Reprinted fmm THE JOURNAL OF Paa~raco~ocv IND EXPERI~~ENTAL THERAPEUTICS Vol. 98, No. 2, February, 1950 ASSAY IN MAN OF THE CHEMICAL FRACTIONS OF VERATRUM VIRIDE, AND IDENTIFICATION OF THE PURE ALKALOIDS GER- MITRIKE AXD GERMIDINE AS POTENT HYPOTENSIVE PRINCI- PLES DERIVED FROM THE DRUG' EDWARD D. FREIS, JOSEPH R. STANTON AND F. CORBIN MOISTER Robert Lhzoson Evans Memorial, Massnchusetts Memorial Hospitals, and the Department of Medicine, Boston University School of Medicine, Boston, Mass., the Veterans Admin- istration Hospital, and the Department oj Medicine, Georgetown University School of Medicine, Washington, D. C. Received for publication November 7, 1949 Recent clinical (1) and experimental (2) evidence indicated that the crude powdered roots and rhizomes of Veratrum v&de may produce marked reductions of arterial pressure in patients with essential hypertension. However, therapeutic application of the available preparations has been limited by the frequency of their disturbing side effects and the inconsistency of their action (1). The present investigation was undertaken (1) to identify the hypotensive principle or prin- ciples of Veratrum viride, (2) to determine whether the hypotensive and toxic factors were separable, and (3) to obtain pure crystalline compounds for clinical use which can be standardized by weight rather than by biological assay. METHODS. Chemical fractionation of the crude roots of Veratrum viride was carried out by Drs. Josef Fried, Howard L. White, and 0. Wintersteiner of the Squibb Institute for Medical Research (3). The fractions and pure alkaloids made available to us by these investigators were tested by oral administration in known hypertensive patients admitted to the wards of the Massachusetts Memorial Hospitals and the Veterans Administration Hospital, Washington, D. C. All patients were hospitalized for at least 48 hours prior to study. With the patient in the supine position, arterial pressure was recorded with an arm cuff and mercury manometer, while pulse rate was counted at the wrist. Prior to administer- ing an extract, readings were taken at minute intervals until the arterial pressure and heart ra.te became stabilized. Further determinations of arterial pressure and heart rate were recorded at intervals of one-half hour for three hours following the oral ingestion of an extract. The various fractions were dissolved either in small amounts of 5 per cent.acetic acid or 95 per cent ethanol depending on their solubility, diluted with water and flavored with orange syrup. Over 400 separate oral assays were carried out in testing 102 fractions. The intravenous route was used only with the pure active alkaloids which were dissolved in 2 per cent acetic acid (1 mgm. per cc.) and diluted further to a concentration of 0.025 mgm. per cc. with normal saline. The solutions were sterilized by passage through a glass filt,er and were stored in rubber capped vials. No loss of activity was apparent after storage at, room temperature for more than two months. A hypotensive dose was defined as the quantity of a given substance producing a reduc- tion of the basal mean (systolic + diastolic) 2 arterial pressure of at least 15 per cent. When a * This investigation was aided in part by the Squibb Institute for Medical Research, New Brunswick, New Jersey, and was carried out in collaboration with Drs. Josef Fried, Howard L. White and 0. Wintersteiner of the Squibb Institute, who supplied all of the fractions of Veratrum viride used in this study. 166 VERATRUM ALKALOIDS 167 positive result was obtained, this was always confirmed by retesting in effective doses in two to three additional patients. On the other hand, before discarding a presumably inactive fraction, it was tested in doses six to eight times greater than the amount calculated to be present in an active dose of the crude drug, as judged by the percentage composition. For example, jervine composed 0.67 per cent of the crude drug. The mean effective dose of the crude root powder was 225 mgm. (table 1). Thus, if all of the hypotensive action of the crude drug were due to jervine, one would expect that a dose of 225 X .0067 or 1.5 mgm. of jervine would exhibit hypotensive activity. Nevertheless, this alkaloid was tested in doses up to 9 mgm. prior to discard. RESULTS. All of the previously known crystalline alkaloids of Verutrum viride were inactive in the doses given. These included the benzene-extractable alka- loids jervine, veratramine, rubijervine, isorubijervine, germine (4-6), and the TABLE1 Progress of fractionation of total amorphous alkaloids (1) Crude root powder.. . . . . . . . . . . (2) Total benzene extractable alkaloids (ref. 6). . . . . (3) Amorphous bases (fraction (2) minus crystalline alkaloids listed in text). . . . . . . . (4) Tertiary bases from (3). . . . . . . . . . . (5) Combined plates 1-7 from S-plate Craig distribution of (4)................................................... (6) Benzene-l'% methanol eluate from alumina chromato- gram of (5). . . . . . . . . . . . . . (7) Fraction I (tube 15, K = 1.67, from 25-plate Craig distri- bution of (6)). . . . . . . . . . . . . . . . . . . . . . . . . . . . . (8) Fraction II (tube 6, K = 0.35, from 25-plate Craig distri- bution of (6)). . . . . . . . . . . . . , . . . . . . . . _ . . , . _ . . . . . . . 3.5 .36 3.2 .26 1.8 1.2 $7 5.5 Per cent 100 1.1 .13 .065 .045 .Ou) alcohol-extractable fraction containing the glucosidic alkaloids, pseudojervine and veratrosine (6). However, the amorphous material remaining after removal of the crystalline benzene-extractable alkaloids was active with 3.5 mgm. (mean effective dose in eight patients, range 2-5 mgm.). Table 1 illustrates the progres- sive purification of this active fraction up to the highly potent but still amorphous products (Fractions I and II) from which the crystalline alkaloids were isolated. Following intravenous administration of the various active fractions the ar- terial pressure and heart rate began to decrease two to five minutes after injection of an effective dose (figure 1). Toxic effects such as substernal or epigastric burn- ing sensation, tingling of the fingers and face, nausea and vomiting also occurred at this time. Arterial pressure and heart rate usually fell rapidly over a period of several minutes following which these values remained at low levels for three to ten minutes and then rose slowly over a period of one-half to four hours to the basal values (figure 1). Following oral administration, the arterial pressure and heart rate began to decline in one to two hours, reached a minimum in two to 168 FREIS, STANTON AND MOISTER three hours at which time toxic effects particularly nausea and vomiting and excessive salivation might occur, and then rose slowly over a period of two to HEARTRATE PER MINUTE - GER);;;RINE I I I I I I 0 IO 20 30 40 50 60 70 66 WNuTEe I ARTERIAL PRESSURE MM tt2 I60 t 120 FIQ. 1. Chart of patient J. U., 45-year-old male with essential hypertension illustrating the response of the blood pressure and heart rate to 0.055 mgm. of germitrine. The drug was given slowly over a period of five minutes intravenously, and injection was discontinued when a definite decrease in arterial pressure occurred. There was slight substernal and epigastric burning sensation from the second to the twelfth minute after the beginning of njection, but no other side effects. ARTEHIAL PRESSURE MM. HG 200 16 iA Tll4 120 SO HEART RATE - PER MIN. 60 ET 60 f 0.7 MG. FRACTION I PER OS t I I I I I I I I 0 I 2 3 4 5 6 7 0 HOURS FIG. 2. Chart of patient E. M., 43-year-old female with essential hypertension illustrat- ing the response of the blood pressure and heart rate to an oral dose of 0.70 mgm. of Fraction I. This patient exhibited no toxic side effects. sixteen hours to basal levels (figure 2). In general the duration of the hypotensive response and frequency of side effects were related to the degree of hypotension VERATRUM ALKALOIDS 169 produced although in a few cases striking reductions in arterial pressure occurred without side effects (figure 2). Marked hypotensive reactions could be achieved in almost all patients by increasing the dosage but this also increased the severity TABLE 2 Effect of intravenous administration of the pure aEkaEoids of Veralrum uiride and album on the blood pressure and heart rate of hypertensive patients ALKALolD DOSE Blood Pressure - -- 1. Blood Pressure Fraction I Fraction I Fraction I Fraction I mgnr. 0.05 0.09 0.07 0.06 W. J. w. c. A. R. G. S. mm. Be cr min mm Erg 160198 90 130/80 220/128 95 168/110 230/130 104 192/105 190/110 88 146/94 `W mb 68 78 82 68 Epigastric burning Epigastric burning 0 0 Fraction II 0.50 W. J. 176/108 92 132/88 72 Epigastric burning Fraction II 0.54 G. S. 185/112 88 145190 74 0 Fraction II 0.26 M. C. 168/110 80 132/88 68 Epigastric burning Fraction II 0.60 J. S. 215/98 70 174/78 62 0 Germitrine 0.06 W. J. 190/120 96 110/78 56 Germitrine Germitrine Germitrine B. M. 150/100 80 110/72 68 E. A. 190/100 84 80140 74 J. U. 190/120 88 122/90 68 Nausea and vomit- ing 0 0 0 Germidine Germidine Germidine Germidine Germidine 0.12 0.15 0.09 0.14 0.23 G. S. 205/120 90 180/105 78 0 W. J. 180/115 a4 156/102 72 Epigastric burning M. C. no/135 80 160/104 64 Epigastric burning J. F. 2241145 84 172/120 40 0 E. K. 144/100 74 132/88 62 0 Germerine Germerine Germerine Germerine Germerine Germerine 0.15 0.28 0.10 0.33 0.38 0.30 G. S. 190/115 J. S. 180/94 M. C. 185/115 J. G. 240/150 F. S. 150/96 W. K. w/90 96 140/88 72 72 154/68 56 76 140/90 56 96 180/118 74 96 110/75 88 56 115/75 50 0 Tingling of skin Substernal burning 0 Nausea Epigastric burning Protoveratrine Protoveratrine Protoveratrine Protoveratrine Protoveratrine Protoveratrine F. D. 175/120 64 115/70 J. S. 178/98 68 148/80 G. W. 190/130 64 165/110 W. J. 175/m 84 140188 M. C. 200/130 80 84/50 F. S. 145/98 80 98/60 52 60 56 60 44 56 < - Tingling of skin Substernal burning Epigastric burning Epigastric burning Iingling of skin Nausea, tingling -T- - of the side effects. As observed following use of the crude drug (l), there was little relationship between body weight and size of an effective dose. Fraction I, when administered orally, exhibited hypotensive activity in four patients in doses of 0.6 to 0.7 mgm. (table 3). In the same patients Fraction II 170 FREIS, STANTON AND MOISTER exhibited activity in doses of 4.2 to 6.0 mgm. By the intravenous route Fraction I produced a significant decrease in arterial pressure and heart rate in doses ranging between 0.05 and 0.09 mgm. (mean 0.067 mgm.), while Fraction II pro- duced a response in doses of 0.4 to 0.60 mgm. (mean 0.51 mgm.) (table 2). Germine, the parent crystalline alkaloid obtained after hydrolysis of these amorphous fractions was inactive in doses of 8 mgm. orally. At this point it is necessary to review the present status of the chemical work on the crystalline alkaloids derived from Fractions I and II, and to make refer- ence to some important new facts recently brought to light by the Squibb in- vestigators which considerably add to, and in some respects alter, the picture as it was presented in their preliminary communication (3). In that note they an- nounced the isolation from Verutrum virile of two new crystalline alkaloids, germitrine and germidine, obtained by crystallization of Fractions I and II, respectively. Both compounds were found to be esters of the known alkaloid germine, C~HQO~N. The acids present in the ester groups of germitrine were identified as a-methylbutyric acid and methylethylglycolic acid, and those of germidine as acetic acid and cr-methylbutyric acid. The analytical findings and other observations suggested that the alkaloid named germitrine was a triester of germine containing one cr-methylbutyryl and two methylethylglycolyl radicals. However, it has now been established by the Squibb investigators (7) that this compound is actually the known alkaloid gemnerine, an ar-methylbutyrate- mono- methylethylglycolate of germine which had been isolated in 1937 by Poet&e (8) from Veratrum album, the white or European hellebore. It was furthermore ascertained t'hat germerine does not occur as such in Fraction I, but arises from a labile precursor (which consititues most of this fraction) when the amorphous product is crystallized from aqueous methanol. This precursor, which has now been also obtained in crystalline form, is a triester of germine the acidic compo- nents of which include acetic acid in addition to the two acids present in the ger- merine moiety. The Squibb investigators will propose in their forthcoming de- tailed publication (9) to transfer the name germitrine (denoting a triester of germine) from the compound now identified as germerine to this genuinely new ester alkaloid, and it should be understood that the term is used with this mean- ing henceforth in this paper. Thus three crystalline ester alkaloids were available for our study, namely, the triester germitrine and the diesters germerine and germidine. Germitrine showed about the same activity as the amorphous Fraction I, from which it is derived, by either route of administration (effective dose 0.05-0.06 mgm. intra- venously, and 1.0 and 1.2 mgm. perorally). Its degradation product, germerine, had to be given at levels about twice as great (0.10-0.15 mgm. intravenously, and 2.5 to 3.0 mgm. perorally) to elicit the same response. The activity of germi- dine is of the same order as that of germerine (0.10-0.38 mgm. intravenously, and 2.5-3.5 mgm. perorally). It should be noted that in this case the crystalline compound was considerably more effective than the amorphous Fraction II from which it is derived. It was of interest to compare the active alkaloids obtained from Verutrum VERATRUM ALKALOIDS 171 virile with protoveratrine, a crystalline alkaloid derived from Veratrum album (8) which has been found by Meilman and Krayer (10) to exhibit hypotensive activity on intravenous injection in hypertensive patients. A sample of this com- pound made available to us by the Squibb workers was active in doses varying TABLE 3 Effect of oral administration of the pure alkaloids of Vera&urn viride and album on the blood pressure and heart rate of hypertensive patients ALKALOID Fraction I Fraction I Fraction I Fraction I Fraction II Fraction II Fraction II Fraction II Germitrine Germitrine Germitrine Germitrine Germidine Germidine Germidine Germidine Germerine Germerine Germerine Protoveratrine Protoveratrine Protoveratrine ngnt. 0.7 0.7 0.6 0.65 6.0 5.5 4.2 4.5 1.2 1.2 0.9 1.0 3.25 3.5 2.2 2.8 2.5 3.0 3.0 1.1 0.9 1.5 PATIENT Blood PRSSUre E. M. M. S. F. D. G. P. Blood PIeSSUre mm.Hg 220/110 235/125 160/115 160/85 rer rnil 76 84 75 72 9. / mm. Hg 95/50 85/58 148/K@ 140/74 5.z LI min 60 52 60 56 E. M. 170/98 74 136/8& 60 M. S. 212/98 64 115/68 46 F. D. 170/110 64 135/78 52 G. P. 180/95 74 130/74 58 J. M. 190/108 110 160/98 88 E. D. 170/105 86 152/86 72 B. M. 165/110 80 75/55 54 J. M. 190/100 104 160/100 74 J. G. 212/140 88 go/50 68 F. S. 160/100 76 m/90 70 M. C. 185/118 80 122/84 68 J. S. 205/88 70 170170 60 J. U. 170/125 100 150/100 68 B. M. 185/125 88 140/100 68 E. D. L62/85 88 135/80 64 J. S. hf. c. F. S. 188/80 60 155/65 56 Nausea 170/100 76 140/90 56 Nausea 160/100 80 LX/85 64 Nausea 1 - I -7 - AFTER DB"O I SIDE Emcw 0 Nausea 0 0 Nausea and vomit- ing Nausea and vomit- ing Nausea Nausea and vomit- ing 0 0 Nausea and vomit- ing 0 Nausea and vomit- ing Nausea 0 Nausea 0 0 0 between 0.1 and 0.15 mgm. (table 2), thus confirming the observations of the previous investigators. The side effects observed were similar to those seen after the alkaloids of Vera&urn viride. In addition, protoveratrine was found to be active orally in doses varying between 0.9 and 1.5 mgm. (table 3). DISCUSSION. Previous attempts at chemical separation of the alkaloids of 172 FREIS, STANTON AND MOISTER Vera&urn viride failed to extract purified compounds from the amorphous base fraction (5, 6). In the present work the guidance obtained by assaying the frac- tions of the amorphous bases for hypotensive activity in man proved invaluable in guiding the chemists and eventually led to the isolation of two previously un- described potent hypotensive ester alkaloids, germitrine and germidine. The alkaloid exhibiting the greatest activity was germitrine which exhibited signifi- cant hypotensive effects in some patients after intravenous doses of as little as 50 micrograms and was followed in descending order of activity by protovera- trine, germidine, and germerine. All of these compounds are ester alkaloids and those derived from Veratrum viride yielded germine on hydrolysis, the latter being inactive in man. Thus, the ester linkages seemed to be essential in deter- mining the hypotensive potency of these alkaloids in man. Despite the differences in hypotensive potency, the action of each ester alka- loid was quite similar to the others in the character of the response of the blood pressure and heart rate, the type of side effects observed, as well as the time of onset and duration of these responses after both intravenous and oral adminis- tration. Whether the frequency and severity of the toxic effects varies with the different compounds cannot be decided by this limited study. The effective oral dose was approximately eight to twenty times greater than the effective intra- venous dose. The results of this investigation suggest that the side effects observed in hyper- tensive patients receiving Veratrum vi&de may not be separable from the hypo- tensive principles of the crude drug. The present evidence indicates that the nausea and vomiting are central in origin since such effects may occur following parenteral administration of the alkaloids. Nevertheless, the availability of pure crystalline materials should be of benefit in that it will permit investigation of single pure alkaloids both acutely and on chronic administration in clinical and experimental investigation. Such studies are now in progress. SUMMAiY AND CONCLUSIONS 1. Systematic assay of the various chemical fractions of Veratrum v&de in hypertensive patients revealed that the cardiovascular effects of the crude drug in man are due largely to the presence of two hitherto undescribed crystalline ester alkaloids, germitrine and germidine. Both these compounds exhibit high hypotensive activity in man on intravenous as well as on peroral administration. 2. Germerine, a third crystalline ester alkaloid which probably does not occur as such in the root but arises by partial hydrolysis of germitrine, exhibits similar hypotensive properties. This compound is not new, but has been previously re- ported as an alkaloidal constituent of Veratrum album. 3. These crystalline compounds are comparable in their effects on the circu- latory system to the ester alkaloid protoveratrine derived from Veratrum album, although each alkaloid differs somewhat from the others in potency. 4. Partial hydrolysis of germitrine to germerine results in a reduction in hy- potensive potency, while oomplete hydrolysis of either germitrine, germidine VERATRUM ALKALOIDS 173 or germerine to germine results in loss of hypotensive action. Thus, the pres- ence of ester groups seems to be essential for hypotensive activity. 5. All of these alkaloids were shown to be active after oral administration, the effective dose by mouth being approximately eight to twenty times greater than the effective intravenous dose. G, The toxic effects of Veratrum viride do not appear to be due entirely to con- taminating substances present in the drug, since similar reactions may occur following administration of the pure amorphous and crystalline alkaloids. Acknowledgment: The authors wish to thank Doctors Robert W. Wilkins and Reginald H. Smithwick for helpful suggestions and cooperation in use of patients throughout the period of this study. REFERENCES 1. FREIS, E. D., AND STANTON, J. R.: Am. Heart J., 36: 723, 1948. 2. FREIS,E.D.,STANTON, J.R., CULBERTSON, J.W., HALFERIN,M. H., LITTER, J., BUR- NETT, C. I-l., AND WILKINS, R. W.: J. Clin. Investigat., 28: 353, 1949. 3. FRIED, J., WHITE, H. L., AND WINTERSTEINER, 0.: J. Am. Chem. Sot., 71: 3260, 1949. 4. WRIGHT, C. R. A.: J. Chem. Sot., 36: 421, 1879. 5. SEIFERLE, E. J., JOHR.S, I. B., AND RICHARDSON, C. H.: .J. Econ. Entomol., 35: 35, 1942. 6. JACOBS, W. A., AND CRAIG, L. C.: J. Biol. Chem., 155: 565, 1944; 160: 555, 1945. 7. WINTERSTEINER, 0. : Personal communication. 8. POETHKE, W.: Arch. d. Pharmazie, 276: 571, 1937. 9. FRIED, J., WHITE, H. L., AND WINTERSTEINER, O.:In preparation. 10. MEILMIN, K., AND KRAYER, 0.: J. Clin. hvestigat., 28: 798, 1949.