the response increasing with dosage so that three daily doses of 1 mg. evoked a maximal response similar to that with 0.2 mg. cortisone daily. .5. Methods of treatment which caused substantial decrease in thymus weight also decreased body growth. .
Other
showed that a high total dose in was almost as effective in one wax-oil (3 mg.) injection (thymus weight 91 mg. per 100 g. body weight) as in three (thymus weight 50 nig. per 100 g. body weight) ; a medium dose was much less so (thymus weights 189 mg. per 100 g. body weight, and 89 mg. per 100 g. body weight). In keeping with this result it was found that with a moderate dose there was no great delay after the last injection in the appearance of the maximal response. With larger doses, however, the maximal effect on the thymus did not appear for some days after injection. This finding is in keeping with the well-established principle of depot administration-that, with the same compound in the same medium, duration of effect is related to dose. It may also be related, through threshold levels, to body size. Several similar experiments on adult rats gave essentially similar results, and further showed that : of
experiments A.C.T.H.
1. Even in rats of 90 g.
size was produced wax-oil medium.
no
detectable stress effect on thymus and daily injection of the
by handling
2. A single injection of 8 mg. A.C.T.H. in wax-oil was almost as effective as 4 daily injections of 2 mg. 3. Reduction of the beeswax content of the medium from 5% to 2% decreased effectiveness greatly. DISCUSSION
than one-tenth maximal efficiency. It need hardly be pointed out that any easy method of potentiating by ten times the clinical effectiveness of A.C.T.H. preparations would be equivalent to increasing the supply by ten times and decreasing. the cost similarly. SUMMARY
1. A test based on decrease of thymus weight in the rat (particularly the nestling rat) has been used to study
the influence of medium on the effectiveness of A.C.T.H. 2. Suspension of the hormone in a medium consisting of 5% beeswax in arachis oil, as at one time used for penicillin, increased the effectiveness of daily injections by ten times or more as compared with solution in saline or suspension in oil alone. 3. Large doses of A.C.T.H. given as a single injection in the wax-oil medium produced an effect for several days. REFERENCES
Buckwalter, F. H., Dickison, H. L. (1948) J. Amer. pharm. Ass. 37, 472. Clayton, B. E., Prunty, F. T. G. (1951) Analyst, 76, 474. Deanesly, R., Parkes, A. S. (1937) Proc. roy. Soc. 124, 279. Floyd, J. C. (1949) J. Pharm. Pharmacol. 1, 747. Jailor, J. W. (1950) Endocrinology 46, 420. Moon, H. D. (1940) Proc. Soc. exp. Biol., N.Y. 43, 42. Prunty, F. T. G. (1951) Practitioner, 166, 33. Brooksbank, B. W. L., Clayton, B. E., McSwiney, R. R. (1951) J. Endocrinol. 7, 75 P. Robson, J. M., Sharaf, A. A. (1951) J. Physiol. 114, 11 P. Romansky, M. J., Rittman, G. E. (1944) Science, 100, 196. Wolfson, W. Q., Thompson, R. E., Robinson, W. D., Duff, I. F., Cohen, C., Lewis, L.. Hant, H. D. (1951) Proceedings of Second Clinical A.C.T.H. Conference. New York; p. 1. —
This work on the potentiation of A.C.T.H. by the use of a wax-oil medium, taken in conjunction with the American work on adsorbates, tannates, and aluminium stearate suspensions, leaves little doubt as to the potential value of slow-release preparations. At least one point, Beeswax, however, will require further attention. which is solid at body-temperature, presumably acts by forming a relatively insoluble coat round the particles of active material.Particle size, therefore, may play some part in determining the effectiveness of A.C.T.H. administered in the wax-oil medium, as it has been found to do in the case of penicillin (see Floyd 1949, for review of this matter). Also, the fluffy preparations of A.C.T.H. obtained by freeze-drying might behave differently from the dense gritty preparations obtained by other methods. Owing to the negligible effect of A.C.T.H. in the thymus test when it is given in saline once daily, we are not able to offer figures for the potentiation effected by the use of a slow-release medium, but it can hardly be less than ten times. A.c.T.H. is used essentially in clinical practice to stimulate the adrenal cortex over at least some days, and it is normally given dissolved in saline, in not more than 2 injections per day. According to Prunty’s figures, A.C.T.H. thus given may well have less At the suggestion, and by courtesy, of 31r. C. J. Eastland of Messrs. Allen & Hanburys, we have been able to try a medium composed of 2% aluminium stearate and4.5% beeswax in arachis oil. This medium, as received by us, was of a creamy consistence at room-temperature, and was used in that form without reheating for the suspension of A.C.T.H. powder. Preliminary results were much less good than with A.C.T.H. suspended in the warmed 5 % waxroil medium described above, and it is likely that the solid particles of A.C.T.H. are coated less adequately when incorporated in a cold medium.
TOXICITY OF DIHYDROSTREPTOMYCIN SULPHATE FOR THE AUDITORY NERVE JOAN GREGORY CONWAY DON M.B. Lond., M.R.C.P.
M.R.C.S.
MEDICAL
ASSISTANT SURGICAL REGISTRAR, EAR, NOSE, AND THROAT
REGISTRAR
DEPARTMENT
LATELY ASSISTANT
UNIVERSITY COLLEGE
HOSPITAL, LONDON
THE efficacy of streptomycin in the treatment of tuberculosis is well recognised, but unfortunately it has toxic effects on the vestibular apparatus. Search for a less toxic remedy led to the preparation of dihydrostreptomycin (Peck et al. 1946), and this was shown by Donovick and Rake (1947) to resemble streptomycin biologically. Further laboratory investigations (Rake et al. 1948, Feldman et al. 1948, Edison et al. 1948) clearly showed that, both in vitro and in laboratory’ animals, the action of dihydrostreptomycin on the tubercle bacillus closely resembled that of streptomycin. Moreover it was thought to be less toxic to-the vestibular apparatus, and Edison et al. (1948) wrote : "
significant fact is that, in the conversion of strepto-r mycin dihydrostreptomycin, the neurotoxic action has been reduced without sacrificing the antimicrobial efficacy." The
to
H. M. BRUCE B.Sc. Lond.
A. S. PARSES M.A., Sc.D. Camb.,
D.Sc. Lond., F.R.S. From the National Institute for Medical Research, Mill Hill, London
IT is the paradoxical fate of many therapeutic preparations to become less effective as they are made more This is particularly true of extracts of some pure. of the endocrine organs, the steady continuous effects of which are not easily reprbduced by relatively infrequent injections of material rendered rapidly absorbable by purification. A number of techniques, including addition of other substances, esterification, and implantation in the dry state, have been used to delay absorption from the site of administration and thus level up the supply of hormone to the animal (see Deanesly and Parkes 1937). In this way a given total dose can be made as effective by infrequent as by frequent injection. Such problems are not, of course, restricted to steroids or to hormones, and they have been successfully dealt with in the case of insulin and of penicillin, and many other therapeutic substances. Difficulties due to over-rapid absorption after parenteral administration have now arisen in connection with adrenocorticotrophic hormone (A.C.T.H.). Acute effects of A.C.T.H., such as discharge of cortical steroids, depletion of cortical ascorbic acid, and changes in blood morphology, can readily be obtained by the short sharp stimulus given by intravenous injection or by the parenteral administration of a rapidly absorbed preparation. Study of these acute effects shows that A.C.T.H. remains in the circulation for less than 30 minutes, and that adrenal stimulation may be ended within an hour of the A.C.T.H. being absorbed. It follows that once-daily injection of a rapidly absorbed preparation may produce effects for only 1 hour out of the 24. It is therefore not surprising that many workers with A.C.T.H. have reported greater effectiveness when a given total dose is subdivided into frequent injections. The record seems to be held by Clayton and Prunty (1951), who in experiments on wound healing in mice gave no less than 25 injections over 30 hours. In clinical use the administration of A.c.T.H. is probably highly inefficient. Prunty (1951), for instance, reports that dosing every 6 hours gives good results, but that continuous intravenous administration increases effectiveness six to ten times as compared with subcutaneous injection four times daily. Nevertheless, comparatively little attention has been given to adjusting the rate of absorption of A.C.T.H. so as to produce maximal effectiveness. Wolfson et al. (1951) recently reported that A.C.T.H. adsorbed on aluminium and suspended in polyvinylpyrrolidone (P.v.P.) had a clinical effect lasting at least 24 hours, and good results were also obtained with A.C.T.H. tannate. It appears from the report of these workers that the use of aluminium stearate in oil, as introduced for penicillin (Ruckwalter and Dickison 1948), had a similar but somewhat lesser effect in delaying absorption. Prunty et al. (1951) record that the use of P.V.P. as a solvent for A.c.T.H. did little to improve its effectiveness. In experimental animals solid implants containing A.C.T.H. and magnesium stearate diluent have been used with some success (Robson and
to be used for assay purposes, some way would have to’ be found of increasing the effectiveness of modern preparations of A.C.T.H. when given by subcutaneous
was
injection. The first method tried
a once-daily injection of in arachis oil. This gave results little better than those obtained with saline. Twice-daily injection of the oil suspension increased the but not sufficiently to hold out any promise effect of a usable test. The next medium tried, 5% beeswax in arachis oil as used at one time for penicillin (Romansky and Rittman 1944), gave remarkable results in increasing the effectiveness of A.C.T.H., and the present report deals with some of the problems associated with the use of the wax-oil medium.
A.C.T.H.
was
powder suspended
slightly
METHODS
experiments were carried out on nestling days old, used for 3-day experiments in which
The main rats 4-10
distributed between the various group. Subsidiary ones were carried out on adults. The thymuses were dissected out and weighed fresh 24 hours after the last of a series of injections, or as otherwise stated. The normal thymus of the young rat is surprisingly regular in size, and at body weights between 10 g. and 100 g. averages 200-300 mg. per 100 g. body weight. In preparing the wax-oil medium, 5 g. of beeswax was dissolved in 95 g. arachis oil by heating to about 70°C. At room-temperature the mixture is semi-solid but can be reduced to a thick cream by shaking violently. The A.C.T.H. powder was incorporated into the required amount of heated medium by grinding with a small part of it and then with the whole. The suspension was reheated and stirred vigorously before each injection. The concentration was usually 10 mg. per ml. ; Armour’s The Preparation K 29901 was used throughout. cortisone acetate was in the form of Merck’s ’Cortone already suspended in proprietary aqueous medium.
litter-maters
were
treatments,
7-10
Effect of
nature
per
RESULTS
carried out of A.C.T.H.
ofmedium.—Several experiments
were
rats to compare the effectiveness dissolved in saline, suspended in oil, or suspended in wax-oil medium ; cortisone was used at the start as a reference substance. The results of one experiment are shown in the accompanying figure, and of two others in the table, and they can be summarised as follows : 1. Cortisone in daily doses of 0’2 mg. reduced the thymus to diffuse residual tissue, which is very difficult to dissect, on
nestling
phosphate
Sharaf 1951).. In considering possible methods of assaying A.C.T.H. we were led to correlate Moon’s (1940) work on the effect of
the thymus of the nestling rat with Jailor’s (1950) report that rats up to 8 days old did not show a The assay problem will be dealt with stress response. elsewhere. It need only be mentioned here that it verv soon became obvious that if an organ like the with a high threshold and requiring a chronic stimulation,
(
ICM.
A.C.T.H. on
thymus,
Thymus glands of nestling A.C.T.H. in 0 B AN AB
rats 8 aays , after 3 days’ treatment witFI6 various media. Utter-mates in horizontal lines. No treatment. 0-1 ml. 5°o beeswax in oil. A.C.T.H.I mg. in 0-1 mi. oil. A.C.T.H. I mg. in 01 mi. 5%, beeswax in oil.
72
averaged
about but -which in the animals listed in the table This destruction of the thymus may be 5 mg. in weight. regarded as maximal. (A smaller dose of cortisone,. 0-1 mg. daily for 3 days, had much less effect.) 2. A.C.T.H. in saline or arachis oil had no certain effect on
thymus weight.
THE EFFECT OF A.C.T.H..A2iD CORTISONE NESTLING RATS
ON THE THYMUS OF
_
3. Administration of the wax-oil medium alone had no effect on thymus weight, and therefore evoked no stress response detectable by this criterion. 4. A.C.T.H. in the wax-oil medium was highly effective, the response increasing with dosage so that three daily doses of 1 mg. evoked a maximal response similar to that with 0.2 mg. cortisone daily. .5. Methods of treatment which caused substantial decrease in thymus weight also decreased body growth. .
Other
showed that a high total dose in was almost as effective in one wax-oil (3 mg.) injection (thymus weight 91 mg. per 100 g. body weight) as in three (thymus weight 50 nig. per 100 g. body weight) ; a medium dose was much less so (thymus weights 189 mg. per 100 g. body weight, and 89 mg. per 100 g. body weight). In keeping with this result it was found that with a moderate dose there was no great delay after the last injection in the appearance of the maximal response. With larger doses, however, the maximal effect on the thymus did not appear for some days after injection. This finding is in keeping with the well-established principle of depot administration-that, with the same compound in the same medium, duration of effect is related to dose. It may also be related, through threshold levels, to body size. Several similar experiments on adult rats gave essentially similar results, and further showed that : of
experiments A.C.T.H.
1. Even in rats of 90 g.
size was produced wax-oil medium.
no
detectable stress effect on thymus and daily injection of the
by handling
2. A single injection of 8 mg. A.C.T.H. in wax-oil was almost as effective as 4 daily injections of 2 mg. 3. Reduction of the beeswax content of the medium from 5% to 2% decreased effectiveness greatly. DISCUSSION
than one-tenth maximal efficiency. It need hardly be pointed out that any easy method of potentiating by ten times the clinical effectiveness of A.C.T.H. preparations would be equivalent to increasing the supply by ten times and decreasing. the cost similarly. SUMMARY
1. A test based on decrease of thymus weight in the rat (particularly the nestling rat) has been used to study
the influence of medium on the effectiveness of A.C.T.H. 2. Suspension of the hormone in a medium consisting of 5% beeswax in arachis oil, as at one time used for penicillin, increased the effectiveness of daily injections by ten times or more as compared with solution in saline or suspension in oil alone. 3. Large doses of A.C.T.H. given as a single injection in the wax-oil medium produced an effect for several days. REFERENCES
Buckwalter, F. H., Dickison, H. L. (1948) J. Amer. pharm. Ass. 37, 472. Clayton, B. E., Prunty, F. T. G. (1951) Analyst, 76, 474. Deanesly, R., Parkes, A. S. (1937) Proc. roy. Soc. 124, 279. Floyd, J. C. (1949) J. Pharm. Pharmacol. 1, 747. Jailor, J. W. (1950) Endocrinology 46, 420. Moon, H. D. (1940) Proc. Soc. exp. Biol., N.Y. 43, 42. Prunty, F. T. G. (1951) Practitioner, 166, 33. Brooksbank, B. W. L., Clayton, B. E., McSwiney, R. R. (1951) J. Endocrinol. 7, 75 P. Robson, J. M., Sharaf, A. A. (1951) J. Physiol. 114, 11 P. Romansky, M. J., Rittman, G. E. (1944) Science, 100, 196. Wolfson, W. Q., Thompson, R. E., Robinson, W. D., Duff, I. F., Cohen, C., Lewis, L.. Hant, H. D. (1951) Proceedings of Second Clinical A.C.T.H. Conference. New York; p. 1. —
This work on the potentiation of A.C.T.H. by the use of a wax-oil medium, taken in conjunction with the American work on adsorbates, tannates, and aluminium stearate suspensions, leaves little doubt as to the potential value of slow-release preparations. At least one point, Beeswax, however, will require further attention. which is solid at body-temperature, presumably acts by forming a relatively insoluble coat round the particles of active material.Particle size, therefore, may play some part in determining the effectiveness of A.C.T.H. administered in the wax-oil medium, as it has been found to do in the case of penicillin (see Floyd 1949, for review of this matter). Also, the fluffy preparations of A.C.T.H. obtained by freeze-drying might behave differently from the dense gritty preparations obtained by other methods. Owing to the negligible effect of A.C.T.H. in the thymus test when it is given in saline once daily, we are not able to offer figures for the potentiation effected by the use of a slow-release medium, but it can hardly be less than ten times. A.c.T.H. is used essentially in clinical practice to stimulate the adrenal cortex over at least some days, and it is normally given dissolved in saline, in not more than 2 injections per day. According to Prunty’s figures, A.C.T.H. thus given may well have less At the suggestion, and by courtesy, of 31r. C. J. Eastland of Messrs. Allen & Hanburys, we have been able to try a medium composed of 2% aluminium stearate and4.5% beeswax in arachis oil. This medium, as received by us, was of a creamy consistence at room-temperature, and was used in that form without reheating for the suspension of A.C.T.H. powder. Preliminary results were much less good than with A.C.T.H. suspended in the warmed 5 % waxroil medium described above, and it is likely that the solid particles of A.C.T.H. are coated less adequately when incorporated in a cold medium.
TOXICITY OF DIHYDROSTREPTOMYCIN SULPHATE FOR THE AUDITORY NERVE JOAN GREGORY CONWAY DON M.B. Lond., M.R.C.P.
M.R.C.S.
MEDICAL
ASSISTANT SURGICAL REGISTRAR, EAR, NOSE, AND THROAT
REGISTRAR
DEPARTMENT
LATELY ASSISTANT
UNIVERSITY COLLEGE
HOSPITAL, LONDON
THE efficacy of streptomycin in the treatment of tuberculosis is well recognised, but unfortunately it has toxic effects on the vestibular apparatus. Search for a less toxic remedy led to the preparation of dihydrostreptomycin (Peck et al. 1946), and this was shown by Donovick and Rake (1947) to resemble streptomycin biologically. Further laboratory investigations (Rake et al. 1948, Feldman et al. 1948, Edison et al. 1948) clearly showed that, both in vitro and in laboratory’ animals, the action of dihydrostreptomycin on the tubercle bacillus closely resembled that of streptomycin. Moreover it was thought to be less toxic to-the vestibular apparatus, and Edison et al. (1948) wrote : "
significant fact is that, in the conversion of strepto-r mycin dihydrostreptomycin, the neurotoxic action has been reduced without sacrificing the antimicrobial efficacy." The
to