Influence of Serotonin Antagonists on Nitrogen Mustard Intoxication'

JOHN B. FIELD, EDWARD C. DOLENDO, ANNIE MIRELES, AND BENJAMIN H. ERSHOFF (Wester-n Institute for Cancer and Leukemia Research, Lo8 Angeles, California)

SUMMARY The administration of a pyrazolone compound before and after a single I.P. LD@-1@dose of nitrogen mustard produced a marked reduction of the expected mor tality in mice. The pyrazolone was most effective I.P. but it was also effective P.O. This protection was also observed in mice receiving the nitrogen mustard I.V. When the pyrazolone was given in multiple daily doses a greater degree of protection was obtained than with a single dose. The protective effect of the pyrazolone was ob served at different toxic levels of nitrogen mustard. The leukopenia induced by the nitrogen mustard was less in extent and duration in pyrazolone-treated mice. A number of agents with antiserotonin activity were tested ; several of these corn pounds were found to have some ability to reduce the toxicity of nitrogen mustard.

The use of many anticancer drugs is limited by their with the Simonsen mice were also observed when using the toxicity, particularly their bone marrow depressing action. pyrazolone compound in female CF1 mice obtained from Epinephnine and norepinephrine have protected animals Carworth, Inc., N. Y. HN2 was given in a single dose, as (11) and human beings (3, 4) from the lethal and pancyto the hydrochloride, usually 5 mg/kg or as otherwise indi penic effects of the alkylating agent, nitrogen mustard. cated. At a dose of 5 mg/kg a virtually uniform mortality In a previous report it was indicated that an unexpected of 90-100 % was attained by the 6th day, with survivors and significant reversal of the intoxication of nitrogen resuming normal health after this time. The serotonin mustard in mice was achieved with a pyrazolone compound antagonist 1-(N-methyl-piperidyl-4')-3-phenyl-4-benzyl (KB-95) (5, 6). pyrazolone-5 (Jfl@95)2 was suspended in 5 % gum acacia This communication deals with further studies con solution and given I.P. Certain other serotonin antag cerrnng this observation in mice and has been extended to onists were dissolved in physiologic saline : d-2-brornolyser include a survey of other serotonin antagonists, evaluated gic acid diethylamide (BOL-148)2; Methysergide bimaleate in a similar manner in mice treated with near-lethal doses (UML-491)2; d-lysergic acid diethylamide tartrate (LSD of nitrogen mustard. In more recent studies it has been 25)2 ; cypropheptadine hydrochloride (Peniactin) furnished found that antihistamines also can ameliorate the toxic by Merck, Sharp & Dohrne (10) ; N-p-chlorobenzhydryl action of nitrogen mustard (7). N'-methylhomopiperazine dihydrochloride (Homochior cyclizine) (SA-97) furnished by Abbott Laboratories, Inc. METHOD (8); and N-benzyl-N-(3-dimethylaminopropyl)-benzamide hydrochloride (RO-29102) furnished by Hoffrnann-La The nitrogen mustard, mechlorethamine hydrochloride Roche, Inc. Some serotonin antagonists were suspended (HN2) was freshly prepared from the standard commercial vials (generously furnished by Dr. Elmer Alpert, Merck & in 5 % gum acacia: 3 ,4-dihydro-6-methoxy-1-methyl-9H Co., Inc., Rahway, N. J.) and given I.P. to rested, 18â€” pynido (3 ,4-b) indol hydrochloride (TJ-10 266A)@ (9); 22 gm, adult white Swiss female mice (Sirnonsen Labora DL-5-hydroxy-a-methyl tryptophan (U-7994)1 ; and a- rnethylserotonin creatinine sulfate (U-7729).' tories, Gilroy, Calif.). Results similar to those obtained Until December 1963, it was our custom to obtain a 1 Communication No. 11. This study was supported by grants predictable uniform mortality of 90-100 % with 5 mg/kg from the following foundations: The Milheim Foundation for of the commercial preparation of mechiorethamine hydro Cancer Research, R. C. Baker, Conrad N. Hilton, Joseph Lowitz, Lidow, William Morris Agency, and Robert H. and Clare M. chloride as furnished. However, since this date we have Avnet. Also the Horace E. Altfeld, Susan Gorshow, Abe Yaras, observed that the material was more toxic than before. Leo Cholodenko,and Harold Rappaport MemorialFunds, North Thus, in studies since that time, the usual dose has been American Aviation Employees Donate Once Club, and the Inter 4.375 mg/kg, producing the same mortality end point and national Rectifier Corporation. Received for publication May 25, 1964; revised November 13, I Furnished by Sandoz, Inc. 1964. 3 Furnished by The Upjohn Company. 382

otherwise undistinguishable from the previous 5 mg/kg. TABLE 1 All of the present studies were conducted with 5 mg/kg EFFECT OF DIFFERENT I.P. DOSES@ OF KB-95 ON and were completed prior to December, 1963. SURVIVAL AND WBC OF MICE TREATED WITH Daily blood samples were taken from the tip of the tail 5 MG/KG HN2 for the WBC and daily body weights were recorded for all o, mice. Control groups of mice received only the HN2 or DoSS AlTER NADIR OF (MG/KG)T@ HN2 STATEMENTaAvzi@ozLEUXOPENIA the serotonin antagonists and an HN2 control was in MM)100 (si.)SURVIVALNo. %PROBABILITY (WBC/cu eluded in every assay of HN2 versus a serotonin antagonist. Mice were usually treated in groups of 8 or 10 and several tests were usually done on each group ; the average results 2 7/14 50 <0.01 3,300 are reported here. 8 10/14 71 <0.01 3,800 Surviving mice were observed for about 1 year. After 400 2 18/30 60 <0.01 4,560 the initial deaths, surviving mice lived indefinitely and 8 28/40 70 <0.01 3,990 the data given refer to prolonged survival. 24 26/50 52 <0.01 3,930 A statistical evaluation of the survival data was done 2,000 242/14 0/8 0 >0.053,150 1,000 Controls12,400a 5/5414 9>0.05 by the chi-square test. In a relatively small number of

cases, a Fisher exact probability test was applied when Chi-square with Yate's correction. expected values were insufficient to meet requirements of the chi-square test. TABLE 2 RESULTS EFFECT OF P.O. KB-95 ON SURVIVAL AND WBC OF HN2DOSEMICE TREATED WITH 5 MG/KG The I.P. administration of 400 mg/kg of the pyrazolone

derivative (KB-95) increased the survival of mice treated OF NADIR AFTER OF LEU with 5 mg/kg of HN2 from 6 % to 43 % when given just (MG/KG)TIME HN2 STATIMENTaAvzi@oz EOPENIA (ai.)Suivxv*z@No. %[email protected]@v (WBC/cu before the 11N2 and to 79 % when the KB-95 was given MM)100 I.P. 24 hr. after the HN2; there was a reduction in both the leukopenia and weight loss of the treated mice (5, 6). The effect of different doses of KB-95 in mice treated with 8 5/10 50 <0.01 3,870 5 mg/kg HN2, including total survival and WBC levels 400 2 7/16 44 <0.01 4,200 attained, is summarized in Table 1. With 100 mg@kg 4 6/10 60 <0.01 3,740 KB-95 given 8 hr. after the HN2, the survival was 71 %; 8 9/18 50 <0.01 â€¢ 3,650 with 400 mg/kg at 8 hr. after the HN2, the survival was 24 5/16 31 0.05>P>0.Olb 3,150 70 % ; and with 2,000 mg/kg given 24 hr. after the HN2, 2,000 242/100/8 0 >0.052,625 1,010 Control HN2 2/34 6 a dose which produced diarrhea and unkemptness in the 1,450 Control KB-95 8/820 100>0.05â€• 5,400 mice, the survival was 0 %. It is obvious that this dose 4004 may have been toxic and contributed to the severe leu kopema. By comparison, control HN2-treated mice a Chi-square with Yate's correction. showed a 9 % survival. S Fisher exact probability test. Effect of KB-95 given P.0.â€”When 1(13-95 was given by stomach tube feeding in suspension with 5 % gum acacia, dose of KB-95.â€”A study was made of the effect of a single the results were as given in Table 2. A significant in dose of 400 mg/kg of KB-95 in mice treated with different crease in survival to 50 % was achieved with a single dose doses of HN2. In Table 4 are summarized the results of 100 mg/kg given 8 hr. after the 11N2 whereas a single when the KB-95 was given 4 hr. after the HN2. As might dose of 400 mg/kg produced a 60 % survival 4 hr. after be expected, survival rate was lower with the larger dose the HN2 and 50 % survival 8 hr. after the HN2. At 24 hr. of HN2. Thus when 6.35 mg/kg HN2 were given, the after the 11N2 the survival was 31 %. A dose of 2,000 survival was 38 %, with 5.62 mg/kg the survival was 44%, mg/kg given 24 hr. after the HN2 gave a 0 % survival. and with 5 mg/kg the survival was 67 %. An appropriate Effe4 ofmultiple dosesofKB-95.â€”Theeffectof repeated control was used in each case; the survival of HN2-treated daily doses of KB-95 on mice treated with a single 5 mg/kg' mice not given KB-95 was 0-6%. dose of HN2 was determined. The serotonin antagonist Effectofotherserotonin antagoni@3tsonHN@ intoxication.â€” was given I.P. or P.O. in a dose of 400 mg/kg twice daily A number of other agents showing anti-serotonin activity and the results are given in Table 3. When the drug was (1, 2, 8â€”10),as demonstrated in bioassay, were obtained started the day before the HN2 was given and continued from several pharmaceutical houses. All were tested in for 4 days, a survival of 94 % resulted, as compared with a survival of 15 % for HN2 controls treated twice daily with mice to determine their lethal dose; approximately half injections of saline. When the KB-95 injections were the minimum lethal dose was used in the HN2 assay. begun on the day the HN2 was given, the survival was The antiserotonins were given routinely at several in @ 88 %, and when the KB-95 was given P.O., starting the tervals: and 2 hr. before the HN2, and 4, 8, and 24 hr. day before the HN2, the survival was 38%. after the HN2. However, only representative values ob Effect of different dosesof HN@ in mice treatedwitha single tamed at 8 hr. after the 11N2 are stated in Table 5. In no

TABLE 3 EFFECT OF MULTIPLE DOSES OF KB-95 ON SURVIVAL, WEIGHT (GM) AND WBC op MICETREATEDwim 5 MG/KGHN2 (SINGLEDOSE) Mice were given 400 mg/kg KB-95 I.P., twice daily for 4 days.

7SurvivalI.P.1RouteDrug begunDay 1Day 2Day 3Day 4Day SDay 6Day

day before HN2 Wt. WBC20.7 5,40020.13,85020.75,10019.75,90019.58,10018.79,35019.9 â€”P15/16 94% 0.OlbI.P.Same <

day as HN2 Wt. WBC21.7 4,38021.83,40021.94,59020.34,90018.76,79019.87,22520.2 â€”P7/8 88%. 0.OlbP.O.1 <

day before HN2 Wt. WBC20.1 5,20019.44,50018.93,95018.33,85016.37,00016.88,90017.711,300P6/1638% 0.05bControlâ€•No >

KB-95 Wt. WBC21.0 4,90019.53,85018.82,75018.03,15015.52,75015.53,80015.0â€”5/32 15%

a Includes 24 mice given 5% gum acacia I.P. and 8 mice given the gum acacia P.O. b Chi-square with Yate's correction.

TABLE 4 EFFECT OF DIFFERENT LEVELS OF HN2 ON SURVIVAL, WEIGHT (GM) AND WBC OF MICE TREATED wim A SINGLB DOSE OF KB-95 Mice were given 400 mg/kg KB-95 I.P., 4 hr after HN2

Dose 7Survival6.35TreatedHN2 1Day 2Day 3Day 4Day 5Day 6Day (mgJkg)MiceDay

Wt. WBC 4,600 2,650 3,000 3,700 5,400 6,10017.69,3006/1638% Control Wt. 21.2 19.5 18.0 16.1 16.1 0/16 WBC21.5 4,10019.9 3,50018.92,40017.81,85017.32,20016.7 0 <0.Ola5.62Treated P

Wt. WBC 4,000 3,700 3,600 4,400 5,800 9,90017.712,9007/1644% Control Wt. 19.6 18.9 17.6 16.2 15.7 1/16 WBC21.6 4,30020.2 2,80018.9 2,75016.92,40016.72,15015.9 6% 0.01.5.00Treated 0.05> P >

Wt. WBC 4,000 3,900 3,700 4,900 6,300 9,600 12,20016/2467% Control Wt. 20.5 20.0 18.8 17.4 16.5 14.0 0/16 WBC21.0 4,30020.0 3,80019.1 3,40017.6 2,30017.72,07518.42,35019.6 P < 0.Olb

a Fisher exact probability test.

b Chi-square with Yate's correction. case was the protective effect against HN2 greater than The most effective agents appeared to be U-b 266A (75% that observed with the KB-95. Many of the serotonin survival), and U-7994 (55 % survival). antagonists possessed some inhibitory activity but only Effect of KB-95 in mice [email protected].â€”Inall pre some appeared to produce a survival greater than 50%. vious tests, the HN2 had been given routinely to the mice

TABLE 5 The validity of the basic observation of the anti-HN2 EVALUATION OF AGENTS WITH ANTI-SEROTONIN ACTIVITY capacity of the serotonin antagonist KB-95 was established ON SURVIVAL, AND WBC OF MICE TREATED WITH by testing increasing doses of HN2 in mice given a fixed 5 MG/KGHN2 (SINGLE DOSE) dose of KB-95. At the highest dose of HN2 (6.35 mg/kg) All drugs were given I.P., 8 hr after HN2. protection was least, with a survival of 38 %, as compared to 67 % at the lowest level of HN2 (5 mg/kg) ; the injected op NADIR HN2 controls had a 0-6 % survival. STATEMENT@' OF LETJ A GENTDOSE (MG/LG)SUIVIVAL (F)AVERAGE KOPENIA When KB-95 was given in repeated doses twice daily, No. % (WBC/cu MM)SA-97 the most successful protection from HN2 mortality was â€˜PROBABILITY seen, with survival up to 94 % compared to 15 % for the control mice. With single doses of KB-95 the survival BOL-148 15 9/18 50 <0.01 2,600 was increased roughly up to 70 %; remarkably, as was Periactin 7.5 23/44 54 <0.01 3,100 shown previously (5, 6), the most significant effects induced U-b 266A 20 4/10 40 <0.01 2,800 by KB-95 occurred when the drug was given 4, 8, or 24 hr. 30 6/10 60 <0.01 2,500 after the HN2. This was demonstrated both with 100 40 6/8 75 <0.01 4,600 mg/kg and 400 mg/kg doses, by both P.O. and I.P. routes. 12/22 <0.01 4,000 U-7994 500 55 This demonstration of increased effectiveness of drug ac U-7729 400 2/10 20 >0.05â€• UML-491 125 10/28 36 <0.01 2,950 tion in combating a toxic sequence of events induced by an LSD 25 15 12/25 48 <0.01 2,300 otherwise lethal agent, when the drug is given as late as RO-29102 125 3/10 30 0.05> P > 0.01â€•4,0003,738 24 hr. after the toxic agent, appears to be unparalleled in Control25 â€”7/18 5/7839 6<0.01 2,200 pharmacologic annals. However, the full significance of this observation will be realized only when the mechanism a Chi-square calculations in all cases except in 2. of the reaction is understood. b Fisher probability test. The effectiveness of KB-95 given P.O. was greatest at about 4 hr. after the HN2, while the I.P. dose was most I.P. Because of the possibility that some local changes effective at a later time, consistent with the time required induced in the peritoneal surfaces by the HN2 might have for absorption from the gastrointestinal tract. The pos evoked the favorable response to KB-95, several groups of sibility that the serotonin antagonists, P.O. or I.P., directly mice were given the HN2 I.V. Thrombosis induced in the interfere with HN2, when the latter is given I.P., was ruled tail vein of the mouse by the HN2 was undoubtedly associ out by the demonstration that KB-95 also inhibits HN2 ated with some local tissue fixation of the HN2, so that administered I.V. mortality in the intravenously treated mice at 5 mg/kg Based on other studies with HN2 toxicity in untreated was actually somewhat lower than in the I.P.-treated mice, it can be stated that the LD50 is approximately 4.3 groups. Of 30 mice given 5 mg/kg HN2, 10 (33 %) sur mg. From the data in Table 4, a single dose of KB-95 vived while of 30 given 6.25 mg/kg HN2, 6 (20 %) sur gave an LD@ with 5.6 mg of HN2. Thus the apparent vived. When treated with 400 mg/kg KB-95 I.P., 24 hr. increase produced by a single dose of KB-95 is approxi after the HN2, the survival was 24/30 (80 %) at 5 mg/kg of mately 1.3 mg/kg, a 30 % increase in the tolerated doses of HN2, and 17/30 (57 %) for the mice treated with 6.25 HN2. mg/kg of HN2. Although no effort was made to correlate the anti Response of WBC.â€”The WBC of the HN2-treated mice serotonin bioassay activity of the agents studied here with steadily fell to about 2,000/cm before death. A consider the anti-HN2 activity, there does not appear to be any able leukopenia was observed with KB-95, but, at its nadir, it never fell to the low levels of the control mice. direct relationship.4 From the published information The KB-95 HN2-treated mice showed a faffing WBC, available (1, 2, 8â€”10),KB-95appears to be less active than similar to but not as extensive as the HN2-treated mice. several other agents listed in Table 5 ; among the agents Within 4â€”5days the count reached a plateau and then studied, it appears that the antiserotonin activity was not returned rapidly to pretreatment levels (Tables 1â€”3). of critical importance to the anti-HN2 activity. Further Normal white cell levels were almost always reattained studies will be designed to study the possible correlation about 5 days after the KB-95 was given. With repeated of anti-serotonin and anti-HN2 activity. daily doses of KB-95 the drop in WBC was negligible and The reversal of the lethal action of nitrogen mustard by this is associated with the excellent survival in this group serotonin antagonists appears to be a unique example of (Table 3). The WBC of surviving mice have been ob the ability of an active biologic agent to interrupt the served for many months and have remained constant. progress of even a well-established lethal process at some DISCUSSION fundamental level and effectively counteract and reverse it. 4 It is of interest to record that serotonin itself possessed con In the mouse the lethal and, to a lesser extent, the siderable ability to reverse the action of nitrogen mustard. Thus leukopenic effect of nitrogen mustard, can be consistently in the same assay with 5 mg/kg of HN2, 500 mg/kg of serotonin in reduced and inhibited by serotonin antagonists. The saline solution, given I.P. to mice, produced 73% survival (48/66) most effective of these in the present evaluation has been when given 8 hr after the HN2 and a 56% survival (37/66) when the pyrazolone agent KB-95. Other agents known in given 24 hr after. Serotonin was also effective to about the same degree when given I.V. This is a further indication that the bioassay to be serotonin antagonists are effective to a lesser anti-serotonin activity of the compounds reported herein is prob or negligible degree. ably of no conseanence in the anti-HN2 activity.

The clarification of the interrelationships suggested here Questions of Oncology (Russian Government publication), may provide fruitful and significant clinical findings. 9.@65-68,1963. 6. . Reversal of Nitrogen Mustard Intoxication by a Se REFERENCES rotonin Antagonist. Proc. Soc. Exptl. Biol. Med., 111:1â€”3, 1962. 1. CERLETTI,A.; BERDE,B.; NEUHOLD,K.; ANDTAESCHLER,M. 7. FIELD, J. B.; MIRELES, A.; DOLENDO, E. C.; ANDERSHOFF, Ceratterizzione Farmacologica di un Nuovo A.ntagonista della B. H. Reversal of Nitrogen Mustard Intoxication by Anti Serotonina con Proprieta Analgesico Antifiogistico. Boll. Histamines. Ibid., 115:1060â€”62,1964. Chim. Farm., 102:602-16, 1963. 8. K@MunA,E. T.; YOUNG, P. R.; AND RICHARDS,R. K. Phar 2. ERSPAMER, V. Recent Research in the Field of 5-Hydroxy macologic Properties of N-p-chloro-Benzhydryl-N'Methyl tryptamine and Related Indolealkylaniines. Fortsch. Arznei Homopiperazine Dihydrochioride (Homochiorcycizine; SA mittelforsch. (Progress in Drug Research, Basel), 3:154â€”367, 97), A Serotonin Antagonist. J. Allergy, 31.237-47, 1960. 1961. 9. McI&t@c, W. M.; Kn.@[email protected],P. A.; AND PAGE, I. H. 10- 3. FIELD, J. B. Increased Human Tolerance for Nitrogen Mus Methoxyharmalan, A Potent Serotonin Antagonist which tards with Norepinephrine; A Preliminary Report. Clin. Re Affects Conditioned Behavior. Science, 134:674â€”75,1961. search, 9:103, 1961. 10. STONE, C. A.; WENGER,H. C.; LUDDEN, C. T.; STAvo@s@i, 4. . Protection of Bone Marrow Inhibition by Nitrogen J. M.; ANDRoss, C. A. Anti-Serotonin-Anti-Histaminic Prop erties of Cyproheptadine. J. Pharmacol. Exptl. Therap., 131: Mustards (HN2) with Norepinephrine. Proc. Am. Assoc. 73â€”84,1961. â€˜Cancer Res., 3:224, 1962. 11. Wmr.is, L. P. Experiments on the Therapeutic Index of Nitro 5. FIELD, J. B.; MIRELES,A.; ANDDOLENDO,E. C. Reversal of gen Mustard. Acta TJnio Intern. Contra Cancrum, 16800-06, Nitrogen Mustard Intoxication by a Serotonin Antagonist. 1960.

John B. Field, Edward C. Dolendo, Annie Mireles, et al.

Cancer Res 1965;25:382-386.

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