Biologic Availability and Therapeutic Equivalence JOHN D. ARNOLD, M.D., and JEAN S E E , Ph.D.
Kansas City, Mo.
HE concepts of biologic or physiologic Tamilability, or therapeutic or generic equivalence, are not likely to have the same meanings for a scientific investigator, a regulatory official, a pharmaceutical representative, or a c1inician.l Some understand these concepts as the study of blood, serum, saliva, o r urine drug levels, while others think of these concepts as the effect of formulations on efficacy; a few may even mention pharmacokinetics. Yet, these expressions, despite their ambiguity, have become part of the current pharmacological jargon, and they are commonly used, pot always appropriately, in many different ways. Is this one of the fads that bialogists may occasionally contrive, or are these concepts useful in therapeutics ? And if they are important, what are their pitfalls? It is to these questions that this paper will be addressed. . Twenty years ago, E. K. Marshall reported that serum levels of the sulfonamides correlated directly with their antibacterial effect, and yet this novel and remarkable conclusion did not attract much attention in the scientific community for three decades. It was not doubt or ignorance that hindered therapeutic progress, since few drugs until recently could be quantified in serum or plasma so easily as with the Bratton-Marshall reaction for sulfonamides.2 What actually revived
Marshall’s idea was the clinical observation that all preparations of the same active substance are not necessarily therapeutically equal. Therapeutic responses and blood drug levels occasionally reveal considerable differences among analogous products or even lots of the same product! Such inconsistencies were traced to differences in certain physical characteristics of drugs, interference from added excipients, or artifacts introduced by the compounding processes. Still, the topic of bioavailability was largely considered esoteric until companies producing generic products began to compete with brand manufacturers? Bib arailability finally had found a cause, which unfortunately diverted the attention of scientists and clinicians from what should have been the main focus. For instance, much remained to be done toward improving the gastrointestinal absorption of drugs5 or studying the rate at which drugs are released from subcutaneous or intramuscular sites of injection.6 A definition of bioavailability grew out of the development of this work. Bib arailability and its synonyms are now understood to describe the rate at which, and the extent to which, active drugs reach the systemic circulation1 or are excreted. This essentially defines the absorption of the drug at its site of entry and not the concentration of the drug at its site of acFrom the Quincy Research Center, Kansas City, tion, which is the important aspect of thin Mo. Presented at a Symposium on Bioavailability concept. Strictly speaking, a highly and Clinical Pharmacokinetics, held at the Fifth albumin-bound drug should have a low Annual Meeting of the American College of Clinical Pharmacology in Philadelphia, Penn., on bioavailability. Similarly, antibiotics that dn not penetrate the nervous system should April 30, 1976. 546
from the SAGE Social Science Collections. All Rights Reserved
The Journal of Clinical Pharmacology
AVAILABILITY A N D THERAPEUTIC EQUIVALENCE
be described as poorly bioavailable in the treatment of cerebrospinal infections. If re extend or refine the basic definition to include other situations, however, we might confuse the issue. If we now tu rn our attention to the rentral issue, therapeutic effects of drugs, w find that bioavailability is not a main determinant because blood drug levels do not always correlate directly with therapentic effect. Two main theories have been advanced so far to explain such anomalies. The agent of the pharmacologic effect may be, in part, a metabolite of the original drug instead of the unchanged drug.? The d ~ may g also be exchanged at an excessively slow rate, o r not at all, between the rireulation and the intracellular compartments where its site of action lies. Also, there are many cases of dissociation between blood drug levels and therapeutic effect for which no explanation has yet been found. Let us look at two specific instances of this dissociation. These cases have been taken from over 300 cases of our clinical investigations. These data offer a rare opportunity to correlate a wide variety of trperimental findings to establish optimum dosages, to interpret therapeutic &ects, and to compare these two variables. The first example illustrates how akain chemicals can sequester nonrecoverable and nonregenerating receptors. The receptor in this case is cholinesterase, Ed the agent, a phosphorothioate, is one of its noncompetitive inhibitors.* In rats, the receptor effect in erythrocytes lasts five days at the most with this substance. In man, a single low dose inhibits erythmc.yte cholinesterase within half an hour, but this effect is reduced by 24 hours. High doses in man, however, inhibit erythmeyte cholinesterase for about 50 days, he life span of red cells. The anticholinesterase effect of phosphorothioate, therefore, is radically difkrent between the red cells of rats and Ottober, 1976
men. The reason for the difference between the two species is attributed to three dependent variables : the drug effect lasts for the life of the erythrocytic enzyme, which in turn depends on the life of the red cell (mature erythrocytes do not produce new enzymes), and the life span of erythrocytes in man is longer than in rats. Thus, the pharmacologic effect is primarily controlled by the receptor, and neither the metabolic transformation nor the epcretion of the drug has a decisive influence on the response of the receptor. If human erythrocytes compensated for the inhibition of their cholinesterase by regenerating their enzyme (as it occurs in serum), the kinetics of phosphorothioate effect could not be determined so clearly. When serum cholinesterase is considered, indeed the irreversibility of its kinetics is not so apparent. This example demonstrates that there is no connection between pharmacodynamic response and the in vivo fate of the drug. The administration of the drug to man, in this case, has to be guided by the duration of the receptor e f f e c t t h e erythrocyte life span. If the receptor effect could not have been measured, or if we looked a t the fate of the substance, we would have encountered serious difficulties in determining the posology of this material. A dosing schedule used in a patient that is based on conventional plasma or serum drug halflife alone can cause serious cumulative toxic effects. The second case of dissociation between drug plasma levels and pharmacologic response was observed with quinine in the treatment of malaria. This case is particularly important because the proper range of plasma concentrations of the alkaloid and the accuracy of the end point of its effect is crucial in treatment. With repeated daily oral doses of 1.6 Gm qEinine sulfate, plasma levels peaked on the third day of treatment and then fell Moreover, if the medica547
ARNOLD A N D SICE
tion is interrupted for a few days and then resumed, the new drug plasma levels a r e lower than the first time it was administered. With this information, the stage was set for comparing the pharmacodynamics and pharmacokinetics of quinine in the treatment of standardized, experimental malaria. We were surprised, however, to find that therapeutic effect was correlated more closely with daily dose rather than with plasma drug levels.1° When we examined the effect of repeat doses of quinine, which induced relatively low plasma drug levels, dosage again turned out to be the better indicator of therapeutic effect. The same is true of erythrocyte concentration of the alkaloid rather than its plasma concentration. I n fact, the second dosage course of quinine, with its relatively low plasma drug levels, succeeded more in eradicating the infection than the first course, with its relatively high plasma levels. One cannot help but feel awkward when having to concede that drug dosage is a more valid indicator of therapeutic success than is its blood level. How can the results of these studies help clinicians who must decide which dose, schedule, or route of administration to use and consider the potential applications and dangers of yet untested substances, new drugs, and sometimes even old drugs? With hindsight, it seems that we could have predicted the prolonged plasma protein binding of iophenoxic acid, the lymphatic localization of thorium dioxide, the retinal concentration of chloroquine and chlorpromazine, and the cardiac effects of phenytoin or lidocaine. But we are still not able to discover some of the biologic actions of many substances. The delay of six to ten weeks which it takes to show a n antiarthritic effect when using gold salts o r penicillamine, for instance, might easily lead us to reject these useful drugs if they were tested under routine 548
procedures. Let us also recognize that a new thalidomide episode could again occur, despite the current screening for mutagenesis. Let us look a t what can be done in the present state of the art. This comprises two categories of information. The first to be considered is pharmacokinetic criteria, which can help a clinician follow the drug as it travels through the host. Pharmacokinetic criteria may be divided into two groups. One group of criteria is composed of variables which indicate the general in vivo fate of the material: rate and efficacy of absorption from sites of administration, modes and rates of biochemical transformation, plasma levels, tissue or organ distribution, storage of the sub stance of its active metabolites, and rates and routes of excretion of the drug and its derivatives. The other group is composed of variables which defme the specific fate and interactions of the substance and its active metabolites at their effector sites : rates of entry into the blood, quality and quantity of concentration and binding, modes and rate of reaction with the receptors, and rates of elimination. One also needs to know what the drug does a t the effector site; this constitutes the pharmacodynamic criteria, which also can be divided into two groups of dataone comprised of therapeutic and the second, toxicity information. The gathering of pharmacodynamic data presents difficulties and uncertainties which often are much more considerable than those of pharmacokinetic work-ups. If pharmacokinetics and pharmacodynamics are to be related to one another and be applicable, they should be used in the same or similar patient populations. Monitoring such a complex system of variables in man undoubtedly presents enormous technical and economic difficulties. As a consequence, we have few ex. amples that would permit thorough comparisons of pharmacokinetics to pharmaThe Journal of Clinical PharmacologJ
AVAILABILITY AND THERAPEUTIC EQUIVAZENCE
These comments certainly are not ineodynamics. A large effort will have to be made to analyze the kinetic pattern of im- tended to deny that all current dosage portant drugs when these factors are schedules are useful, but they are meant deemed important. As it now stands, we to indicate a need to reassess the posology are making difficult and momentous de- of drugs and to develop more accurate tiisions about drug usage with data based methods of drug administration. Perhaps on few principles, most of which are re- the greatest contribution of pharmacokinetics to therapy will be less in providferred to as bioequivalence. Bioequivalence has come to mean how ing automatic formulas for dosage and well a drug satisfies certain operational or more in offering a means of monitoring regulatory requirements with respect to absorption, elimination, and compliance. plasma level, therapeutic response, and But in the final analysis, the criterion of excretion patterns. I n common meaning, determining effective dosage regimens still therefore, it has come to suggest therapeu- remains the response of each patient. tic equivalence. Actually, bioequivalence References 1. Chasseaud, L. F., and Taylor, T.: Bioshould more properly be called metabolic availability of drugs from formulations equivalence. Therapeutic equivalence, on after oral administration. A m . Rev. the other hand, can only be determined by Pharmacol. 14 :35 (1974). 2. Bratton, A. C., and Marshall, E. H.: A rigorous clinical comparisons; bioequivanew coupling component for sulfanilamide lence and therapeutic equivalence may comdetermination. J . Biochem. 28 :537 (1939. plement, but cannot be substituted for, one 3. Reinhold, J. G., Phillips, F. J., and Flippin, H. F.: A comparison of the behavior of another. microcrystalline sulfadiazine with that of Discrepancies between the metabolic beordinary sulfadiazine in man. Amer. J . krior of a drug and its therapeutic endNed. Sci. 210:141 (1945). organ effect can have important conse- 4. Glazko, A. J., Kinkel, A. W., Alegnani, W. C., and Holmes, E. L.: An evaluation quences. The same formulation of a drug of the absorption characteristics of difgiven to differently conditioned hosts can ferent chloramphenicol preparations in normal human subjects. Clin. P h a r m o l . be bioinequivalent as determined by the 9:472 (1968). plasma levels of the drug. I n the case of 5. Mallis, G. I., Schmidt, D. H., and Lindenquinine, the drug and its formulation baum, J. : Superior bioavailability of digodn solution in capsules. Clin. Pharrere identical. The difference was the way macol. Therap. 18 :761 (1975). in which the individual metabolized the 6. Wilder, B. J., and Ramsay, R. E.: Oral and drug. The effect of plasma levels did make intramuscular phenytoin. Clin. Pharzacol. Therap. 19 :360 (1976). 8 difference, but opposite to what ac7. Elson, J., Strong, J. M., Lee, W. K., and apted theory predicted. Atkinson, A. J., Jr.: AntiarrhytMic poThe physiologic or chemical characteristency of N-acetylprocainamide. Clin. Pharmcol. Therap. 17:134 (1975). ties of certain tissues can also play a critiJ., Cross, R. Nelson, D., Arnold, J., eal role in therapeutic response. The limit- 8. Doull, and Martin D.: Effect of a phosphorothioing measure of noncompetitive inhibition ate on human plasma and RBC cholinesterase activity. Unpublished results. of human host cells is the ability of such 9. Trenholme, G. M., Williams, R. L., Rieckells to repair physiochemical drug alteramann, K. H., Frischer, H., and Carson, tions. In such cases, the physiologic or P. E.: Quinine disposition during malaria and during induced fever. Clin. Pharmbiochemical life of the cell is of major incol. Therap. 19:4 (1976). hence relative to the plasma life of the 10. Arnold, J., and Edgcomb, J.: Quinine blood h g . In fact, the response would appear levels in malaria therapy. Unpublished resulta. bbe cumulative in spite of the fact that 11. Azamoff, D. L.: Application of blood level he drug itself and its metabolites' might data to clinical trials. Clin. Pharmaool. lave been already altered. Therap. 16:183 (1973). hber, 1976
549
Kansas City, Mo.
HE concepts of biologic or physiologic Tamilability, or therapeutic or generic equivalence, are not likely to have the same meanings for a scientific investigator, a regulatory official, a pharmaceutical representative, or a c1inician.l Some understand these concepts as the study of blood, serum, saliva, o r urine drug levels, while others think of these concepts as the effect of formulations on efficacy; a few may even mention pharmacokinetics. Yet, these expressions, despite their ambiguity, have become part of the current pharmacological jargon, and they are commonly used, pot always appropriately, in many different ways. Is this one of the fads that bialogists may occasionally contrive, or are these concepts useful in therapeutics ? And if they are important, what are their pitfalls? It is to these questions that this paper will be addressed. . Twenty years ago, E. K. Marshall reported that serum levels of the sulfonamides correlated directly with their antibacterial effect, and yet this novel and remarkable conclusion did not attract much attention in the scientific community for three decades. It was not doubt or ignorance that hindered therapeutic progress, since few drugs until recently could be quantified in serum or plasma so easily as with the Bratton-Marshall reaction for sulfonamides.2 What actually revived
Marshall’s idea was the clinical observation that all preparations of the same active substance are not necessarily therapeutically equal. Therapeutic responses and blood drug levels occasionally reveal considerable differences among analogous products or even lots of the same product! Such inconsistencies were traced to differences in certain physical characteristics of drugs, interference from added excipients, or artifacts introduced by the compounding processes. Still, the topic of bioavailability was largely considered esoteric until companies producing generic products began to compete with brand manufacturers? Bib arailability finally had found a cause, which unfortunately diverted the attention of scientists and clinicians from what should have been the main focus. For instance, much remained to be done toward improving the gastrointestinal absorption of drugs5 or studying the rate at which drugs are released from subcutaneous or intramuscular sites of injection.6 A definition of bioavailability grew out of the development of this work. Bib arailability and its synonyms are now understood to describe the rate at which, and the extent to which, active drugs reach the systemic circulation1 or are excreted. This essentially defines the absorption of the drug at its site of entry and not the concentration of the drug at its site of acFrom the Quincy Research Center, Kansas City, tion, which is the important aspect of thin Mo. Presented at a Symposium on Bioavailability concept. Strictly speaking, a highly and Clinical Pharmacokinetics, held at the Fifth albumin-bound drug should have a low Annual Meeting of the American College of Clinical Pharmacology in Philadelphia, Penn., on bioavailability. Similarly, antibiotics that dn not penetrate the nervous system should April 30, 1976. 546
from the SAGE Social Science Collections. All Rights Reserved
The Journal of Clinical Pharmacology
AVAILABILITY A N D THERAPEUTIC EQUIVALENCE
be described as poorly bioavailable in the treatment of cerebrospinal infections. If re extend or refine the basic definition to include other situations, however, we might confuse the issue. If we now tu rn our attention to the rentral issue, therapeutic effects of drugs, w find that bioavailability is not a main determinant because blood drug levels do not always correlate directly with therapentic effect. Two main theories have been advanced so far to explain such anomalies. The agent of the pharmacologic effect may be, in part, a metabolite of the original drug instead of the unchanged drug.? The d ~ may g also be exchanged at an excessively slow rate, o r not at all, between the rireulation and the intracellular compartments where its site of action lies. Also, there are many cases of dissociation between blood drug levels and therapeutic effect for which no explanation has yet been found. Let us look at two specific instances of this dissociation. These cases have been taken from over 300 cases of our clinical investigations. These data offer a rare opportunity to correlate a wide variety of trperimental findings to establish optimum dosages, to interpret therapeutic &ects, and to compare these two variables. The first example illustrates how akain chemicals can sequester nonrecoverable and nonregenerating receptors. The receptor in this case is cholinesterase, Ed the agent, a phosphorothioate, is one of its noncompetitive inhibitors.* In rats, the receptor effect in erythrocytes lasts five days at the most with this substance. In man, a single low dose inhibits erythmc.yte cholinesterase within half an hour, but this effect is reduced by 24 hours. High doses in man, however, inhibit erythmeyte cholinesterase for about 50 days, he life span of red cells. The anticholinesterase effect of phosphorothioate, therefore, is radically difkrent between the red cells of rats and Ottober, 1976
men. The reason for the difference between the two species is attributed to three dependent variables : the drug effect lasts for the life of the erythrocytic enzyme, which in turn depends on the life of the red cell (mature erythrocytes do not produce new enzymes), and the life span of erythrocytes in man is longer than in rats. Thus, the pharmacologic effect is primarily controlled by the receptor, and neither the metabolic transformation nor the epcretion of the drug has a decisive influence on the response of the receptor. If human erythrocytes compensated for the inhibition of their cholinesterase by regenerating their enzyme (as it occurs in serum), the kinetics of phosphorothioate effect could not be determined so clearly. When serum cholinesterase is considered, indeed the irreversibility of its kinetics is not so apparent. This example demonstrates that there is no connection between pharmacodynamic response and the in vivo fate of the drug. The administration of the drug to man, in this case, has to be guided by the duration of the receptor e f f e c t t h e erythrocyte life span. If the receptor effect could not have been measured, or if we looked a t the fate of the substance, we would have encountered serious difficulties in determining the posology of this material. A dosing schedule used in a patient that is based on conventional plasma or serum drug halflife alone can cause serious cumulative toxic effects. The second case of dissociation between drug plasma levels and pharmacologic response was observed with quinine in the treatment of malaria. This case is particularly important because the proper range of plasma concentrations of the alkaloid and the accuracy of the end point of its effect is crucial in treatment. With repeated daily oral doses of 1.6 Gm qEinine sulfate, plasma levels peaked on the third day of treatment and then fell Moreover, if the medica547
ARNOLD A N D SICE
tion is interrupted for a few days and then resumed, the new drug plasma levels a r e lower than the first time it was administered. With this information, the stage was set for comparing the pharmacodynamics and pharmacokinetics of quinine in the treatment of standardized, experimental malaria. We were surprised, however, to find that therapeutic effect was correlated more closely with daily dose rather than with plasma drug levels.1° When we examined the effect of repeat doses of quinine, which induced relatively low plasma drug levels, dosage again turned out to be the better indicator of therapeutic effect. The same is true of erythrocyte concentration of the alkaloid rather than its plasma concentration. I n fact, the second dosage course of quinine, with its relatively low plasma drug levels, succeeded more in eradicating the infection than the first course, with its relatively high plasma levels. One cannot help but feel awkward when having to concede that drug dosage is a more valid indicator of therapeutic success than is its blood level. How can the results of these studies help clinicians who must decide which dose, schedule, or route of administration to use and consider the potential applications and dangers of yet untested substances, new drugs, and sometimes even old drugs? With hindsight, it seems that we could have predicted the prolonged plasma protein binding of iophenoxic acid, the lymphatic localization of thorium dioxide, the retinal concentration of chloroquine and chlorpromazine, and the cardiac effects of phenytoin or lidocaine. But we are still not able to discover some of the biologic actions of many substances. The delay of six to ten weeks which it takes to show a n antiarthritic effect when using gold salts o r penicillamine, for instance, might easily lead us to reject these useful drugs if they were tested under routine 548
procedures. Let us also recognize that a new thalidomide episode could again occur, despite the current screening for mutagenesis. Let us look a t what can be done in the present state of the art. This comprises two categories of information. The first to be considered is pharmacokinetic criteria, which can help a clinician follow the drug as it travels through the host. Pharmacokinetic criteria may be divided into two groups. One group of criteria is composed of variables which indicate the general in vivo fate of the material: rate and efficacy of absorption from sites of administration, modes and rates of biochemical transformation, plasma levels, tissue or organ distribution, storage of the sub stance of its active metabolites, and rates and routes of excretion of the drug and its derivatives. The other group is composed of variables which defme the specific fate and interactions of the substance and its active metabolites at their effector sites : rates of entry into the blood, quality and quantity of concentration and binding, modes and rate of reaction with the receptors, and rates of elimination. One also needs to know what the drug does a t the effector site; this constitutes the pharmacodynamic criteria, which also can be divided into two groups of dataone comprised of therapeutic and the second, toxicity information. The gathering of pharmacodynamic data presents difficulties and uncertainties which often are much more considerable than those of pharmacokinetic work-ups. If pharmacokinetics and pharmacodynamics are to be related to one another and be applicable, they should be used in the same or similar patient populations. Monitoring such a complex system of variables in man undoubtedly presents enormous technical and economic difficulties. As a consequence, we have few ex. amples that would permit thorough comparisons of pharmacokinetics to pharmaThe Journal of Clinical PharmacologJ
AVAILABILITY AND THERAPEUTIC EQUIVAZENCE
These comments certainly are not ineodynamics. A large effort will have to be made to analyze the kinetic pattern of im- tended to deny that all current dosage portant drugs when these factors are schedules are useful, but they are meant deemed important. As it now stands, we to indicate a need to reassess the posology are making difficult and momentous de- of drugs and to develop more accurate tiisions about drug usage with data based methods of drug administration. Perhaps on few principles, most of which are re- the greatest contribution of pharmacokinetics to therapy will be less in providferred to as bioequivalence. Bioequivalence has come to mean how ing automatic formulas for dosage and well a drug satisfies certain operational or more in offering a means of monitoring regulatory requirements with respect to absorption, elimination, and compliance. plasma level, therapeutic response, and But in the final analysis, the criterion of excretion patterns. I n common meaning, determining effective dosage regimens still therefore, it has come to suggest therapeu- remains the response of each patient. tic equivalence. Actually, bioequivalence References 1. Chasseaud, L. F., and Taylor, T.: Bioshould more properly be called metabolic availability of drugs from formulations equivalence. Therapeutic equivalence, on after oral administration. A m . Rev. the other hand, can only be determined by Pharmacol. 14 :35 (1974). 2. Bratton, A. C., and Marshall, E. H.: A rigorous clinical comparisons; bioequivanew coupling component for sulfanilamide lence and therapeutic equivalence may comdetermination. J . Biochem. 28 :537 (1939. plement, but cannot be substituted for, one 3. Reinhold, J. G., Phillips, F. J., and Flippin, H. F.: A comparison of the behavior of another. microcrystalline sulfadiazine with that of Discrepancies between the metabolic beordinary sulfadiazine in man. Amer. J . krior of a drug and its therapeutic endNed. Sci. 210:141 (1945). organ effect can have important conse- 4. Glazko, A. J., Kinkel, A. W., Alegnani, W. C., and Holmes, E. L.: An evaluation quences. The same formulation of a drug of the absorption characteristics of difgiven to differently conditioned hosts can ferent chloramphenicol preparations in normal human subjects. Clin. P h a r m o l . be bioinequivalent as determined by the 9:472 (1968). plasma levels of the drug. I n the case of 5. Mallis, G. I., Schmidt, D. H., and Lindenquinine, the drug and its formulation baum, J. : Superior bioavailability of digodn solution in capsules. Clin. Pharrere identical. The difference was the way macol. Therap. 18 :761 (1975). in which the individual metabolized the 6. Wilder, B. J., and Ramsay, R. E.: Oral and drug. The effect of plasma levels did make intramuscular phenytoin. Clin. Pharzacol. Therap. 19 :360 (1976). 8 difference, but opposite to what ac7. Elson, J., Strong, J. M., Lee, W. K., and apted theory predicted. Atkinson, A. J., Jr.: AntiarrhytMic poThe physiologic or chemical characteristency of N-acetylprocainamide. Clin. Pharmcol. Therap. 17:134 (1975). ties of certain tissues can also play a critiJ., Cross, R. Nelson, D., Arnold, J., eal role in therapeutic response. The limit- 8. Doull, and Martin D.: Effect of a phosphorothioing measure of noncompetitive inhibition ate on human plasma and RBC cholinesterase activity. Unpublished results. of human host cells is the ability of such 9. Trenholme, G. M., Williams, R. L., Rieckells to repair physiochemical drug alteramann, K. H., Frischer, H., and Carson, tions. In such cases, the physiologic or P. E.: Quinine disposition during malaria and during induced fever. Clin. Pharmbiochemical life of the cell is of major incol. Therap. 19:4 (1976). hence relative to the plasma life of the 10. Arnold, J., and Edgcomb, J.: Quinine blood h g . In fact, the response would appear levels in malaria therapy. Unpublished resulta. bbe cumulative in spite of the fact that 11. Azamoff, D. L.: Application of blood level he drug itself and its metabolites' might data to clinical trials. Clin. Pharmaool. lave been already altered. Therap. 16:183 (1973). hber, 1976
549
