ESTERS AND SODIUM NITRITE IN MAN

Transcription

1 ACQUIRED TOLERANCE TO AND CROSS TOLERANCE BETWEEN THE NITROUS AND NITRIC ACID ESTERS AND SODIUM NITRITE IN MAN L. A. CRANDALL, JR., C. D. LEAKE, A. S. LOEVENHART AND C. W. MUEHLBERGER From the Pharmacological Laboratory of the University of Wisconsin Received for publication October 6, 1930 The pharmacology of glyceryl trinitrate and allied substances is seldom discussed without reference to the tolerance developed by their prolonged use. During that period when glyceryl trinitrate was more commonly employed in conditions of high blood pressure than at present, the decreasing response which followed the continued administration of the drug was recognized as an obstacle to its successful therapeutic use. Tolerance to glyceryl trinitrate and other drugs of the nitrite group has been discussed by several writers. Stewart (1) reports the case of a patient in whom 1 minim of a 1 per cent alcoholic solution of glyceryl trinitrate was the beginning dose; after the continuous use of the drug over a period of six months, this patient was obtaining less effect from 16 minims of a 10 per cent solution than with the original dose of 1 minim of a 1 per cent solution. In this case the dose was increased to 160 times that originally used without increasing its effectiveness. Matthew (2) observed acquired tolerance to glyceryl trinitrate in patients, but no acquired tolerance to erythrol tetranitrate, mannitol hexanitrate, or sodium nitrite. Laws (3), Ebright (4), and Heitz (5) all report tolerance to glyceryl trinitrate on the part of men engaged in the manufacture of dynamite. Nerertheless, no care- This contribution is part of an extended study of the pharmacology of the nitrates and nitrites undertaken under the direction of Professor A. S. Loevenhart ( ). It is affectionately dedicated to his memory.-l. A. C., C. D. L., and C. W. M. 103

2 104 CRANDALL, JR., LEAKE, LOEVENHART, MUEHLBERGER fully controlled experimental study of acquired tolerance to glyceryl trinitrate and allied substances has been made. Nor does there seem to be adequate recognition of the existence of cross tolerance within the group of substances commonly referred to as the nitrites, which general term includes the inorganic nitrites and the esters of nitrous and nitric acids, in spite of the report of Myers and Austin (6).. METHODS We have studied the development of tolerance in man to sodium nitrite, amyl nitrite, methyl nitrate, ethylene glycol diitrate, glyceryl trinitrate, and erythrol tetranitrate. Our criterion of tolerance was a decrease in the headache effect of these esters. For this purpose we determined what we term the headache dose of our subjects for the various substances. This headache dose was taken to be that dose which would consistently cause a moderate headache in a given subject. The headache dose was repeatedly tested in each subject used, both before and after tolerance development, and although there are marked individual variations the subjective symptoms from a given dose were found to be remarkably constant in any one subject, which confirms observations previously made by Oltman (7). We realize the objections inherent in any method of standardization which employs subjective phenomena as criteria; therefore, blood pressure and pulse-rate determinations were also made. The reason for taking the headache effect of these drugs as the criterion for tolerance, instead of the blood pressure or pulse-rate effect, was that we found the headache effect of a given dose in any one subject to be more constant than any other effect of the drug. The duration of the moderate headache from one headache dose varied with the different substances; it was approximately eight hours in the case of erythrol tetranitrate, six hours for glyceryl trinitrate, four hours for ethylene glycol dinitrate, one-half to one and one-half hours for methyl nitrate, and two to three minutes for amy! nitrite. The headache dose of sodium nitrite could not be determined due to the fact that marked nausea appeared prior to any symptoms of headache.

3 TOLERANCE TO NITRIC ACID ESTERS 105 The erythrol tetranitrate was given by mouth in tablet form. The glyceryl trinitrate and ethylene glycol dinitrate were administered by application to the skin of the forearm of 1 or 10 per cent alcoholic solutions. It had been shown in this laboratory (Herrman and Muehlberger, unpublished work) that glyceryl trinitrate has not a sufficiently high vapor pressure at room temperature to produce any effect by inhalation, and that the effect of this substance applied to the skin results solely from absorption through the skin. The vapors of ethylene glycol dinitrate will produce an effect but the subjects were instructed always to keep the part of the arm, to which the drug was applied, away from the nose. +We do not believe the inhalation was a factor in this case, especially since the person who applied the ester did not develop any symptoms. Methyl nitrate and amyl nitrite were administered as follows: a measured quantity of air was saturated with methyl nitrate or amyl nitrite vapor by passage through a U-tube containing pure methyl nitrate or amyl nitrite at room temperature, the saturated vapor being collected in a balloon, and diluted with air; the subject then inspired the contents of the balloon, plus an additional inspiration sufficient to draw into the lungs such of the vapor as might remain in the trachea. The breath was then held for ten seconds. The amount of methyl nitrate or amyl nitrite which a given quantity of air would take up was determined by weighing the U-tube before and after passing air through it; we found that 100 cc. of saturated air contained 86 mgm. of methyl nitrate and 21.6 mgm. amyl nitrite at room temperature, with a variation in case of ± 10 per cent which was probably due to temperature variation. The effect of one headache dose, as measured in this way, was remarkably constant in a given subject in spite of the error in the concentration of the vapors. Sodium nitrite was given to man by mouth, both in the form of a solution and in salol.-coated capsules. The headache dose of sodium nitrite was not determined because of the production of unpleasant symptoms, notably nausea, before the headache dose was reached. We therefore used the blood pressure and pulse rate effect of sodium nitrite as the criterion of tolerance for this substance.

4 106 CRANDALL, JR., LEAKE, LOEVENHART, MUEHLBERGER We further studied cross tolerance between the members of this group, and also between the nitrites and histamin.2 Blood pressure and pulse rate determinations were made in all cases except where methyl nitrate and amyl nitrite were used; in the case of amy! nitrite the changes were so fleeting and the effect of holding the breath was so great, that blood pressure and pulse rate measurements were of no significance. Any changes in blood pressure or pulse rate which one headache dose of methyl nitrate may have caused were within the limits of error of such measurements. Whenever tests of the amount of tolerance were to be made, or the subjects headache dose of any substance was to be determined, the subject was required to sit quietly in a chair for at least one hour before the drug to be tested was administered; this was to allow the blood pressure and pulse rate to reach their basal levels. Just prior to the administration of the drug, the blood pressure and pulse rate were taken, and these determinations were repeated every fifteen or thirty minutes during the next two or three hours (longer in the case of erythrol tetraitrate), the subject not being allowed to get up from his chair or to move vigorously during this period. Since the development of tolerance took as long as three days in some cases, and the doses were given hourly throughout the day and every three hours during the night, the subjects were required to be at the laboratory only a part of the day and were given the solutions and pipettes with which to administer the doses to themselves when they were not at the laboratory. All the subjects were university students or graduates and cooperated well. Control applications of pure alcohol were frequently applied to the skin and any subject who reported symptoms from such a control was eliminated from the study. During the development of tolerance, we attempted to keep the subject at the point of an incipient headache throughout the whole period. The dosage of whatever substance was being used would be so adjusted that the subject would always have a slight fullness of the head. In this manner, the tolerance would, we 2 The histamin was used in the form of the hydrochloride, and was obtained from the Special Chemicals Company, Highland Park, Illinois.

5 TOLERANCE TO NITRIC ACID ESTERS 107 believed, be developed in the minimum time compatible with the comfort of the subject, and the periods necessary for tolerance development by the various nitrites could be compared. RESULTS Erythrol tetranitrat8 Tolerance to the headache effects of one headache dose of erythrol tetranitrate was established in one subject (G. B.). The headache dose of erythrol tetranitrate given in tablet form by mouth was 45 mgm. for this subject; this dose produced a moderate headache lasting about eight hours. Changes in blood pressure or pulse rate were within the limits of error in such work. In this same subject, the headache dose of ethylene glycol dinitrate by skin application of 1 per cent alcoholic solution was 3.5 cc. (35 mgm.), and the headache dose of glyceryl trinitrate as a 1 per cent alcoholic solution was 1.8 cc. (18 mgm.). The tolerance was developed by giving 15 mgm. erythrol tetranitrate by mouth every three hours, day and night. As the subject became tolerant, this was gradually increased to 30 mgm. every three hours. When the erythrol tetranitrate had been administered in this manner for fifty-four hours, the administration of one headache dose of ethylene glycol dinitrate caused only a moderate fullness; the erythrol tetranitrate was given for six hours longer, at which time a total of 390 mgm. had been given. At that time the administration of one headache dose of erythrol tetranitrate produced no symptoms. The development of a complete tolerance to the headache effects of one headache dose of erythrol tetraitrate thus consumed sixtyhours. On the next day, one headache dose of glyceryl trinitrate was applied, which caused a slight headache lasting about ten minutes, whereas normally the headache dose of glyceryl trinitrate caused a moderate headache lasting about five hours. The administration of erythrol tetranitrate was then continued for five days at end of which time there was a complete tolerance to the headache effects of 2.6 headache doses of erythrol tetranitrate. Twenty-four hours after this dose of erythrol tetranitrate an amount of ethylene glycol dinitrate equal to 2.6 headache doses was applied to the skin, with a

6 108 CRANDALL, JR., LEAKE, LOEVENHART, MUEHLBERGER resulting slight fullness of the head lasting eight minutes. The tolerancewas then allowed to lapse and after twelve days one headache dose of erythrol tetranitrate had almost all of its usual effect. Glyceryl trinit rate Tolerance to at least one headache dose of glyceryl trinitrate was acquired once in each of four subjects. The headache doses of these subjects for glyceryl trinitrate were the same in all four cases, viz., 1.0 cc. of 1 per cent solution (10 mgm.) ; three of these subjects were tested for cross tolerance to ethylene glycol dinitrate, and in each of these 3 cases the headache dose of ethylene glycol dinitrate was 1.8 cc. of 1 per cent solution (18 mgm.). The periods required for tolerance development in these 4 subjects were thirty-six, thirty-nine, thirty-four, and forty-four hours, or an average of thirty-eight hours. The total amount of glyceryl trinitrate necessary to develop a tolerance to the headache effects of one headache dose of glyceryl trinitrate averaged 52.5 mgm. in these 4 subjects. Cross tolerance toward the headache effects of one headache dose of ethylene glycol dinitrate was tested in 3 of these subjects, and it was found that when a subject was tolerant to the headache effects of one headache dose of glyceryl trinitrate, he was also tolerant to the headache effects of one headache dose of ethylene glycol dinitrate. In one subject the tolerance was increased during a further period of forty-four hours from a tolerance to the headache effects of one headache dose to a tolerance to the headache effects of two headache doses of glyceryl trinitrate; during this period 51 mgm. of glyceryl trinitrate were applied. In two subjects we demonstrated that the tolerance could be maintained at the level of a tolerance to one headache dose by the daily application of this dose; the tol erance was thus maintained in one subject for six days, and in the other for eight days. In two subjects the tolerance had completely disappeared at the end of ten days, while in one subject a small amount of tolerance could still be detected eight days after the administration of glyceryl trinitrate had been discontinued; the fourth subject was not studied in regard to the duration of tolerance.

7 TOLERANCE TO NITRIC ACID ESTERS 109 Ethylene glycol dinitrale Tolerance to ethylene glycol dlinitrate was studied once in each of three subjects, and twice in a fourth subject. The headache doses of these subjects for ethylene glycol dinitrate varied from 1.8 to 3.5 cc. of 1 per cent solution; the headache doses of glyceryl trinitrate varied from 1.0 to 1.8 cc. of 1 per cent solution. The time necessary to develop tolerance to the headache effects of one headache dose varied from twenty-four to thirty-six hours, with an average of thirty-two hours ; the amount of ethylene glycol dinitrate applied in the development of this tolerance averaged 170 mgm. Cross tolerance was tested toward glyceryl trinitrate in 3 subjects, and it was found that when a subject was tolerant to the headache effect of one headache dose of ethylene glycol dinitrate, he was also tolerant to the headache effect of one headache dose of glyceryl trinitrate. Cross tolerance toward erythrol tetranitrate was tested in one subject who was tolerant to the headache effects of one headache dose of ethylene glycol dinitrate and he was also found to be tolerant to the headache effects of one headache dose of erythrol tetranitrate. In one subject it required a further twenty-eight hours to increase from a tolerance to one headache dose of ethylene glycol dinitrate to a tolerance to the headache effects of two headache doses of ethylene glycol dinitrate. It was found that in an individual tolerant to the headache effects of one headache dose of ethylene glycol diitrate, this level of tolerance could be maintained by the daily application of one headache dose; this was carried out for a period of seven days in one subject and for five days in another. In two subjects the application of the ethylene glycol dinitrate during the development of tolerance was confined to one arm and the test doses were applied to the other arm; these cases exhibited as much tolerance as those in which the test doses were applied to the same areas which had been used when tolerance was being produced. This shows that the tolerance is not due to a change in the absorbability of the ester by the skin, i.e., it is not a local tolerance. The tolerance developed by ethylene glycol dinitrate lasted from ten to thirteen days after administration had been discontinued.

8 1 10 CRANDALL, JR., LEAKS, LOEVENHART, MUEHLBERGER The longer durations were found in those subjects in whom the headache doses were highest. Methyl nitrate Tolerance was produced in two subjects by the inhalation of methyl nitrate vapor from a vial every half hour. The inhalation was so regulated that the subjects had a slight to moderate fullness throughout the period of tolerance production. The subjects were not required to waken during the night for the purpose of inhaling the drug, but with the exception of eight hours for sleep, 5 to 10 deep inhalations from the vial were taken by each subject every half hour until tolerance had been established. The periods during which the subjects actually inhaled the drug were twenty-three hours in one case and twenty-six in the other. At the end of these periods the subjects were found to be tolerant to the headache effects of their previously established headache doses of methyl nitrate (117 and 470 mgm., respectively). The tolerance of the first subject to her normal headache doses of ethylene glycol dinitrate (0.48 cc. 10 per cent alcoholic solution) and amy! nitrite (11.0 mgm. by inhalation) was then tested and she was found to be completely tolerant to the headache effects of these doses. The second subject had no symptoms following the administration of her normal headache dose of ethylene glycol dinitrate (0.38 cc. of 10 per cent alcoholic solution). In the first subject no detectable tolerance remained at the end of ten days; in the second subject there was a barely perceptible tolerance after ten days without administration of any nitrite. Amyl nitrite Tolerance to the headache effects of one headache dose of amyl nitrite was developed in three subjects. The first subject inhaled sufficient amy! nitrite from a vial every fifteen minutes to cause moderate fullness; after thirteen and one-half hours of such inhalation no symptoms resulted from the application of one headache dose (1.8 cc. ipercent alcoholic solution) of ethylene glycol dinitrate. There was a marked tolerance present after six days had elapsed without nitrite administration. The second

9 TOLERANCE TO NITRIC ACID ESTERS 111 subject inhaled sufficient amyl nitrite every five minutes for three and one-half hours to cause a moderate fullness ; three hours later the application of one headache dose of ethylene glycol diitrate (3.8 cc. 1 per cent solution) caused only a very slight fullness lasting ten minutes. Nine days later a scarcely perceptible tolerance was present. A more careful study was made in the case of the third subject, whose headache dose of amy! nitrite was found to be 11 mgm. by inhalation. Measured quantities of vapor were administered to this subject every five minutes, beginning with a dose somewhat less than the headache dose. At first this subject responded with increasing symptoms and the dose had to be reduced; after thirty-five minutes it was possible to increase the dose by degrees, and 1 hour after the administration was begun it was difficult to raise the dose rapidly enough to produce marked symptoms. At the end of two hours the subject was receiving 2 to 3 times her headache dose and the administration was discontinued. The next day the subject was given one headache dose of ethylene glycol dinitrate (0.48 cc. 10 per cent solution) with no symptoms; and the day following no symptoms were produced by headache doses of methyl nitrate (117 mgm. by inhalation) or amyl nitrite given 1 hour apart. Twelve days later the tolerance had completely disappeared. Sodium nitrite It was not practicable to determine the headache dose of sodium nitrite due to unpleasant side actions, chiefly nausea, although headaches have been reported in the literature resulting from sodium nitrite administration. The blood pressure effect was taken as the criterion of action. We attempted to produce tolerance in 3 subjects by sodium nitrite administration. The first subject took 480 mgm. sodium nitrite in aqueous solution in the course of four days, and on the fifth day one headache dose of ethylene glycol dinitrate (1.8 cc. 1 per cent solution) produced a typical moderate headache. In the second subject we found that 150 mgm. of sodium nitrite in solution caused a fall of 3 mm. in blood pressure and a rise of 10 beats per minute in pulse rate. During the course of two days this subject took 940 mgm. sodium

10 1 12 CRANDALL, JR., LEAKE, LOEVENHART, MUEHLBERGER nitrite. At the end of this period the application of 1 headache dose (1.8 cc. 1 per cent solution) of glyceryl trinitrate produced a typical headache, and later 150 mgm. of sodium nitrite caused a 3 mm. fall in blood pressure and an 8 beat per minute rise in pulse rate. In the third subject 210 mgm. sodium nitrite in salolcoated capsules by mouth caused a fall of 13 mm. in the blood pressure and a rise of 6 beats per minute in the pulserate. In the course of four days this subject took grams of sodium nitrite, and at the end of this period 1 headache dose of ethylene glycol dinitrate (3.4 cc. 1 per cent solution) caused a typical moderate headache, while the administration of 210 mgm. sodium nitrite resulted in a 13 mm. fall in blood pressure and a 12 beat per minute rise in pulse rate. We then attempted to produce tolerance to sodium nitrite in a dog. Blood pressure was taken, using a specially made cuff on the fore-leg and the auscultatory method for determining blood pressure; the dog was trained to lie quietly on a table for hours at a time. We determined that the subcutaneous injection of 3 cc. of a 10 per cent solution of sodium nitrite (0.3 gram) in this animal caused a fall in blood pressure of 24 mm. and a rise of 22 beats per minute in the pulse rate. This dose of 0.3 gram of sodium nitrite was given the animal 2 to 3 times a day for a period of six days without any indication of the development of tolerance. Two subjects were made completely tolerant to one headache dose of ethylene glycol dinitrate, and the effect of doses of sodium nitrite, for which the normal blood pressure and pulse rate effects were known, was tested. In the first subject it was found that 180 mgm. of sodium nitrite caused an 8 mm. fall in the blood pressure and a 7 beat per minute rise in the pulse rate. This subject was then made tolerant to the headache effect of 1 headache dose of ethylene glycol dinitrate. After tolerance had been established 180 mgm. of sodium nitrite produced no change in the blood pressure and an increase of only 3 beats per minute in pulse rate. In the second subject 240 mgm. sodium nitrite caused a 6 mm. fall in the blood pressure and 12 beats per minute rise in pulse rate. After the subject was tolerant to ethylene glycol dinitrate this dose caused no fall in blood pressure and no change in pulse rate.

11 TOLERANCE TO NITRIC ACID ESTERS 113 Histamin This substance was chosen because, while it is chemically unrelated to the members of the nitrite group, it lowers the blood pressure by peripheral dilatation, causes a rise in the pulse rate secondary to the lowering of the blood pressure, and produces a headache. In other words, its pharmacological action is similar to that of the nitrites, while it differs widely from them chemically. Two subcutaneous injections of 1 mgm. histamin in one subject caused a moderate headache lasting fifteen to twenty minutes, a fall of 3 to 4 mm. in blood pressure, and an increase of 22 to 28 beats per minute in pulse rate. The subject was then made tolerant to the headache effects of 1 headache dose of ethylene glycol dinitrate, and two subsequent subcutaneous injections of 1 mgm. of histamin caused moderate headache lasting fifteen to forty-five minutes, a fall of 3 to 5 mm. in blood pressure, and a rise of 16 to 25 beats per minute in the pulse rate. This experiment was repeated in a second subject who was made tolerant by means of methyl nitrate, and no difference was noted in the results of histamin injection before and after tolerance to methyl nitrate was established. TOLERANCE TO BLOOD PRESSURE AND PULSE RATE Blood pressure and pulse rate determinations were made in all cases except where methyl nitrate or amyl nitrite was used. In general, the blood pressure and pulse rate respond to the administration of one headache dose of any of the nitrites varies somewhat in a given subject, while the headache response to this dose is comparatively constant. A further advantage of using headache as the criterion of tolerance is that certain of the drugs studied, notably methyl nitrate and erythrol tetranitrate, cause typical headaches in doses which produce little or no perceptible change in pulse rate or blood pressure, while the objective changes produced by amy! nitrite are so rapid that accurate determinations are difficult. Therefore, we have taken the headache effect as our criterion wherever possible. We have observed, however, that when a subject has been made tolerant to the headache effect of one headache dose of a member of the nitrite group,

12 114 CRANDALL, JR., LEAKE, LOEVENHART, MUEHLBERGER there is also a certain degree of tolerance established to the blood pressure and pulse rate effects. Tolerance to the blood pressure and pulse rate effects is not as readily established as is tolerance to the headache effect ; that is, when an individual is made tolerant to the headache effect of one headache dose of a nitrite, he has also acquired a certain amount of tolerance to the blood pressure and pulse effects, but this tolerance is not complete. In studying cross tolerance we have found that no matter by which member of the group the tolerance is developed, approximately the same degree of tolerance to blood pressure and pulse rate effects is present when the subject is tolerant to the headache effect of one headache dose of any nitrite or nitric acid ester. DISCUSSION We have shown that tolerance may be established by the periodic administration of erythrol tetranitrate, glyceryl trinitrate, ethylene glycol dinitrate, methyl nitrate, and amyl nitrite. We have also shown that it is very difficult, if not impossible, to develop a tolerance by the administration of sodium nitrite. The existence of cross tolerance has been established in the following cases: tolerance to erythrol tetranitrate confers tolerance to glyceryl trinitrate and ethylene glycol dinitrate; tolerance to glyceryl trinitrate confers tolerance to ethylene glycol dinitrate; tolerance to ethylene glycol dinitrate confers tolerance to glyceryl trinitrate and erythrol tetranitrate; tolerance to methyl nitrate confers tolerance to amyl nitrite and ethylene glycol dinitrate; tolerance to amyl nitrite confers tolerance to ethylene glycol dinitrate and methyl nitrate. We feel that this demonstration of cross tolerance is sufficient evidence to warrant the statement that there is probably complete cross tolerance between all these members of the nitrite group. While we have not succeeded in developing tolerance to sodium nitrite or any of these esters by repeated administration of sodium nitrite, yet tolerance developed by any ester also confers tolerance to sodium nitrite. Since the completion of the experiments on which these conclusions are based, Myers and Austin (6) have reported the production of tolerance to the depressor action of sodium nitrite and glyceryl trinitrate

13 TOLERANCE TO NITRIC ACID ESTERS 115 following the repeated subcutaneous administration of sodium nitrite in rabbits. Under the direction of one of us (C. W. M.) Durrant and Smith (unpublished work) attempted to confirm these studies and came to the conclusion that if any tolerance was produced by sodium nitrite it was not of appreciable magnitude even after four weeks administration of sodium nitrite in doses comparable to those used by Myers and Austin. This work in connection with that previously performed on man and dog by oral administration leads us to believe that if any tolerance is developed by the administration of sodium nitrite it occurs very much more slowly than with the other members of the nitrite group. TABLE 1 Time required for production of tolerance to headache effects of one headache dose SUBSTANCE ROUTE TOTAL TThIE Sodium nitrite Oral No tolerance Erythrol tetranitrate Oral 60 Glyceryl trinitrate Skin absorption 38 Ethylene glycol dinitrate Skin absorption 32 Methyl nitrate Inhalation 24 Amyl nitrite Inhalation 2-3 It is very significant that no matter which member of this group is used in the development of tolerance, the tolerance, once developed, is the same as the tolerance produced by any other member of this group. No matter by which substance produced, the tolerance invariably remains for approximately ten days (varying somewhat with the individual) after the drug is withdrawn; there is always the same degree of tolerance to blood pressure and pulse rate, within the limits of error; and finally there is a complete cross tolerance to the headache effects of one headache dose. In the production of the tolerance, however, there is a marked difference between the esters used, so far as the time required is concerned (see table 1). If we compare the ease of hydrolysis of these substances, as given by Herrman, Leake, Loevenhart, and Muehlberger (8), with the rate at which they hours

14 116 CRANDALL, JR., LEAKE, LOEVENHART, MUEHLBERGER produce tolerance, we see that tolerance is produced most readily by those members of the group which hydrolyze most slowly. These authors showed that the nitric acid esters, in the order of ease of hydrolysis, range from mannitol hexanitrate which is readily hydrolyzed, to methyl nitrate which is hydrolyzed very slowly, indeed, under the conditions obtaining in their experiments. With sodium nitrite, which yields the NO2 group directly, we were unable to produce any tolerance. The rate of hydrolysis of amyl nitrite has not been determined; but qualitative tests have shown that amyl nitrite hydrolyzes very rapidly on contact with water which agrees with the observations of Leech (9). All the esters of nitric acid, however, follow the TABLE 2 Amounts of nitrites required for production of tolerance to headache effects of one headache dose, calculated on the basis of one headache dose glyceryt trinitrate = 10 mgm. SUBSTANCE ROUTE AMOUNT Sodium nitrite Oral No tolerance Erythrol tetranitrate Oral 216 Glyceryl trinitrate Skin absorption 52 Ethylene glycol dinitrate Skin absorption 160 Methyl nitrate Inhalation Not measured Amyl nitrite Inhalation 102 general rule that tolerance is produced most readily by those substances which hydrolyze most slowly. It would seem that nitrous acid, as such, is incapable of producing tolerance or, at best, produces it very slowly. While the time required to develop tolerance to a given ester is remarkably constant, the amount of the ester taken during the production of tolerance varies with the individual. Those subjects with the highest headache doses require the largest amounts of esters for the development of tolerance. We cannot compare the amounts of the various esters taken because the same subjects were not used in developing tolerance to all the esters. We may reduce the amounts actually used in tolerance production mgm.

15 TOLERANCE TO NIThIC ACID ESTERS 117 by the various esters to a common basis by multiplying them by the ratio between a fixed or standard dose and the headache dose of the subject for glyceryl trinitrate. We have taken 1 cc. of 1 per cent alcoholic solution of glyceryl trinitrate (10 mgm.) as this standard dose. Thus, if the headache dose of a subject were 2.0 cc. and this subject used 200 mgm. of erythrol tetranitrate in tolerance development, we should say that on the basis of the standard dose this subject would have used 100 mgm. of erythrol tetraitrate. Average amounts required for tolerance production by the various esters were calculated in this manner and are given in table 2. It must be borne in mind that erythrol tetranitrate was administered by mouth, glyceryl trinitrate and ethylene glycol dinitrate by skin application, and amyl nitrite by inhalation. The only comparable figures, therefore, are those of glyceryl trinitrate and ethylene glycol dinitrate. The tolerance is evidently due to some chemical factor. This is indicated by the fact that there is no cross tolerance to histamin. Apparently then, the tolerance is not developed toward a particular type of physiological response but toward a certain type of chemical agent. That the tolerance is not produced locally in the tissues to which the drug is applied is shown by the fact that if the drug is applied to a certain skin area throughout the production of tolerance, and the test application of the headache dose is made on an area which has not previously been used, the tolerance is undiminished. This is further substantiated by the fact that tolerance to erythrol tetraitrate given by mouth may be produced by the skin application of ethylene glycol diitrate. Very few attempts were made to increase the tolerance beyond one headache dose, since we were chiefly interested in studying cross tolerance within this group. Such experiments as were made indicate that it is not more difficult to increase from a tolerance to the headache effects of one headache dose to a tolerance to two headache doses than from a condition of no acquired tolerance to a tolerance to the headache effects of one headache dose. It is interesting that we were able to keep the tolerance at the level of a tolerance to one headache dose for a period of one week merely by a daily application of one headache dose.

16 118 CRANDALL, JR., LEAKE, LOEVENHART, MUEHLBERGER This report is in the nature of a preliminary study of tolerance in this most interesting group of substances. The nitrite group appears well adapted for work upon the elucidation of the nature of tolerance and cross tolerance since, unlike morphine and other tolerance-developing drugs, there appears to be no danger of chronic poisoning. Laws (3) states that men working in dynamite have poor appetites, lose weight, and show a tendency to mental dullness. Ebright (4) and Heitz (5) do not confirm Laws statements but, on the contrary, report that examination of men working with glyceryl trinitrate in dynamite manufacture show no evidence of ifi-effects, although some of the men examined had been employed as long as twenty years. The nitrite group is especially adapted to tolerance studies because of the simple chemical constitution of its members, the purity in which they can be obtained, the ease and accuracy of their quantitative chemical determination, the fact that they can be administered by skin absorption, inhalation, and by the gastro-intestinal tract, and by the marked and readily measurable effects which they produce in the body. SUMMARY 1. By the repeated administration to normal humans of erythrol tetraitrate, glyceryl trinitrate, ethylene glycol dinitrate, methyl nitrate, and amyl nitrite, we have developed tolerance to the headache producing action of these drugs, and to the changes which they produce in blood pressure and pulse rate. 2. Tolerance to headache producing action is more easily established than is tolerance to effects on blood pressure and pulse rate. 3. Cross tolerance has been shown to exist between most of the above substances, and it is believed that there is a complete cross tolerance between all members of this group. 4. It has been impossible to develop tolerance to sodium i- trite by repeated administration of large doses over four days in man, or over five days in the dog. When tolerance has been produced by a nitric acid ester the blood pressure and pulse rate response to a moderate dose of sodium nitrite is greatly decreased.

17 TOLERANCE TO NITRIC ACID ESTERS There is no cross tolerance between these drugs and histamin, a substance producing similar effects but which is chemically unrelated to the nitrites. 6. Tolerance is most readily developed by amyl nitrite, then with increasing difficulty by methyl nitrate, ethylene glycol diitrate, glyceryl trinitrate, and erythrol tetranitrate. A partial relationship between ease of tolerance development and ease of hydrolysis by alkali is pointed out. 7. A tolerance to the headache effect of a dose which wifi produce a moderate headache decreases gradually and usually disappears completely in ten to twelve days. 8. It was found that the tolerance could be maintained at a constant level by the daily administration of the dose to which the individual was tolerant. 9. As the degree of tolerance was increased there was no change in the ease or difficulty of its production. 10. The tolerance is not due to decreased absorption, but is present to equal degree no matter what the route of administration of the nitrite. REFERENCES (1) STEWART, D. D.: Tolerance to nitroglycerin. Jour. Amer. Med. Assoc., 1905, xliv, (2) MATTHEW, E.: Vaso-dilators in high blood pressure. Quart. Jour. Med., , ii, 261. (3) LAWS, G. C.: Effects of nitroglycerin on those who manufacture it. Jour. Amer. Med. Assoc., 1898, xxxi, 793 (4) EBRIGHT, G. E.: The effects of nitroglycerin on those engaged in its manufacture. Jour. Amer. Med. Assoc., 1914, lxii, 201. (5) HEITZ, J.: De l etat cardio-vasculaire des ouvriers fabriquant ou maniant habituellement la nitroglycerin. Arch. des Mal. du Coeur, 1924, xvii, 578. (6) MYERS, H. B. AND AUSvIN, V. T.: Nitrite toleration. Jour. Pharmacol. and Exper. Therap., 1929, xxxvi, 227. (7) OLTMAN, T.: Master s Thesis, University of Wisconsin, (8) HERRMAN, R. F., LEAKE, C. D., LOEVENHART, A. S., AND MUEHLBERGER, C. W.: The relation between rate of alkaline hydrolysis of certain nitric acid esters and their power to lower the blood pressure. Proc. Amer. Soc. for Pharmacol. and Exper. Therap., 1926, xxvii, 259. (9) LEECH, D. J.: The Croonian lectures on the pharmacological action and therapeutic uses of the nitrites and allied compounds. Brit. Med. Jour., 1893, i, 1305, also Lancet, 1893, i, 1499.