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An improved assay for the simultaneous determination of the biological activities of anti-inflammatory steroids
VOLUME I
FEBRUARY 1963
S T E R 0 1D S
163
AN IMPROVED ASSAY FOR THE SIMULTANEO[B ~ETERMINATION OF THE BIOLOGICAL ACTIVITIES OF ANTI-INFLAMMATOEY STEROIDS
BY Sanford L. Steelman, Evan R. Morgan and Robert H. Silber Merck Institute for Therapeutic Research Rahway, New Jersey Heceived January 15. 1963 Numerous assays have been described for the determination of the varied activities of the adrenal cortical hormones.
In most in-
stances only one or two indices, such as adrenal weight decrease (ACTH suppression) and thymic involution were simultaneously quantitated. Meier, et al.l described a method for the measurement of antiinflammatory activity by the inhibition of granuloma formation.
This
has become the basis of many anti-inflammatory assay procedures. Dorfman 2 has recently reviewed the various assay methods for antiinflammatory steroids.
It has been repeatedly demonstrated that valid
comparisons of potencies are difficult to make when assays are conducted under different conditions.
A preliminary report has been made by
Silber3 indicating the feasibility of the concurrent estimation of effects on body, adrenal, and thymus weights in the intact rat after feeding steroids in the diet.
A procedure has now been developed to quantita-
tively measure simultaneously in the rat, four separate indices of biological activity--body weight change, thymic involution, adrenal suppression and granuloma inhibition.
By the use of such methods it
should be easier to select anti-inflammatory steroids with meaningful differences in their biological activities.
164
S T E R 0 I D S
FEBRUARY 1963
VOLUME 1
METHOIB
Male rats (Holtzman) weighing approximately 125 grams were fed for seven days a diet containing the steroid.
The night prior to
the start of the experiment and the one before autopsy, food was withdrawn from the animals so that fasted body weights could be obtained. A 24-hour food consumption was determined between the 4th and 6th days of treatment.
All animals were individually caged for ease of handling.
The diets were prepared by adding a solution of the steroid in 50-100 ml of ethanol to the required quantity of ground Purina Chow. Thorough mixing and evaporation of the ethanol was accomplished with a laboratory model rotary diet mixer.
Rats were anesthetized with ether and two cotton pellets weighing 45-50 + 1 mg each were individually implanted subcutaneously in the ventral abdominal area.
Before implantation, each pellet was
moistened with O.1 ml of a penicillin G-streptomycln sulfate solution (lO mg/ml of each).
In addition, the animals were given subcutaneously
O.1 ml of the above solution on days 1 and 3 of the test.
If this
regimen was followed no infections of the implantation areas were incurred.
After one week of treatment the animals were weighed and then killed with carbon dioxide.
The cotton pellets, together with the
surrounding tissue, were carefully dissected from the subcutaneous tissue.
The adrenal and thymus glands were removed, trimmed and weighed
immediately.
The pellets were air dried overnight at 60°C. prior to
VOLUME 1
weighing.
S T E R 0 1D S
FEBRUARY 1963
165
The net increase in pellet weight was determined by sub-
traction of the tare weight.
RESULTS
Table I lists the experimental data obtained in a single three dose level assay comparing hydrocortisone with cortisone.
For all four
indices of activity, highly significant regression curves were obtained with no slope divergence between any of the dose response curves of the two compounds.
The various indices produced log dose response curves
and standard statistical methods for the treatment of such data were employed to calculate potencies.
In the case of thymic weights the
variation of response at the various dosage levels was not constant so that a logarithmic transformation was used for calculation of the data. The mean index of precision (~ - s) for each index was excellent except for the adrenal weight which was still in a very acceptable range.
The
precision of the granuloma potency is comparable or superior to that reported by other authors for both granuloma pouch and pellet methods. Bush and Alexander 4 recommended the use of carrageenin to improve the precision of the granuloma pellet assay.
The authors' experience has
been that carrageenin does indeed increase granuloma formation but it changes neither the slope nor the variation of the individual responses, and, as a result, no advantage is obtained in its use.
Statistical analyses of assays showed that all four indices of activity were more accurately measured when the steroid was incorporated into the diet rather than given as a single daily oral dose.
.The
166
S T E R 0 I D S
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FEBRUARY 1963
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VOLUME 1
FEBRUARY1963
S T E R O I D S
167
total amount of steroid required for potency determination was also less.
As a reference standard doses of 25, 50 and i00 mg or 3Oj 60, 120 mg/kg diet of hydrocortisone have been used with good results.
The
former dosage schedule was preferable since in some instances the thymus weight curve gave significant curvature at the highest level.
In the evaluation of systemic anti-lnflammatory steroids, it should be kept in mind that there are a number of factors which can alter the potency of a compound.
For instance, hydrocortisone acetate
given subcutaneously gives abnormally low values because of its limited solubility and absorbability (Hershberger and Calhoun5 and Silber3). The latter author has sho~n that use of the oral route of administration minimizes or prevents such absorption differences.
A number of 21
esters and similar derivatives have been compared with the parent steroid and it has been determined that, after correcting for differences in molecular weight, the method herein described gave the theoretical potency for the derivative.
DISCUSSION
Using the method described above, four indices of biological activity can be determined simultaneously with good precision.
The
graded decrease in body weight is generally regarded as a reflection of catabolic action.
Since ,11 compounds used in the test should be of
known purity and toxicity, adrenalectomy is not required.
Selected
steroids have been assayed in adrenalectomized rats using the conditions and steroid concentration herein described, and it has been found that
168
S T E R 0 I D S
FEBRUARY 1963
VOLUME 1
both thymic and anti-inflammatory potencies are in excellent agreement with those in intact animals.
It will be noticed in Table i that food consumption decreases with increasing dosage of the steroid.
Statistical analysis has shown
that attempts to correct calculations for dosage from food consumption data were, in general, not meaningful.
Obviously, if one had a compound
which did not decrease food consumption at anti-inflammatory doses a correction would he in order.
To date, no such anti-inflammatory
steroidal compound has been detected.
In fact, no significant divergence
of body weight and granuloma potencies has been seen, substantiating the assumption of many that the general catabolic action of glucocorticoids may be intimately associated with anti-inflammatory activity.
Many anti-inflammatory steroids have been evaluated with this method.
The potencies obtained were consistent with those reported by
other methods.
Even steroids with potencies in excess of one hundred
times hydrocortisone have given excellent results with little or no evidence of divergence of the slopes of the various indices.
Table 2
summarizes the data of a single large experiment in which three compounds of widely differing potencies were compared.
These 16~ methyl deriva-
tives of hydrocortisone have previously been described by Arth et. al. 6. It can be seen that highly precise potencies were obtained and that there were no instances in which there was a significant potency difference between the various parameters measured for a given compound. Other compounds have been found which give significant separations.
In
general, progesterone derivatives give a high adrenal suppression activity in relation to granuloma inhibition.
This has been previously
VOLUME
FEBRUARY 1963
I
ST E R 0 I D S
169
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VOLUME 1
FEBRUARY1963
S T E R 0 I D S
171
reported by Glenn, et al.7 for 6a methyl 17a acetoxy progesterone (MAP). In the authors t experience MAP has a slight anti-inflammatory activity. However, the 17a methoxy derivative of 6a methyl 17 hydroxy progesterone (MMP) has been shown to have a highly significant suppression of adrenal weight without other significant biological activity. the data of a typical experiment.
Table 3 summarizes
Other examples of activity separation
with anti-inflammatory steroids will be published in the near future.
The method herein reported has also been found to be useful for the evaltmtion of non-steroidal anti-inflammatory compounds. Phenylbutazone at doses of 200-2000 ~ / k g of diet gave highly significant graded granuloma inhibitions without affecting body, thymus or adrenal weights.
At levels above 2000 m ~ k g diet, toxicity appears in the form
of body and thymic weight decreases and adrenal weight increase. These changes indicate adrenocortical stimulation and provide a built in method of separating anti-inflammatory activity from toxicity.
The
granuloma inhibition dose response curves of non-steroidal compounds are not as steep as with anti-inflammatory steroids, so that precision is significantly decreased.
SUMMARY A method has been described which simultaneously measures four biological indices of anti-inflammatory steroids with excellent precision.
Its applicability for selection of both steroidal and
non-steroidal anti-inflammatory compounds has been illustrated.
172
S T E R 0
I D S
FEBRUARY 1963
VOLUME 1
RE~CES
.
Meier, R., Schuler, W., and Desaulles, P., EXPERIENTIA 6, 469, (195o).
.
Dorfman, R. I., METHODS IN HORM~E RESEARCH, Vol. II, Academic Press, New York, 1962, p. 325.
3.
Silber, R. H., ANN. N.Y. ACAD. SCI. 82, 821 (1959).
4.
Bush, E. I., and Alexander, R. W., ACTA ENDOCRINOL. 35,
268 (196o). .
Hershberger, L. D., and Calhoun, D. L., ENDOCRINOLOGY60, 153 (1957).
.
Arth, G. E., Fried, J., Johnston, D. B. R., Hoff, D. R., Sarett, L. H., Silber, R. H., Stoerk, H. C., and Winter, C. A., J. AM. CH~. SOC. 80, 3161 (1958).
.
Glenn, E. M., Richardson, S. L., and Bowman, B. J., METABOLISM ~, 185 (1959).
ACKNOWL~DGEMEN TS
The authors are indebted to Maryann Petraitis, Raymond L. Primka, Mary E. Regn and Walter Worosila for valuable technical assistance in carrying out the many experiments. The compounds were generously supplied by members of the Fundamental Research Department, Merck Sharp and Doh~e Research Laboratories, Rahway, New Jersey. Messrs. J. L. Ciminera and A. G. Itkin provided valuable assistance by performing statistical analyses of the biological data.
FEBRUARY 1963
S T E R 0 1D S
163
AN IMPROVED ASSAY FOR THE SIMULTANEO[B ~ETERMINATION OF THE BIOLOGICAL ACTIVITIES OF ANTI-INFLAMMATOEY STEROIDS
BY Sanford L. Steelman, Evan R. Morgan and Robert H. Silber Merck Institute for Therapeutic Research Rahway, New Jersey Heceived January 15. 1963 Numerous assays have been described for the determination of the varied activities of the adrenal cortical hormones.
In most in-
stances only one or two indices, such as adrenal weight decrease (ACTH suppression) and thymic involution were simultaneously quantitated. Meier, et al.l described a method for the measurement of antiinflammatory activity by the inhibition of granuloma formation.
This
has become the basis of many anti-inflammatory assay procedures. Dorfman 2 has recently reviewed the various assay methods for antiinflammatory steroids.
It has been repeatedly demonstrated that valid
comparisons of potencies are difficult to make when assays are conducted under different conditions.
A preliminary report has been made by
Silber3 indicating the feasibility of the concurrent estimation of effects on body, adrenal, and thymus weights in the intact rat after feeding steroids in the diet.
A procedure has now been developed to quantita-
tively measure simultaneously in the rat, four separate indices of biological activity--body weight change, thymic involution, adrenal suppression and granuloma inhibition.
By the use of such methods it
should be easier to select anti-inflammatory steroids with meaningful differences in their biological activities.
164
S T E R 0 I D S
FEBRUARY 1963
VOLUME 1
METHOIB
Male rats (Holtzman) weighing approximately 125 grams were fed for seven days a diet containing the steroid.
The night prior to
the start of the experiment and the one before autopsy, food was withdrawn from the animals so that fasted body weights could be obtained. A 24-hour food consumption was determined between the 4th and 6th days of treatment.
All animals were individually caged for ease of handling.
The diets were prepared by adding a solution of the steroid in 50-100 ml of ethanol to the required quantity of ground Purina Chow. Thorough mixing and evaporation of the ethanol was accomplished with a laboratory model rotary diet mixer.
Rats were anesthetized with ether and two cotton pellets weighing 45-50 + 1 mg each were individually implanted subcutaneously in the ventral abdominal area.
Before implantation, each pellet was
moistened with O.1 ml of a penicillin G-streptomycln sulfate solution (lO mg/ml of each).
In addition, the animals were given subcutaneously
O.1 ml of the above solution on days 1 and 3 of the test.
If this
regimen was followed no infections of the implantation areas were incurred.
After one week of treatment the animals were weighed and then killed with carbon dioxide.
The cotton pellets, together with the
surrounding tissue, were carefully dissected from the subcutaneous tissue.
The adrenal and thymus glands were removed, trimmed and weighed
immediately.
The pellets were air dried overnight at 60°C. prior to
VOLUME 1
weighing.
S T E R 0 1D S
FEBRUARY 1963
165
The net increase in pellet weight was determined by sub-
traction of the tare weight.
RESULTS
Table I lists the experimental data obtained in a single three dose level assay comparing hydrocortisone with cortisone.
For all four
indices of activity, highly significant regression curves were obtained with no slope divergence between any of the dose response curves of the two compounds.
The various indices produced log dose response curves
and standard statistical methods for the treatment of such data were employed to calculate potencies.
In the case of thymic weights the
variation of response at the various dosage levels was not constant so that a logarithmic transformation was used for calculation of the data. The mean index of precision (~ - s) for each index was excellent except for the adrenal weight which was still in a very acceptable range.
The
precision of the granuloma potency is comparable or superior to that reported by other authors for both granuloma pouch and pellet methods. Bush and Alexander 4 recommended the use of carrageenin to improve the precision of the granuloma pellet assay.
The authors' experience has
been that carrageenin does indeed increase granuloma formation but it changes neither the slope nor the variation of the individual responses, and, as a result, no advantage is obtained in its use.
Statistical analyses of assays showed that all four indices of activity were more accurately measured when the steroid was incorporated into the diet rather than given as a single daily oral dose.
.The
166
S T E R 0 I D S
I. •~
r.-I r.-I
~
FEBRUARY 1963
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VOLUME 1
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VOLUME 1
FEBRUARY1963
S T E R O I D S
167
total amount of steroid required for potency determination was also less.
As a reference standard doses of 25, 50 and i00 mg or 3Oj 60, 120 mg/kg diet of hydrocortisone have been used with good results.
The
former dosage schedule was preferable since in some instances the thymus weight curve gave significant curvature at the highest level.
In the evaluation of systemic anti-lnflammatory steroids, it should be kept in mind that there are a number of factors which can alter the potency of a compound.
For instance, hydrocortisone acetate
given subcutaneously gives abnormally low values because of its limited solubility and absorbability (Hershberger and Calhoun5 and Silber3). The latter author has sho~n that use of the oral route of administration minimizes or prevents such absorption differences.
A number of 21
esters and similar derivatives have been compared with the parent steroid and it has been determined that, after correcting for differences in molecular weight, the method herein described gave the theoretical potency for the derivative.
DISCUSSION
Using the method described above, four indices of biological activity can be determined simultaneously with good precision.
The
graded decrease in body weight is generally regarded as a reflection of catabolic action.
Since ,11 compounds used in the test should be of
known purity and toxicity, adrenalectomy is not required.
Selected
steroids have been assayed in adrenalectomized rats using the conditions and steroid concentration herein described, and it has been found that
168
S T E R 0 I D S
FEBRUARY 1963
VOLUME 1
both thymic and anti-inflammatory potencies are in excellent agreement with those in intact animals.
It will be noticed in Table i that food consumption decreases with increasing dosage of the steroid.
Statistical analysis has shown
that attempts to correct calculations for dosage from food consumption data were, in general, not meaningful.
Obviously, if one had a compound
which did not decrease food consumption at anti-inflammatory doses a correction would he in order.
To date, no such anti-inflammatory
steroidal compound has been detected.
In fact, no significant divergence
of body weight and granuloma potencies has been seen, substantiating the assumption of many that the general catabolic action of glucocorticoids may be intimately associated with anti-inflammatory activity.
Many anti-inflammatory steroids have been evaluated with this method.
The potencies obtained were consistent with those reported by
other methods.
Even steroids with potencies in excess of one hundred
times hydrocortisone have given excellent results with little or no evidence of divergence of the slopes of the various indices.
Table 2
summarizes the data of a single large experiment in which three compounds of widely differing potencies were compared.
These 16~ methyl deriva-
tives of hydrocortisone have previously been described by Arth et. al. 6. It can be seen that highly precise potencies were obtained and that there were no instances in which there was a significant potency difference between the various parameters measured for a given compound. Other compounds have been found which give significant separations.
In
general, progesterone derivatives give a high adrenal suppression activity in relation to granuloma inhibition.
This has been previously
VOLUME
FEBRUARY 1963
I
ST E R 0 I D S
169
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FEBRUARY 1963
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VOLUME 1
FEBRUARY1963
S T E R 0 I D S
171
reported by Glenn, et al.7 for 6a methyl 17a acetoxy progesterone (MAP). In the authors t experience MAP has a slight anti-inflammatory activity. However, the 17a methoxy derivative of 6a methyl 17 hydroxy progesterone (MMP) has been shown to have a highly significant suppression of adrenal weight without other significant biological activity. the data of a typical experiment.
Table 3 summarizes
Other examples of activity separation
with anti-inflammatory steroids will be published in the near future.
The method herein reported has also been found to be useful for the evaltmtion of non-steroidal anti-inflammatory compounds. Phenylbutazone at doses of 200-2000 ~ / k g of diet gave highly significant graded granuloma inhibitions without affecting body, thymus or adrenal weights.
At levels above 2000 m ~ k g diet, toxicity appears in the form
of body and thymic weight decreases and adrenal weight increase. These changes indicate adrenocortical stimulation and provide a built in method of separating anti-inflammatory activity from toxicity.
The
granuloma inhibition dose response curves of non-steroidal compounds are not as steep as with anti-inflammatory steroids, so that precision is significantly decreased.
SUMMARY A method has been described which simultaneously measures four biological indices of anti-inflammatory steroids with excellent precision.
Its applicability for selection of both steroidal and
non-steroidal anti-inflammatory compounds has been illustrated.
172
S T E R 0
I D S
FEBRUARY 1963
VOLUME 1
RE~CES
.
Meier, R., Schuler, W., and Desaulles, P., EXPERIENTIA 6, 469, (195o).
.
Dorfman, R. I., METHODS IN HORM~E RESEARCH, Vol. II, Academic Press, New York, 1962, p. 325.
3.
Silber, R. H., ANN. N.Y. ACAD. SCI. 82, 821 (1959).
4.
Bush, E. I., and Alexander, R. W., ACTA ENDOCRINOL. 35,
268 (196o). .
Hershberger, L. D., and Calhoun, D. L., ENDOCRINOLOGY60, 153 (1957).
.
Arth, G. E., Fried, J., Johnston, D. B. R., Hoff, D. R., Sarett, L. H., Silber, R. H., Stoerk, H. C., and Winter, C. A., J. AM. CH~. SOC. 80, 3161 (1958).
.
Glenn, E. M., Richardson, S. L., and Bowman, B. J., METABOLISM ~, 185 (1959).
ACKNOWL~DGEMEN TS
The authors are indebted to Maryann Petraitis, Raymond L. Primka, Mary E. Regn and Walter Worosila for valuable technical assistance in carrying out the many experiments. The compounds were generously supplied by members of the Fundamental Research Department, Merck Sharp and Doh~e Research Laboratories, Rahway, New Jersey. Messrs. J. L. Ciminera and A. G. Itkin provided valuable assistance by performing statistical analyses of the biological data.