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The Determination of Analgetic Potency by a Quantal Method*†
404
JOURNAL OF THE
AMERICANPHARMACEUTICAL ASSOCIATION
(11) Conrad M. ibid. 356 24(1907). (12) Thunbkg, ?., Shond: Arch. Phrsiol., 33, 217(1916), Chcm. Abstr. 11,458(1917). (13) Boeshken, J., Sturm. W., and Goettsch. G., Rec. frou. chim. 37 144(1918). (14) Fhilmann E. and Bentzon J. Ber. 51.522(1918). (15) Biilmann’ E’ and Lund ’HI A&. chim.. 19, 137 (1923): Richard&n,’k. M.. and kann’an. R. K., Biochem. J . .
23,68(1929). (16) Richardson G. M. ibid. 26 1959(1932). Science 78, 485(1933). (17) Hill E. S. ’and MiChadis (18) Mddoch ). and Doebnk O:, Ber., 9; 1102(1876). (19) Deniges, b.,’Bull. sot. phirm. Bordeaux, 66, S(1928); Chem Abstr 23 4160(1929). (20) Doja: Id. Q., and Mokeet, A , J . Indian Chem. Soc., 13,542(1936). (21) Nightingale D. Org. Syntheses, 23, 6(1943); ff., Fischer E. (translated ’by Stanford, R. V.) Introductlon to the ’Preoaration of Organic Comoounds’:. Williams and
k.
Vol. XL, No. 8
(26) Heyroth, F. F., and Loofbourow. J. R., J . Am. Chem. SOC.56 1728(1934). (27) bavidson, D., and Epstein, E., J . Org. Chem., 1, 305
(1938). and Wertheimer, E., J . B i d . Chem., (28) Briickmann, 0.. 168,241(1947). (29) Patterson, J. W., Lazarow, A., Lemm, P. J., and Levey, S., ibid., 177,187(1949). (30) Biilmann, E.. and Mygind. H. G.. Bull. SOC. chim., 47,532(1930). (31) Biltz H. Ber 45 3659(1912). (32) Tipsdn, R. S.‘,’anh Cretcher, L. H., Anal. Chem., 22, 822( 1950). (33) Pregl, F.. (translated by Pylernan, E.), “Quantitative Organic Microanalysis,” Churchill, London, 1924, p. 118, Fig. 26. (34) Tipson, R. S.. and Cretcher, L. H., J . Org. Chem., in press. (35) W6hler F. andLiebig J. Ann. 38 357(1841) (36) Hartle;, W.N., J . Chcrn.’Soc.,b7, ’1796(1905).. (37) Marchlewski, L., and Wierzuchowska, J., Bull. intern. ha.1929A, , p. 85. abes, R.. and Reisburger, H.. Arch. exptl. Pafh. Pharmokol., 156,228(1930).
The Determination of Analgetic Potency by a Quanta1 Method*,t By BERNARD V. CHRISTENSEN and ARTHUR TYE A modification of the Hardy-Wolff-Goodellradiant-heatmethod into a quantal procedure is described. Patterned after EDw bioassays, this modification determines the fifty per cent analgesic dose (ADSO)of an analgetic when groups of white rats are exposed forty minutes after treatment with the analgetic to a selected stimulus (320 millicalories/sec./cm.*) for the standard period of three seconds. Analgesic potency is measured by com aring the ADa of an analgetic with the ADm of morphine. The method is rapi8and convenient and appears to be well suited for the screening and evaluation of analgetics.
Hardy-Wolff-Goodell (1) radiant-heat method has won wide acceptance as a reliable procedure for analgesimetric studies. However, it has generally been used to obtain data based on graded responses. Involving, as it does, the tedious step-wise determination of pain thresholds with varying successive intensities of stimulus both before and after treatment of the test animal with the analgetic, the procedure for obtaining this type of response is slow and time-consuming. Moreover, the data so obtained are difficult to evaluate because the exact relationship between rise in pain threshold and analgetic potency is subject to various interpretations. The modification of the Hardy-Wolff method from a graded response procedure to a quantal one patterned after an ED60 quantal assay seems HE
*
Received April 9, 1951, from the College of Pharmacy, Ohio State University. t Fesented before the Subsection on Pharmacy, American Association for the Advancement of Science, Scientific Section, Cleveland, Ohio, December, 1950.
to offer a means of avoiding these drawbacks. This modification would involve the treating of successive groups of test animals with graded doses of an analgetic, exposing them, after a certain time interval, to a certain selected intensity of radiant-heat stimulus for the standard period of three seconds, and determining the percentage of each group that has attained analgesia as shown by failure to react to the stimulus. From the data so obtained a dose-per cent effect curve can be constructed and the dose of the analgetic that would cause analgesia in 50 per cent of animals determined. The 50 per cent analgesic dose (ADw)of the analgetic can then be compared with the ADmof a standard analgetic like morphine, and estimation of its potency can be made in terms of morphine. One advantage of such a procedure is that it effects a considerable saving of time and labor by not determining the pretreatment thresholds, as these are known to be normally distributed. Secondly, the actual determination of the after-treatment
August, 1951
SCIENTIFIC EDITION
response is simple and clear-cut, requiring only a single observation, in contrast to the tedious step-wise determination of a pain threshold. Finally, the method of evaluation of results, the comparison of the ED60 of the best preparation with that of a reference standard, is one that is widely used in pbarmacological assay work. It is true that a quantal response gives less information per animal than a graded response and that, therefore, more animals are required to give an assay of equal reliability. But if, as in this case, the graded response determination involves complex measurement and computation, the drawback of requiring a greater number of animals is more than compensated for by the simplification of experimental procedure and interpretation of results. In designing such a quantal procedure, the first essential is the selection of a suitable intensity of stimulus. For measuring analgesic potency, it would Seem reasonable to choose one that would cause a degree of pain such as is commonly encountered clinically and of the order that analgetics are generally required to combat. Hardy, et al. (2), found that with radiant heat stimuli the pain threshold is reached a t about 220 millicalories/sec./c.2 and that stimuli greater than 680 millicalories/sec./cm.2 evolve ceiling pain. A stimulus of 320 millicalories/sec./ c m . 2 causes medium to severe pain and should therefore be a suitable stimulus. Hardy, et al., found that the quality of pain sensations changes a t about this point from a definite pricking sensation to one with an added burning quality. Another advantage of a stimulus of 320 millicalories/sec./am2 is that it is in the rangeofmaximum discriminatory sensitivity, which extends approximately from 220 to 320 millicalories/sec./ cmP2 The second essential in designing a quantal assay is the selection of the optimum time interval between treatment and observation of re-
405
sponse. In analgesimetry the response is probably best measured a t the time of maximum effect. Time-action curves of various analgetics show that the peak effect generally occurs twenty minutes to one hour after injection. Experiment with various intervals during this period showed that forty minutes was the most efficient time interval. The final procedure adopted was the application by a modified Hardy-Wolff-Goodell pain threshold apparatus of a stimulus of 320 millicalories/sec./cm.2 to the tails of white rats forty minutes after injection of an analgetic. Doseper cent effect curves were obtained for various analgetics using five dose-groups of 12 rats each. The curves when plotted on log-probit paper were found to be satisfactory when tested for linearity and parallelism and gave satisfactory estimates of the potency of various analgetics and morphine solutions treated as unknowns. Using the same number of animals a three dosegroup cross-over design appeared t o be slightly more efficient and gave the following potency ratios with 19/20 confidence limits: Drug
Potency Ratio
Morphine sulfate Codeine phosphate Methadone HCl Metopon HCI Meperidine HC1
.o
1
0.14 (0.13 to 0.15) 2 . 2 (2.0 to 2.4) 3 . 9 (3.6 to4.3) 0 . 2 4 (0.22 to 0.27)
The EDw quantal method here described is a rapid and convenient procedure which appears suitable for the routine screening and evaluation of analgetics. It might also be used in the investigation of various problems associated with analgesia such as potentiation and addiction. REFERENCES (1) Hardy. J. D., Wolff, H. G., and Goodell, H.. J . Clin. Invest. 19 640(1040). (2) Hddy, J. D., Wolff. H. G., and coodell, H., ibid., 27. 380(1948).
JOURNAL OF THE
AMERICANPHARMACEUTICAL ASSOCIATION
(11) Conrad M. ibid. 356 24(1907). (12) Thunbkg, ?., Shond: Arch. Phrsiol., 33, 217(1916), Chcm. Abstr. 11,458(1917). (13) Boeshken, J., Sturm. W., and Goettsch. G., Rec. frou. chim. 37 144(1918). (14) Fhilmann E. and Bentzon J. Ber. 51.522(1918). (15) Biilmann’ E’ and Lund ’HI A&. chim.. 19, 137 (1923): Richard&n,’k. M.. and kann’an. R. K., Biochem. J . .
23,68(1929). (16) Richardson G. M. ibid. 26 1959(1932). Science 78, 485(1933). (17) Hill E. S. ’and MiChadis (18) Mddoch ). and Doebnk O:, Ber., 9; 1102(1876). (19) Deniges, b.,’Bull. sot. phirm. Bordeaux, 66, S(1928); Chem Abstr 23 4160(1929). (20) Doja: Id. Q., and Mokeet, A , J . Indian Chem. Soc., 13,542(1936). (21) Nightingale D. Org. Syntheses, 23, 6(1943); ff., Fischer E. (translated ’by Stanford, R. V.) Introductlon to the ’Preoaration of Organic Comoounds’:. Williams and
k.
Vol. XL, No. 8
(26) Heyroth, F. F., and Loofbourow. J. R., J . Am. Chem. SOC.56 1728(1934). (27) bavidson, D., and Epstein, E., J . Org. Chem., 1, 305
(1938). and Wertheimer, E., J . B i d . Chem., (28) Briickmann, 0.. 168,241(1947). (29) Patterson, J. W., Lazarow, A., Lemm, P. J., and Levey, S., ibid., 177,187(1949). (30) Biilmann, E.. and Mygind. H. G.. Bull. SOC. chim., 47,532(1930). (31) Biltz H. Ber 45 3659(1912). (32) Tipsdn, R. S.‘,’anh Cretcher, L. H., Anal. Chem., 22, 822( 1950). (33) Pregl, F.. (translated by Pylernan, E.), “Quantitative Organic Microanalysis,” Churchill, London, 1924, p. 118, Fig. 26. (34) Tipson, R. S.. and Cretcher, L. H., J . Org. Chem., in press. (35) W6hler F. andLiebig J. Ann. 38 357(1841) (36) Hartle;, W.N., J . Chcrn.’Soc.,b7, ’1796(1905).. (37) Marchlewski, L., and Wierzuchowska, J., Bull. intern. ha.1929A, , p. 85. abes, R.. and Reisburger, H.. Arch. exptl. Pafh. Pharmokol., 156,228(1930).
The Determination of Analgetic Potency by a Quanta1 Method*,t By BERNARD V. CHRISTENSEN and ARTHUR TYE A modification of the Hardy-Wolff-Goodellradiant-heatmethod into a quantal procedure is described. Patterned after EDw bioassays, this modification determines the fifty per cent analgesic dose (ADSO)of an analgetic when groups of white rats are exposed forty minutes after treatment with the analgetic to a selected stimulus (320 millicalories/sec./cm.*) for the standard period of three seconds. Analgesic potency is measured by com aring the ADa of an analgetic with the ADm of morphine. The method is rapi8and convenient and appears to be well suited for the screening and evaluation of analgetics.
Hardy-Wolff-Goodell (1) radiant-heat method has won wide acceptance as a reliable procedure for analgesimetric studies. However, it has generally been used to obtain data based on graded responses. Involving, as it does, the tedious step-wise determination of pain thresholds with varying successive intensities of stimulus both before and after treatment of the test animal with the analgetic, the procedure for obtaining this type of response is slow and time-consuming. Moreover, the data so obtained are difficult to evaluate because the exact relationship between rise in pain threshold and analgetic potency is subject to various interpretations. The modification of the Hardy-Wolff method from a graded response procedure to a quantal one patterned after an ED60 quantal assay seems HE
*
Received April 9, 1951, from the College of Pharmacy, Ohio State University. t Fesented before the Subsection on Pharmacy, American Association for the Advancement of Science, Scientific Section, Cleveland, Ohio, December, 1950.
to offer a means of avoiding these drawbacks. This modification would involve the treating of successive groups of test animals with graded doses of an analgetic, exposing them, after a certain time interval, to a certain selected intensity of radiant-heat stimulus for the standard period of three seconds, and determining the percentage of each group that has attained analgesia as shown by failure to react to the stimulus. From the data so obtained a dose-per cent effect curve can be constructed and the dose of the analgetic that would cause analgesia in 50 per cent of animals determined. The 50 per cent analgesic dose (ADw)of the analgetic can then be compared with the ADmof a standard analgetic like morphine, and estimation of its potency can be made in terms of morphine. One advantage of such a procedure is that it effects a considerable saving of time and labor by not determining the pretreatment thresholds, as these are known to be normally distributed. Secondly, the actual determination of the after-treatment
August, 1951
SCIENTIFIC EDITION
response is simple and clear-cut, requiring only a single observation, in contrast to the tedious step-wise determination of a pain threshold. Finally, the method of evaluation of results, the comparison of the ED60 of the best preparation with that of a reference standard, is one that is widely used in pbarmacological assay work. It is true that a quantal response gives less information per animal than a graded response and that, therefore, more animals are required to give an assay of equal reliability. But if, as in this case, the graded response determination involves complex measurement and computation, the drawback of requiring a greater number of animals is more than compensated for by the simplification of experimental procedure and interpretation of results. In designing such a quantal procedure, the first essential is the selection of a suitable intensity of stimulus. For measuring analgesic potency, it would Seem reasonable to choose one that would cause a degree of pain such as is commonly encountered clinically and of the order that analgetics are generally required to combat. Hardy, et al. (2), found that with radiant heat stimuli the pain threshold is reached a t about 220 millicalories/sec./c.2 and that stimuli greater than 680 millicalories/sec./cm.2 evolve ceiling pain. A stimulus of 320 millicalories/sec./ c m . 2 causes medium to severe pain and should therefore be a suitable stimulus. Hardy, et al., found that the quality of pain sensations changes a t about this point from a definite pricking sensation to one with an added burning quality. Another advantage of a stimulus of 320 millicalories/sec./am2 is that it is in the rangeofmaximum discriminatory sensitivity, which extends approximately from 220 to 320 millicalories/sec./ cmP2 The second essential in designing a quantal assay is the selection of the optimum time interval between treatment and observation of re-
405
sponse. In analgesimetry the response is probably best measured a t the time of maximum effect. Time-action curves of various analgetics show that the peak effect generally occurs twenty minutes to one hour after injection. Experiment with various intervals during this period showed that forty minutes was the most efficient time interval. The final procedure adopted was the application by a modified Hardy-Wolff-Goodell pain threshold apparatus of a stimulus of 320 millicalories/sec./cm.2 to the tails of white rats forty minutes after injection of an analgetic. Doseper cent effect curves were obtained for various analgetics using five dose-groups of 12 rats each. The curves when plotted on log-probit paper were found to be satisfactory when tested for linearity and parallelism and gave satisfactory estimates of the potency of various analgetics and morphine solutions treated as unknowns. Using the same number of animals a three dosegroup cross-over design appeared t o be slightly more efficient and gave the following potency ratios with 19/20 confidence limits: Drug
Potency Ratio
Morphine sulfate Codeine phosphate Methadone HCl Metopon HCI Meperidine HC1
.o
1
0.14 (0.13 to 0.15) 2 . 2 (2.0 to 2.4) 3 . 9 (3.6 to4.3) 0 . 2 4 (0.22 to 0.27)
The EDw quantal method here described is a rapid and convenient procedure which appears suitable for the routine screening and evaluation of analgetics. It might also be used in the investigation of various problems associated with analgesia such as potentiation and addiction. REFERENCES (1) Hardy. J. D., Wolff, H. G., and Goodell, H.. J . Clin. Invest. 19 640(1040). (2) Hddy, J. D., Wolff. H. G., and coodell, H., ibid., 27. 380(1948).