The Vinca Alkaloids : a New Class of Oncolytic Agents

The Vinca Alkaloids : A New Class of Oncolytic Agents

IRVING S. JOHNSON, JAMES G. ARMSTRONG, MARVIN GORMAN, AND J. PAUL BURNETT, JR.

(Lilly Laboratoriesfor Research and the Lilly Laboratotiesfor Clinical Research, Indianapolis, Indiana)

SUMMARY A phytochemical investigation of the plant [email protected] demonstrated that a number of alkaloidal substances can be obtained with antitumon activity. Over 30 alkaloids have been obtained, of which fourâ€”vinbiastine, vinleunosine, vincnistine, and vinrosidineâ€”are known definitely to be active. Chemically these compounds are closely related to one another and to two monomeric alkaloids, vindoline and catharan thine. The structure of these latter two compounds has been determined, and partial structures for the biologically active alkaloids have been proposed. They represent a new class of large complex dimenic alkaloids containing both indole and dihydnoindole moieties. Experimentally, a strain-specific, transplantable, acute, lymphocytic leukemia (P-1534) carried in DBA/@mice served as a bioassay for obtaining these compounds and for predicting their clinical activity. Vinblastine, vincnistine, and vinrosidine are capable of prolonging and/on â€œcuringâ€•miceof the P-1534 leukemia even when therapy is delayed until a near-terminal state of generalized disease. Resistance to an additional challenge of leukemic cells has been observed in these â€œcuredâ€•animals. Parenteral ad ministration of vincnistine has been demonstrated to â€œcureâ€•micegiven intracranial implants of the P-1534. The experimental tumor spectrum and toxicological studies are presented and discussed. Biochemical studies performed to date do not reveal any effect on cellular nespira tion, glycolysis, protein or nucleic acid synthesis. The mechanisms of action of these compounds, which may differ within the group as well as from those of other known agents, remain to be determined. Only two of these compounds, vinbiastine and vincnistine, have received extensive clinical evaluation. In spite of their close similarity, chemically, a somewhat different group of human neoplasms responds to these compounds, and there has been a singular lack of cross-resistance between these two drugs and any other oncolytic drug now in wide use. Vinblastine has proved effective in chonioepithelioma, Hodgkin's disease, and other lymphomas, and a number of beneficial results have been obtained in car cinoma of the breast and bronchus. In addition, there have been smaller numbers of a variety of other neoplasias reported as responding to this compound. Vincnistine has been striking in its ability to induce complete hematological remission of the acute leukemias of childhood, both lymphocytic and myelogenous in type. Re sponses have also been reported in a number of other malignancies. The problems and obstacles encountered in obtaining a full realization of the clinical efficacy of these new types of oncolytic compounds are discussed in addition to areas of clinical application other than those previously reported.

The plant Vinca rosea Linn. (periwinkle) of the the natural state pink and white color varieties are family Apocynaceae is an ever-blooming pubescent found, and hybrids such as blush pink with red herb on sub-shrub which is widely cultivated as an eye, crimson, and white with red eye are commen ornamental in gardens throughout the world. In cially available. In a recent review (77) of current 1390

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1963 American Association for Cancer Research. JOHNSON et al.â€”Vinca Alkaloids . 1391 phytochemical research on this plant, Svoboda has that â€œindefiniteâ€•survival of animals implanted pointed out that the probable correct botanical with the P-1534 leukemia was being obtained by name for this plant is Catharanthus roseus G. Don, tneatment with crude fractions of the plant which but, owing to the frequent and prevalent use of were chemically free of both leurosine and VLB Vinca ro8ea, the latter name will be used synony (38). Further investigation of those fractions ne mously. sulted in Svoboda's obtaining still two other new This plant has enjoyed a popular reputation in active alkaloids, leurocnistine and leurosidine (74). indigenous medicine in various parts of the world. The A.M.A. Council on Drugs has approved yin Peckholt (63) in 1910 described the use in Brazil blastine' (VLB), vinleurosine (VLR), vincnistine2 of an infusion of the leaves to control hemorrhage (VCR), and vinnosidine (VRD) as generic names and scurvy, as a mouthwash for toothaches, and for these alkaloids; and they will be referred to by for the healing and cleaning of chronic wounds. In these names in the balance of this report. the British West Indies it has been used to treat The purpose of this report is to summarize our diabetic ulcer (86) and in the Philippines has been current knowledge of this new class of antineoplas reported as being an effective oral hypoglycemic tic agents, including the chemistry, pharmacology, agent (fl). The plant is also used in South Africa possible mechanisms of action, and experimental as a hypoglycemic, and, in fact, a preparation un and clinical activity. der the name of â€œCovincaâ€•ismarketed for this purpose. In England the plant has been sold for ISOLATION AND CHEMISTRY diabetic treatment under the name â€œVin-q-lin.â€• Following the initial observation that extracts The folklore reputation which this plant enjoyed of Vinca rosea Linn. produced prolongation of life as an oral hypoglycemic agent independently stim in mice with P-1534 leukemia, a detailed fractiona ulated its phytochemical investigation in two dif tion of the plant was undertaken. It was shown ferent laboratories, unknown to each other. One of (41) that the activity was found entirely in the these groups included Noble, Beer, and Cutts, then alkaloidal constituents of this material, and that at the Collip Laboratories, University of Western the alkaloids of the leaves were far more active Ontario, and the other a group in the Lilly Re than those contained in either the stems or the search Laboratories including Svoboda, Johnson, roots (73). The leaf material was therefore used for Neuss, and Gonman. Although neither group could the preparation of the compounds described hero substantiate the hypoglycemic activity, the Ca in, and a procedure of differential extractions was nadian group observed a peripheral granulocyto developed (78) which separated the alkaloids into penia and bone marrow depression in rats asso several groups, as shown in Chart 1, according to ciated with certain fractions (15, 18). Continued their varying basicities This procedure, which in investigation led to their preparation of vincaleu volves the conversion of the bases to their tartrates koblastine (VLB) sulfate, an alkoloid capable of followed by extraction into organic solvents, was producing severe leukopenia in rats (17, 60, 61). first used by Svoboda (71) to prepare alkaloids from During this period the Lilly group had demon another plant of the same family, ALitonia con strated that certain alkaloidal fractions gave a no stricta F. Muell. It was found that the antitu producible prolongation oflife of DBA/@ mice given mor activity was primarily located in fraction A implants of the acute lymphocytic neoplasm, the (Chart 1). P-1534 leukemia (41). Fraction A was partially purified by chromatog The detection of activity against the P-1534 leu raphy on alumina, deactivated by treatment with kemia was considered particularly significant, ow 10 per cent acetic acid. This yielded, in addition ing to the fact that this tumor system has detected to a number of inactive alkaloids (the results of other clinically useful antitumor agents in our lab purification of fraction A are shown in Chart @), oratory (40) and had been sensitive enough to two pure compounds possessing antitumor activity study structure-activity relationships of active (7@), vinleurosine and VLB (isolated as sulfate). compounds which correlated with the clinical ac When the noncnystalline residues obtained from tivity (@3,46, 53). An intensive phytochemical in this chromatography were tested for antitumor ac vestigation resulted in the obtaining by Svoboda tivity, certain of the post-VLB fractions appeared (7@) of leunosine, a new dimeric alkaloid closely to be more active than either vinleurosine or VLB related chemically to VLB, as well as VLB sulfate. (74). These materials were subjected to a gradient The effectiveness of both of these alkaloids against pH technic involving partition of the mixtures the P-1534 leukemia was first demonstrated in the between benzene and buffers from pH @.8to 7.5

Lilly Laboratories. 1 VelbanÂ® (Vinblastine, Lilly). Early in this investigation it became apparent 2 OncovinTM (Vincristine, Lilly).

1) HCI(2N) 1) 2% Tartarlc Acid 2) NH4OH.CHCI3 2) Benzene

@ii@iy-SolubleI Drug @lkalolds(E)] 1) NH3 2) Benzene

â€œAlkalineBenzeneExtract

1) 2% Tartaric Acid 1) NH4OH 2) EtCI2

Alkaline EtOH Extract

Phase Phenolic Alkaloids 1) NaOH (pH 11) (C,D) 2) EtCI,

CHART 1.â€”Extraction scheme for Vinca rosea L. leaves

[Frac@on@

Ai,03 Chromatography

Benzene Benz-CHCI,(1:1) CHCI, @ I I 1 I CATHARANTHINE LEUROSINE Moth Llq. Al,03 LOCHNERIDINE Combined 1 IVINDOLININE(.2HCl)@ISOLEUROSINBenzBenz(deactivated)FVIROSINE11CHCI,residues @ IA.JMALICINE@jVLB(-H,S04) chromatography I Ai,O,chromatography

. CHCI3 Benz CHCI3 CHCI3 I I ______Benz.CHCI, CHCI3 @ OSINEJ (1:3) Gradient @ GradIent pH 2) chromatography ______I2.1.3.4 @ I Benz@CHCI3 @[email protected] [CATHARICINEJ (1:3) IVINDOUDINEI ICAROSID1N.!JICombined I1:3ResIdues1 â€˜Gradient Gradient IpH(4.4.5.4) Ichromatog. pHofmoth. Iiq. (3.4) [VINCARODINEI CHCI3.CH3OH [itEUROCRI@@j (99:1)

, [NE0LEuR0sIDINE]

CHART 2.â€”Isolation of alkaloids from fraction A

in 0.5 pH unit increments. The resulting materials At the time of our entry into this area a number were then crystallized as the bases or sulfate salts of well-known alkaloids had been reported from as shown in Chart @.Bythis procedure two addi this plant (78) ; these are listed in Table 1. They tional biologically active alkaloids, vincnistine and are all found in other genera and species of the vinrosidine, were obtained pure (74) . These com family Apocynaceae, and their structures are well pounds differed in their activity from the previous documented. No direct chemical relationship could two in that they routinely gave survivors (38), be seen between the alkaloids in Table 1 and the rather than just limited prolongation of life in four active alkaloids. The remaining alkaloids @ P-1534 leukemia. The remaining fractions (Chart which are listed in Table with their tentative 1) were purified as described above for fraction empincal formulae, melting points, rotations, and A, and the results obtained are outlined in Chart pK'@ values were all of unknown structure but 3 (75, 76). In addition to the alkaloids listed in were, by and large, indole and dihydroindole in Chant 3 from fraction E, Moza and Trojanek (54) nature (77). A comparison of physical properties

I Fraction [FractionB1I IFractionE Al,03 Al,O, IAl,03 Chromatography Chromatography IChromatography F I Benzene Benz.CHCI3 Benz@CHCl3 Benzene Benz-CHCI, (3:1) (3:1) I (3:1) IVindolineI I LOCHNERICINEI @ (Ajmalicine] [Tetrahydroalstonine [VlndOJifld ITETRAHYDROALSTONINEI

[FractionB IFractionF] IA120,Chromatography IA120, .L@ I I @J@hromatography Benz-CHCI3Benz@CHCl, CHCI, (3:1) (1:1) CHC1, II LAjmaucinel[LOIHNERI1@iE]______ISERPENTINE(HJ1O,I râ€”1-PERIVINEI @ Moth.Liq. H,S04 Benzene Benz@CHCl3 (2:1) @ IS1TSIRIKINE(Y2H2S04)Catharanthinetlne@1@ndolinejI (3:1) I-CATHARINE IVINDOLICINE

CHART 3.â€”Isolation of alkaloids from fractions A1, B1, B (A + B), E and F

have recently reported two additional compounds TABLE 1 tentatively named vindolidine3 and locherinine. ALKALOIDS FROM Vinca roses LINN. The biological comparison of the four active alkaloids from this plant (38) indicated consider NameFormulaAjmalicine able differences in the nature and scope of their activities, and it was felt that an understanding of the chemistry of this apparently new class of on Tetrahydroalstonine C,1H34N,O, Serpentine C21H,2N20, colytic agents was important to the eventual un Lochnerine C20H24N,02 derstanding of their mode of action as well as to Akuammine* C,@[email protected]@O4 Reserpine*C,1H,4N@O@ C@H@N2O, enable one to study chemical modifications of these compounds which might show enhanced activities. a Akuammine (vincamajoridine) and reser 3 Since the name vindolidine had previously been given to pine have not been encountered in our investiga another alkaloid (Table 2) Prof. Trojanek has suggested the tion, whereas the presence of the other four has alternate name vindorosine for his compound. been confirmed.

such as titration, infrared and ultraviolet spectra, Comparison of these spectra with those of sev and nuclear magnetic resonance spectra of the four eral other Vinca rosea alkaloids, notably catharan active alkaloids indicated first of all that they thine and vindoline (31) (Table @),clearly mdi are closely related chemically, and secondly that cated a close interrelationship between these com they are nonsymmetrical â€œdimericâ€•alkaloids (30). pounds. When the infrared spectrum of a solution Thus, for vinleurosine and VLB titration showed containing equimolar quantities of catharanthine the presence of two basic nitnogens, pK'a 5.5 and and vindoline was compared with those of vinleu 7.5, one of which would quatennize when treated rosine and VLB, it was virtually supenimposable @ with methyl iodide or similar reagents. Analyses of from to 8 and quite similar up to 16@ (Chant

TABLE 2 NEW ALKALOIDS FROM Vinca rosea LINN.

[email protected]., Â°C.*(a)@ CHCIspK'@ in DMFEtOH U.v.)@max.,

(decompn. 72 .5, 7 .5 (H20) 259 Vinblastine C4H@N4O, 211â€”216(decompn. + 42@ S .4, 7 .4 (H,O) 214, 259 Virosine C@H,.N@O4f 258â€”264(decompn. â€”160.5 5 .85 (66%) 226, 270 Perivine CHH@N,O$ 180â€”181 â€”121.4 7.5(66%) 226,314 Catharanthine C@H24N,O, 126â€”128 + 29 .8 6 .8 (66%) 226, 284, 292 Lochnericine C,iHUN@O@ 190â€”193(decompn.) â€”482 4 .2 (66%) 226, 297, 827 Vindolinine-2HC1 C@H@N@O,â€¢2HCl 210-212 (decompn.) â€” 9 (H,O) 3 .8, 7 . 1 (66%) 245, 300 Vindoline C,@HI,N,O 154â€”155 â€”18 5 .5 (66%) 212, 250, 304 Isoleurosine C4@HoN4O,t 202â€”206(decompn. + 61 .2 4 .8, 7.3 (66%) 214,261,287 Lochnenidine C,OHUN,OS@ 211â€”214(decompn. â€”607.5 5 .5 (66%) 230, 298, 328 @ Sitsirikine' 1/2 H@SO4 1/2 H@SO4289-241 (decompn. + 23 (Base) 7 .6 (66%) 224,282,288 Vincamicine (Dimeric) 224â€”228(decompn. +418 4 .80, 5 .85 (66%) 214, 264, 815, 341 Catharine [email protected]@N4O,.CU,OH 271â€”275(decompn. â€”54 .2 5 .34 (66%) 222, 265, 292 Vindolicine ([email protected]) 248-251 (melts, re â€”48 .4 5 .4 (66%) 212,257,308 cryst.) 265â€”267(decompn. Vinrosidine (Dimeric) 208â€”211(decompn. + 55.8 5 .0, 8 .8 (33%) 214, 265 VincristineC4H@N4O,t296Carosidine [email protected]@N4O@0202â€”205 218â€”220(decompn.+ + 17 .0 (EtCh)5 5 .0, 7 .4 (38%)214, 220, 255, 288 (de 89 .8 compn.) Carosine C@H@N4O@0f 214-218 + 6 .0 .4, 5 .5 (88%) 255, 294 Pleurosine C4H,sN4Oiot 191â€”194 (decompn. + 61 .0 4 .4, 5 .55 (33%) 267,308 Neoleurosidine C@4@H@N4Oi@t 219â€”225(decompn. + 41 .6 5 . 1 (88%) 214, 268 @ Vincarodine C441L,N4O@ot 258â€”256(decompn. â€”197.4 5 .8 (66%) 280, 272, 298 Catharicine C@H,,N4O,ot 281â€”284(decompn.) + 34 .8 5 .8, 6 .3 (88%) 214, 268, 293, 315 Vindolidine C4,H.@N4O@ot 244â€”250(decompn.) â€”113.2 5 .3 (83%) 261, 311 Neoleurocristine C4,H,N4O@,t 188â€”196(decompn.) â€”57 .87 4 .68 (38%)212,254,308220, 257, 298 250,302Lochneridinine#C@H,sN@O4163-169424247,Vindolidi@ie#(Dimeric) C,,H,0N@O5263-278, 167â€” â€”814 326

a The melting points were determined on a Kofler microstage. The ultraviolet absorption spectra were obtained with a Cary model 14 spectrophotometer. t Whilethesemolecularformulasagreewellwiththeanalyticalresultsforeachparticularalkaloid,itshouldbenotedthatthey are to be considered as proximate at this time, in light of our experience with the other dimeric alkaloids (57). @ Determined on VLB etherate. Â§OnthebasisofmassspectrometricevidenceobtainedbyH.BudzildewiczandI. M. WilsonofStanford,wenowpreferthe C,@,formulationratherthan the C1,firstreported(54). #SeeRef.58. the free bases, sulfates, dihydrochlonides, and qua 5). It was assumed that the double molecules yin ternary salts indicated that they are C@H@_58N4O9 leurosine and VLB were composed of catharan compounds (57). A study of the infrared spectra thine and vindoline moieties, with minor molecular (Chart 4, Aâ€”D)of the bases further supported this modification, bonded together in some unique contention (thus the intensity of the indole N-H manner (30) . It was therefore possible to investi was approximately one-half that expected for a gate the structures of the more plentiful smaller typical indolic C21 alkaloid) and indicated that molecules and then relate this informatioa to the VLB contains a hydroxyl group (X CHC13 @.8, dimenic compounds. 1O.0@) that is not present in vinleunosine. Aside Cathananthine (31, 56) (I)@ was found to be a from this difference, the infrared spectra are very C21H2@N@O2compound and to form a methiodide similar (Chart 4, Aâ€”D)(30). 4 Catharanthine and derivatives are shown in Chart 8.

WAVELENGTH (MICRONS)

CHART 4.â€”Infrared spectra of biologically active Vines alkaloids A. Vinleurosine C. B. Vinblastine D.

readily. The physical characteristics of the quater double bond at C3-C4â€”viz., voacangine (III), di nary salt showed that the basic nitrogen (pK'a 7.0) hydrocatharanthine (IV) (vide infra)â€”the Ci-C18 was analogous to the one forming this salt in the bond is broken to afford 3-methyl-S-ethyl pynidine dimeric compounds (77). Spectral data indicated (83). that the alkaloid was a simple @,3-substituted pen Mild hydrogenation of I afforded only one iso tacyclic indole containing an isolated double bond, men, dihydrocatharanthine (IV). Its infrared spec ester group, and closely related to the isoquinucli trum was strikingly similar to that of coronar dine alkaloid coronanidine5 (II), C21H26O2N2(@7). idine. The differences in these compounds could The N.M.R. spectrum (Chart 6B) confirmed the be explained by examining Dreiding models which above assignments and indicated that the double showed that the hydrogens could only have en bond contained one vinyl proton at 5.SÃ¶,and that tered the molecule from the side nearest to Nb to a C-ethyl group characterized by a methyl triplet give the axial ethyl group in dihydnocatharanthine. a 1.1Ã¶and an allylic methylene quartet at 1.9Ã¶was The ethyl group in the Iboga alkaloids has been present (56). These data clearly suggested structure shown to be equatorial (@).7 (I) for cathananthine (Chant 8). Supporting cvi The reduction of catharanthine and dihydro dence for the position of the double bond was ob catharanthine with LiA1H4 afforded corresponding

FREQUENCY (CM1)

z 0

WAVELENGTH (MICRONS)

CHART 5.â€”Infrared spectral comparison of vinleurosine and an equimolar solution of catharanthine and vindoline

tamed recently from decoupling experiments per alcohols. The formation of the tetrahydro-1,3- formed on the vinyl proton of the alkaloid.6 oxazine derivative (70) from cathananthinol gave Saturation of this proton led to changes in the additional evidence for the position of canbometh shape of the C-@ proton at 2.6@6, and, in addition, oxyl at C18. small decoupling effects were seen on the protons The Iboga alkaloids possessing a carbomethoxy at 4.18@(C-5 proton) and 1.96 (methylene of ethyl function at C13are known to decarboxylate smooth group). As expected, dehydrogenation of catharan 7 The following evidence can also be correlated with this thine with Pd on carbon yielded 3-ethyl pyridine assignment: by the cleavage of the allylic C1-C2 bond. In iso a) The pK'a values of the axial series are higher than those quinuclidine Iboga alkaloids which do not have a in the equatorial series; dihydrocatharanthine pK'@ 6.4, coronaridine pK'. 6.1 (38% DMF). 6 The correlation of isoquinucidine alkaloids (with no aro b) The rate of methiodide formation has been found to be matic enethexyl groups) is readily found by the observance of a sensitive indicator of the configuration of the C-ethyl group, @ a triplet centered at 6.8 in the infrared spectrum. N. Neuss, the rate being much faster in the axial series (private commu â€œLillyCollection of Physical Data on Indole and Dihydroin nication, Prof. M. Shamma, Penn. State Univ., State College, dole Alkaloids,â€• Eli Lilly and Company, Indianapolis, Indiana Pennsylvania). (1961). c) The Re values of these compounds vary when they are 6 The decoupling experiments were carried out by Mr. Paul run by thin-layer chromatography on silica gel plates. (RT of Landis, Eli Lilly and Company, on a proton-proton decoupler dihydrocatharanthine 0.2, coronaridine 0.8, solvent ethyl ace patterned after that described by Mr. L. F. Johnson, Varian tate-chloroform, 1 : 1.) This difference may be ascribed to the Associates, Palo Alto, California (private communication). acidity of the silica.

JOHNSON et al.â€”Vinca Alkaloids 1397

ly on saponification (@9), followed by mild acid in the orientation of the C-4 ethyl group as pre treatment, or alternately after prolonged refiuxing viously mentioned. The final confirmation of the of the ester with hydrazine (65) in ethanol. Anal nature of the ring system was given by the isola ogously, dihydrocatharanthine afforded the con tion of 4-ethyl-@,6-dimethyl-11-H-indolo (@, 3-C) responding descarbomethoxy base epi-ibogamine quinoline from Se-dehydrogenation of epi-iboga (V), differing from the known ibogamine (VI) only mine. The identical compound has been obtained

â€˜ â€˜ â€˜ I I 5@O 400 300 260 100 0 C.Ps.

CHART 6.â€”N.M.R. spectra in CDC13 (60 mc.) A, vindoline; B, catharanthine

earlier by the same treatment of ibogamine (57). acetyl group. This was further corroborated in the Catharanthine (I) must therefore be &-dehydno infrared spectrum by the appearance of a hydnoxyl coronanidine. band (@.7@i)and corresponding reduction of inten The nature of the second half of the dimeric sity of the canbonyl band. compounds could best be understood by a study The presence of the methyl ester was shown by of the dihydroindole alkaloid vindoline (VII) (@6, lithium aluminum hydride reduction of the base 31), a C@[email protected] basic nitrogen did with simultaneous removal of the acetyl to yield not form a methiodide in analogy to the nitrogen vindolinol, C@H30N2O4. The infrared spectrum of with PK'a 5.4 in the dimenic alkaloids. The nature vindoline, in which a broad band at 3.5â€”4.0@twas of the six oxygens was determined by the forma conspicuous, showed that the fifth oxygen was a tion of suitable derivatives (31). Thus, brief treat hydrogen-bonded hydroxyl. Formation of the di ment of vindoline with acid afforded desacetylvin acetate C27H3@N2O7(acetic anhydnide in pynidine) doline, C2@H30N2O5,corresponding to the loss of an was accompanied by the disappearance of this a Chart 9 shows vindoline and related compounds. broad band in the infrared spectrum. From the

4. VL.B

CHART 7.â€”N.M.R. spectrum vinbiastine in CDC1, (60 mc.)

I R :COOCH3 II R1 : COOCH3; R2: C2H5; R3 H;R4 : H; Coronaridine ill R1 : COOCH3; R2 : C2 H5; R3 H; R4 : OCH3; Voacangine

@ IV R1 COOCH3; R2: H; @ R3 C2H5; R4 H; Dihydro cat haranthine V R1:H;R2:H;R3:C2H5; R4 H; Epi-ibogamine

VI R1 : H; R2 : C2H5; R3 : H; R4 H; lbogamine

CHART 8.â€”Structures of catharanthine and related compounds

study of the ultraviolet and infrared spectra, the (8).Thecleavagecanberationalizedasoccurring @ sixth oxygen was shown to be present as an ano in the manner shown in Chant 10. matic methoxyl on a dihydroindole chromophore. The position of the carbonyl in the ketone (IX) The confirmation of the assignment of the oxygen was assigned on the basis of the presence of the functional groups was obtained from the N.M.R. peak at m/e @98(M-4@ is equivalent with loss of spectrum (Chart 6A). ketene in IX or ethylene in XI) which suggested Vindoline consumed 1 mole of hydrogen at at that this group involved either C-3 or C-4. Position inosphenic pressure, showing it to be a pentacyclic 4 was selected for two reasons; the typical ABX compound and afforded dihydrovindoline (VIII) pattern at low field (3â€”3.58)in the N.M.R. spec which could be converted to an amorphous hy trum which was consistent with structure VIII in groscopic hydrochloride (Chart 9). Pyrolysis of dicated three protons between the canbonyl group this salt at 195Â°â€”@00Â°C.in vacuum gave a distil and nitrogen; and equilibration with CH3OD/ late from which a C21H2@N@O2compound (IX) was methoxide resulted in the introduction of only two obtained in a 15 per cent over-all yield by direct deuterium atoms per molecule (M = 34@); where crystallization from hexane. The methoxydihydno as if the carbonyl was at C-3 there would be three

I DELTA VALUES 2.65 3.4 2.1â€”3.0 5.u 6.91 5.23 6.30

5.43 @ 3.75 __ 900 2.68 3.80 VII

CH)O@@@J1@H CH3 CH3O R p XII VIII x R:CH3C@- XI R=CH3

CH3d''211@ CH3 H COOCH3 XIII XIV

CHART 9.â€”Structures of vindoline and related compounds

indole portion of this compound was the same as B in vindoline, and the second oxygen was found to be present as a ketonic canbonyl (X@fi'c',5.85j@)(@8).

Vindoline (VII) was found to possess the same @N@YD ring system as the alkaloid aspidospermine (X) CH3Q IC (7), obtained from plants of the Aspidosperma CH3 genus which is in the same family as Vinca. This Ix observation was made by comparing the mass XI spectral fragmentation patterns of the pyrolysis CLEAVAGE AT A,C rn/c â€¢174 ketone and dihydnovindoline with that of the aspi B.C rn/c =188 dospermine (X) derivative desacetyl-N-methyl as C.D rn/c =298 (HEAVY LINES) A,D rn/c :124 pidospermine (XI). In each compound the identical CHART 10.â€”Mass spectral fragmentation of the vindoline intense peaks were found at rn/c 1@4,174, 188, and @98(@8).A study by Biemann and co-workers has pyrolysis ketone (IX, Y =C= 0) and desacetyl-Nmethyl shown that these four peaks are uniquely formed by cleavage of this type of a pentacyclic system aspidospermine (Y

exchangeable hydrogens. The other fragmentation still be consistent with the remainder of the spec peaks occurring in these vindoline derivatives (p28) trum). Since in the N.M.R. spectrum of vindoline are consistent with the proposed skeletal structure (Chart 6A) there is a single unsplit proton peak at and are due to more complex fragmentation which S.43Ã´ which shifts on removal of the 0-acetyl will be discussed elsewhere (7) . The similarities in moiety to 4.07Ã´the carbon bearing the acetoxyl the mass spectra of dihydrovindoline and the C21- must contain a single proton and those carbons on ketone indicate that no rearrangements of the ring either side of it must contain no protons. Likewise, system occurred during the pyrolysis. The correla acetylation of the 3-hydroxyl in vindoline does not tion of the mass spectrum of vindoline with dihy result in an analogous shift of any protons to a drovindoline is rendered more complex because of lower field, and it must therefore be tertiary. The the double bond in the former which alters the only arrangement consistent with these data is typical fragmentation patterns somewhat; how that shown for vindoline (VII) with the hydroxyl ever, all other physical measurements are con and carbomethoxyl groups attached to carbon 3 sistent with the simple hydrogenation of an iso and the acetoxyl on carbon 4. lated double bond in the conversion of VII to VIII. The double bond was placed as shown also on There remained then the problem of the place the basis of the N.M.R. spectrum which shows two ment of functional groups and the stereochemistry cis vinyl protons (J = 10 c.p.s.), one of which (5.885) is further split by two nonequivalent adja cent protons with coupling constants J = 5 and @2c.p.s.Since vindoline is stable to both borohy dnide reduction and zinc and acid reduction, the double bond is not present as an enamine (7, S XVII XVIII position) and therefore can only be in the 6, 7 position as shown. It is interesting to note that a. related dihydroindole alkaloid, vindolinine (19) (Tablep2),foundinthesameplant,hasbeenshown to have structure XIV and possess an ester and double bond in the same position as those of yin doline. @ COOCH3 CH3O â€¢OOCH3 The stereochemistry of the ring system of vindo line (VII) is assumed to be as found in aspido XV R=CH3 spermine (X), and this conclusion is supported by XVI R=CHO the observation that the methyl (of the ethyl CHART 11.â€”Structures of dimeric alkaloids and derivatives group) in vindoline yields a 3 proton triplet in the N.M.R. at abnormally high field (0.48Ã¶).One can of the molecule. The position of the aromatic explain this shift on the basis of increased shielding methoxyl was shown to be at either C-is or C-16 due to ring currents from the aromatic ring. Ob by examination of the typical 1,@,4 aromatic pro servation of models suggests that, with the aspido ton pattern (ortho splitting J = 8 c.p.s., meta split spermine stereochemistry, the methyl is in the ting J = @.Sc.p.s.). The final selection of C-16 is proper position to exhibit such enhanced shielding. based on the comparison of the infrared and ultra The stereochemistry of the functional groups at violet spectra of vindoline with 6-methyoxy-N- positions S and 4 is, as yet, not certain, and a. methyldihydroindole (XII) and by the isolation of discussion of this problem will be published else ind-N-methylnorhanmine (XIII), from a soda lime where. distillation of vindoline at 3@50â€¢Thisderivative As mentioned above, spectral evidence strongly also indicates the position of the N-CH3 as being suggested that the dimeric alkaloids were com on the anilino nitrogen. A second product (IX) posed of a molecular combination of catharanthine which was identical to the C21-ketone obtained (I) and vindoline (VII) (59). This assumption above was also isolated from the soda lime reaction. could be further substantiated by the products The formation of the four major fragmentation peaks in the mass spectrum of dihydrovindoline of acid cleavage (concentrated hydrochloric acid, (vide supra) not only indicated the ring system of stannous chloride, tin, refiux) carried out on the the alkaloid but showed that all the oxygen func four active dimenic alkaloids. In each case there tions other than the aromatic methoxyl must be was obtained upon chromatography of the reac located at carbons 3 and 4 (i.e., the peak m/e @98 tion mixture an indole compound (vide infra) , fol could not contain the ester, acetyl, or hydroxyl and lowed by vindoline derivatives (Chart 11). Vin

blastine (XV),9 vinleurosine, and vinnosidine af hydnoxy derivative, C42H5,N406 (XIX). This for forded desacetylvindoline, thus proving the iden mulation is consistent with the reduction of two tity of the dihydroindole portion of these alkaloids. methyl esters, one acetate and an N-fonmyl group The corresponding fraction from the cleavage of in the case of vincnistine. The N.M.R. spectrum vincristine (XVI) yielded des-N(a@-methyldesace of XIX shows, accordingly, only two methyl sig tylvindoline, M = 400, C@[email protected] mass nals, aromatic OCH3 at 3.88 and N-CH3 at @.73Ã¶. spectrum, as well as ultraviolet and infrared spec The N-CHO of vincristine is, therefore, at the tra, demonstrates the relationship of this com anilino nitrogen in place of the N-CH3 in VLB, and pound to desacetylvindoline. Both VLB and yin therefore vincnistine is des-N(a)-methyl N(a)formyl cnistine yield the same tetracyclic indole derivative VLB (57). velbanamine (XVII), C19H2@N2O, the infrared The mode of attachment of the indole portion spectrum of which clearly shows the oxygen to be of the dimenic alkaloids to the dihydroindole frag present as a hydnoxyl (57). ment must be through the C-is aromatic carbon, A related tetracyclic indole derivative, cleava since the N.M.R. spectrum shows only two protons mine (XVIII), C19H24N2, was obtained from the in a 1 :4 relationship in the aromatic portion of the cleavage of vinleurosine (the correct formula of molecule rather than the 1,@,4 three-proton pat vinleurosine is still in doubt owing to difficulty of tern present in vindoline (Charts 6 and 7). preparing a solvent-free sample) . Isolation of cleav From the data presented thus fan one can draw amine (XVIII) demonstrates the relationship of a fairly complete picture of the structures of the the indole portion of C46alkaloids to catharanthine dimenic alkaloids. Vincnistine and vinblastine are (I), since it was also obtained by a similar acid best represented by structures XV and XVI, ne treatment of the latter (@6, @7,@9).Mass spectral spectively.'Â° The point of attachment of the vindo evidence indicates that cleavamine has structure line portion as well as the location of the hydroxyl XVIII and that velbanamine is its dihydrohy in the catharanthine moiety is, as yet, obscure; droxy analog XVIII. Recently, the structure of however, on the basis of the biogenetic considera cleavamine has been unambiguously substantiated tions discussed below, we prefer position 4' for the by an x-ray structure determination (47). The hydroxyl group. An N.M.R. study of the fully structure of the tetracyclic indole derived from acetylated VLB shows that this hydroxyl is ten vinrosidine has not, as yet, been determined. tiary. The point of attachment of vindoline in the Thus, vinleurosine, vinrosidine, and VLB differ other two biologically active alkaloids is probably from one another only with respect to the catha the same as for VLB, but the nature of their oxy ranthine portion of the molecule, whereas VLB genation in this region of the molecule is, as yet, (XV) and vincnistine (XVI) differ only with re unknown and under investigation. @ spect to the vindoline portion. The nature of this The remaining alkaloids in Table can be classi latter difference was demonstrated in the following fled into three general groups on the basis of the way: chemical data available to date. Eleven of the A comparison of the empirical formulae of VLB alkaloids (Table @)are definitely of the dimenic (XV) and vincnistine (XVI) (Table @)shows that indole-indoline class and closely related to the four the former has one less oxygen and two more hy already discussed. These are listed in Table 3 The drogens than the latter. The U.V. spectrum of yin resemblance is seen in their infrared, ultraviolet cnistine is quite different from those of the other and N.M.R. spectra as well as the results of acid three dimeric compounds (Table @),and this dif cleavage of several of them. ference is indicative of a different substitution on The second group (Table 3) which includes ca N(a) of the dihydroindole moiety. The infrared thananthine and vindoline consists of ten mono spectra of VLB and vincristine (Chart 4) are simi menic alkaloids ranging in size from C18to C25corn lar, with the exception of the presence of a strong pounds. The known alkaloids of Table 1 are also @ additional band, S.94j@in the spectrum of all in this category. In addition to vindoline (@8), the latter. The N.M.R. spectra differ in that the catharanthine (56), and vindolinine (19) the struc N-CH3 proton resonance at @.73Ã´presentin VLB tune of lochnenidine (47) and vindonosine (54) (Chart7)ismissinginvincnistine;and,conversely,(desmethoxyl vindoline) have been described, and in place of only one low field proton 9.85 in VLB, partial structures of penivine (78), lochnenicine there are two in vincnistine at 9.5 and 8.9Ã¶. 10 The possibility of the indole portion being in the open Lithium aluminum hydride reduction of the two tetracycic form as in velbanamine (XVII) cannot be entirely alkaloids afforded good yields of the same penta excluded on the basis of analytical (57) or infrared data (58). Note Added in Proof: Recent mass spectral studies have a Chart 11 contains structures of dimeric compounds and shown that the formula of VLB should be CuH58O9N4, and derivatives. thus the indole moiety must be tetracycic as in velbanamine.

(31), sitsirikine (76), and lochneninine (54) have antileukemic properties. LiA1H4 reduction of yin been proposed. leurosine and VLB, yielding the corresponding The third group (Table 3) consists of dimenic carbinols (58), likewise destroyed activity. The hy alkaloids which are not of the indole-indoline type. drogenation of the double bond in these compounds Vindolicine (76) and vindolidine (75) appear to be also reduces activity.â€• dimers of vindoline, and vincanodine (75) seems to An interesting change in the spectrum of bio be related to the alkaloid vincine from V. minor logical activity of the active alkaloids was observed Linn. upon quatennization of the basic nitrogen in the With this background of knowledge concerning catharanthine moiety of these compounds with the chemical nature of the dimenic alkaloids our various alkyl halides (Table 4). Catharanthine (pK'a 7.5) readily forms a series of quaternary salts TABLE 3 from the appropriate alkyl halide in benzene at CLASSIFICATION OF THE ALKALOIDS room temperature, whereas the basicity of the FROM Vines rosea LINN. basic N (pK'a 5.5) in vindoline is such that it does @1Band Leurosine-like Alkaloids: not react under these conditions. In the case of 1. Vincaleukoblastine (Vinblastine) 2. Leurosine (Vinleurosine) vinleurosine the methiodide and ethiodides were 3. Leurocristine (Vincristine) 4. Leurosidine (Vinrosidine) TABLE 4 5. Isoleurosine 6. PER CENT PROLONGATION OF DIFFERENT 7. Carosine ALKYL HALIDES OF LEUROSINE AND 8. VBL IN P.1534LEUKEMIA 9. Catharicine 10. Neoleurocristine 11. Neoleurosidine ActivityAlkyl Monomeric Alkaloids: halides(% prolonga 1. tion)Leurosine:Methyl 2. Virosine 8. Catharanthine 4. Vindoline iodide 5. Lochnericine 64MethylMethyl chloride67-144 6. Vindolinine bromide41Methyl 7. Lochneridine tosylate 8. Sitsirildne Dimethyl sulfate 44 9. 128N-PropylEthyl iodide45 10. iodide33Ally! Miscellaneous Dimeric Alkaloids: iodide71Bencyl 1. Vindolicine bromide 2. Vindolidine Carbethoxymethyl bromide Inactive 8. Vincamicine 45VLB:Methyl@9-Hydroxyethyl bromideInactive 4. Vincarodine 5. Carosidine iodide attention was turned toward the effect of struc 25Propy!Ethyl iodide26 iodide tuna! changes on biological activity. One can con Dimethyl sulfate73 60 sider the four active alkaloids as minor modifica tions of one another, and, as will be shown below, each of these compounds has a somewhat different much more active than the parent base. With antitumor spectrum; as far as is known, they also VLB, vinrosidine, and vincristine, the quaternary exert a varying clinical spectrum and possess dif salts were considerably less active than the parent ferent toxic manifestations (vide infra). A number compounds.'2 These results will be described in of the dimenic alkaloids of Table 3 such as neoleu more detail below. It therefore appears that the rosidine, neoleurocnistine, and cathanine possess requirements for activity in the vindoline portion only one pK'. value (Table @),indicating that the of the molecule are much more rigid than in the basic nitrogen in the catharanthine portion of the catharanthine portion. A number of other deniva molecule has been changed, most probably by tives are currently under investigation. amide formation. None of these derivatives has The nature of the biogenesis of these interesting any activity. new dimeric compounds is of great interest. Wen It is apparent that all the alkaloids possess an kert and others have outlined detailed schemes for acetyl group at C4 (vindoline portion). Removal of the biosynthesis of many monomeric alkaloids of

this group destroyed antileukemic activity in VLB. 1@Private communication, Dr. N. Neuss, Lilly Research VLB contains two hydroxyls, whereas vinleurosine Laboratories.

has only one (58). Complete acetylation of these 12 Personal communication, Dr. G. H. Svoboda, Lilly Or hydroxyls yields the respective acetates devoid of ganic Development Laboratories.

the Iboga (catharanthine) on Aspidosperirta (yin A possibly analogous chemical reaction to the doline) types. These are formed by the interaction biochemical addition of an aromatic radical to a of an appropriately oriented unit with tryptophan, double bond is the Meerwein anylation reaction and it has been shown that tryptophan is an excel (67), in which the aromatic radical formed by de lent precursor for the Vinca alkaloids.'3 The non composition of a diazonium salt adds to the least tryptophan portion is thought to be derived from substituted position of a double bond according to either shikimic and pyruvic acids or, alternately, the reaction in Chart 1@. Further investigations from acetate via mevalonate to cyclopentane mon into the nature of this biosynthetic mechanism are oterpenoid intermediates. The details of these planned. schemes can be found in the paper by Wenkert (87), and we shall discuss only the method of at EXPERIMENTAL BIOLOGICAL tachment. This is most probably photochemical PROPERTIES since, as mentioned above, the major portion of A@rITtmzoR STUDIES the dimenic alkaloids are found in the leaves of the The most striking experimental biological effect plant. The first step can be visualized as an oxida of the four Vinca alkaloids under discussion are tion of vindoline to a free radical by a peroxidase their effectiveness in prolonging life or, in some type of enzyme followed by attack at the double cases, â€œcuringâ€•DBA/@mice given implants of the bond of catharanthine by this radical. Alternately, P-1534 leukemia. The procedures for antitumor

TABLE 5

AcTIvITY OF ORIGINAL EXTRACT OF WHOLE PLANT AND CRUDE FRACTIONS AGAINST P.1534 LEUKEMIA

cent cent wt. change survival increase in MaterialDosage indefinite time (days) TICPer survival . survivorsDefatted (mg/kg/day)Av. (gm) T/CAv. timePer

whole plant .0 1/+2 7 FractionA 0.5 â€”0.6/+1.2 27/19 38 20 FractionB 30.0 â€”1.2/+1.2 24/19 25 40 Totalalkaloids120 6.0 +0.8/+2.9 28/17 61 20 7.5 +0.2/+0.6 30/13 122 0 15.0 â€”2.3/+0.6 20/13 51 60 15.0 +0.3/+0.5 30/13 130 0 75(Oral)+0 â€”1.5/+0.626/15 21/1373 530 0

the vindoline may react in the phenolic state be @ AR-N2C( [AR.] H2-C=CR@ fore methylation, and the cathananthine may be already oxygenated at the double bond (hydnoxy! or epoxide).'4 The resulting intermediate could AR@CH2@CR2@X [AR-CHf CR2] then be reduced to a neutral molecule in various x ways (such as reaction with water) to yield the CHART 12.â€”Meerwein arylation reaction wide variety of dimeric products found in the plant. The large number of dimeric products speaks testing in this laboratory have been previously de for the addition step of the free radical not being scribed (4@) but essentially consist of intrapeni enzymatical!y controlled. The formation of the toneal inoculation of leukemic on ascites cells and N-CHO derivative of VLB (i.e., vincristine) is subcutaneous trocar implantation of solid tumors, analogous to the oxidation of various dimethylani with therapy being initiated the day following im lino compounds by horseradish penoxidase enzyme plantation and continuing for ten daily injections. and hydrogen peroxide where oxidative demethyl Effectiveness is described in terms of per cent pro ation occurs (p24). The peroxidase-catalyzed di longation over saline-treated control groups in the menization of aniline derivatives is known to yield case of leukemic and ascites-beaning animals and compounds similar to vindolicine (a vindoline per cent inhibition in terms of comparison of mean dimer). tumor diameters of treated groups to those of

13 Unpublished data, Drs. R. E. McMahon and M. Gorman, saline-treated controls in the case of solid tumors. Lilly Research Laboratories. The results obtained with the first extracts of

â€˜4This scheme resulted from a discussion with Prof. E. Vinca rosea in December of 1957 against the P Wenkert, Indiana University. 1534 leukemia are seen in Table S. From extracts

with this type of potency and with the P-1534 as TABLE 7 the bioassay, Svoboda obtained vinleurosine and ANTI-P-1534 ACTIVITY OF VINCA ALKALOIDS vinblastine. Results obtained with crude extracts (5 mice/group) chemically free of vinleurosine and vinblastine are seen in Table 6. From extracts of this type of cent i potency Svoboda obtained vincnistine and vinro MaterialDosage prolonga nite (mg/kg/day)ToxicdeathsPer sidine. A comparison of the anti-P-1534 activity of survivorsVinleurosine7 tionJndef the four compounds under discussion is seen in .5 Table 7. In this connection, it must be stated, 10.0 0 32 0 1125 0 41 0 however, that the response of the P-1534 leukemia 20.0 1 76 1 0Vinblastine0.05 150.0(Oral)0 00 460 TABLE 6 ANTI-P-1534 ACTIVITY OF AMORPHOUSFRAC 0.1 0 53 0 TIONS FREE OF VINBLASTINE 0.3 0 70 0 0.45 0 98 0 AND VINLEUROSINE 0.45 0 131 0 (5 mice/group) 0.6 1 150 0 0Vincristine0 1.5(Oral)0 041 700

Dosage 35 cent (mg/kg/ 0.30 2 ? S survivors3.0 deathsPer prolongationIndefinite day)Toxic 0.25 0 226 S 0.20 1 110 0.15 0 55 2 3.0 0 ? 5 0.12 0 49 0 3.0 2 ? 3 0.09 0 32 0 0.6 2 181 0 0Vinrosidine10 0.002 030 241 18.0 2 147 0 4.5 0 151 1 .0 9.0 1 147 2 7.5 0 ? 5 9.0 0 174 1 5.0 0 127 0 6.0 0 123 4 4.0 0 75 0 20.0 0 148 1 3.0 0 30 0 30.00 0? 2385 4 2.01 0? 214 0

TABLE 8 COMPARISON OF ANTITUMOR AcTivITY OF FOUR â€œACTIvEâ€•ALKALoIDs

TUMORHOSTALKALOIDS*ABCDLilly

mammary Sarcoma 180 CAP' â€” â€” â€” â€” Adenocarcinoma 755 C57BL/6 â€” â€” â€” + C-1498 leukemia C57BL/6 â€” â€” â€” â€” P-l534leukemia DBA/2 +++ ++ +++ +++ L1210 leukemia DBA/2 â€” â€” â€” â€” Ridgeway osteogenic sarcoma AKR â€” â€” +++ +++ Mecca lymphosarcoma AKR â€” â€” + + AKR leukemia AKR â€” + - ++ Ehrlich ascites Cox std. +++ +++ â€” â€” Freund ascites CAl?' +++ +++ ++ ++ S-180 ascites CAF@ +++ + +++ B82A leukemia C58 ++ + ++ +++ Walker carcinosarcoma 256 Rat + + + â€” Lilly mammary C3H Gardner lymphosarcoma CSH â€” â€” + â€” S-91melanoma DBA/1 â€” â€” â€” â€” X-5568 myeloma C3H N.D. N.D. + + High malignancy clone CSH N.D. N.D. â€” â€” Lilly rhabdomyosarcomaDBA/1 Rat+ +â€” â€”â€” â€”+ â€”

S A, vinbiastine; B, vinleurosine; C, vinrosidine; D, vincristine. + = 30-50% inhibition of solid tumors or prolongation of survival time in leukemias; ++ = 50â€”100% inhibition of solid tumors or prolongation of leukemias; +++ = 100% inhibition of solid tumors of > 100% prolongation of leukemias; N.D. = not done.

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1963 American Association for Cancer Research. JOHNSON et al.â€”Vinca Alkaloids 1405 as maintained in our laboratories has changed in greater therapeutic effect in the combination then response to vinleurosine and vinblastine since 1958â€” apy. Combinations of the various active Vinca 196@2,but the response to vincristine and vinrosi alkaloids themselves gave no evidence of any dine during the period 1960 to the present has greater activity in this tumor system either, with remained unchanged. Vinb!astine currently gives the possible exception of vinrosidine (Table 9), a lower order of activity (50 per cent prolongation) which seems to be additive with both vinleurosine at a maximum tolerated dose, whereas vinleurosine and vincristine but not vinblastine. gives a somewhat greater and certainly more uni In a different tumor system, the B8@A leukemia, form response than previously. Unfortunately a there was a suggestion of additive effect with yin â€œtumorbankâ€• was not available to us during this leurosine and vincristine under certain regimens of

TABLE 9

COMBINATION THERAPY OF THE P-1534 LEUKEMIA WITH VINIt0SIDINE AND OTHER VINCA ALKALOIDS (5 mice/group)

wt. cent surv. CompoundDosage change prolonga . survivorsVinrosidine (mg/kgX 1 X 10)Av. (days)Per (gm)Av. tionIndefinite

(VRS) 750 9 Vinleurosine (VIAl) 15 .000 +1 .2 18 29 0 Vinblastine (VLB) 0.200 +1 .5 22 61 0 Vincristine (VCR) 0 125 +0 .4 24 74 1 VRS+VLR Asabove â€”0.8 31 128 0 VR@S+VLB â€˜4 a â€”0.6 23 69 3 VRS+VCR U ii+0 29 1090 3 Saline3 â€”0.219 1437 0

TABLE 10 EFFECTS OF DELAYED TREATMENT WITH VINCRISTINE SULFATE ON P-1534

Treatment started surv. cent pro (dayssurvivors1 after Xfreq.Av. (days) T/CToxic deathsPer longationIndefinite implantation)Mg/kg

3 025X1X10 28/14 3/5 108 0/5 6 0.25X1X10 33/14 0/5 146 4/5

1 0.25X1X10 30/15 0/10 93 2/10 1 2.5X1 26/15 3/10 70 4/10 3 2.5X1 23/15 3/10 81 0/10 6 2.5X1 31/15 0/10 98 0/10 100.2SX1X1O2.5X1?/14 33/153/5 0/10? 1122/5 1/10 period, and we are unable to check sensitivity ret therapy. The two regimens in which this sugges roactively. tion might be made was by giving one compound The four â€œactiveâ€•alkaloidshave similar expeni daily for a week followed by a week's therapy with mental tumor spectra but with some significant the other compound, and secondly by giving one differences (Table 8). The most striking difference compound in the A.M. and the other in the P.M. in spectrum is the activity of vinnosidine and yin In the same system vinblastine and vincristine cristine against several AKR tumors. were not additive. A number of studies have been made expeni Vinbiastine has previously been shown to be mentally of combining vinbiastinc or vincnistine effective against the P-1534 leukemia when thera therapy with therapy with other â€œstandard,â€•use py was delayed for several days after implantation ful antitumor compounds against the P-i534 icu (4@). Vincnistine is also effective in this manner kemia. These studies will be described in some (Table 10), More remarkable, perhaps, was the detail elsewhere, but in all cases there was a no fact that â€œcuresâ€•orindefinite survivors could be ticeable lack of any evidence for potentiation or obtained when therapy was initiated late in the

disease. These animals were proved to be free of B-8174 leukemias and to a lessen extent in the leukemia by passage of sp!eens of such â€œcuredâ€•B8@. He has also observed a peculiar relationship animals to normal DBA/@ mice. These animals in terms of cross-resistance between vincnistine remained free of disease but were not resistant to and certain compounds in sensitive and resistant a challenge with P-1534 cells. A typical experi lines of these leukemias (10) . Vincnistine was mac ment is seen in Table 11. tive in P-815 and P-388 leukemias resistant to Resistance to a new challenge to P-1534 cells NSC 38@80 but is not additive to this compound after â€œcuresâ€•has been seen, however, and de in a sensitive line. NSC 38@80 is potentiated in serves some comment in reference to the Vinca these systems by stilbamidine, tniparanol, and alkaloids in general. This resistance, in our hands, amphotericin, whereas vincnistine is not. Cross has not been a regularly predictable phenomenon. resistance of these compounds is difficult to inter It is our impression that this occurs most fre pret in view of the obvious differences in chemo quently in â€œsurvivorsâ€• obtained by delayed treat therapeutic behavior. Burchenal has also observed cross-resistance of vincnistine and vinblastine in a TABLE 11 line of P-815 made resistant to vinblastine. PASSAGE EVIDENCE FOR â€œCURESâ€•OFYIN Dr. Dornis J. Hutchinson'6 has observed a CRISTINE-S04-TREATED ANIMALS marked effect of vinb!astine on several lines of L1@10 leukemia resistant to amethopterin, 6-MP, surviving surv. 5-FU, and all three compounds (Table 1@). These GroupNo. in groupNo. (days)A at 40 daysAv. time TABLE 12 with with 11.5 B 4@, spleensfrom 4@, P-l534leuke EFFECT OF VINBLASTINE ON RESISTANT C @i â€œcuredâ€• 4 [email protected] LINES OF L1210 D4)Challenged4) P-lS34animals@)ChaIIe@@@ed4J miacellS.2 12.5

Four groups of normal DBA/2 mice were given intraperito . Relative . genera centric neat injections of suspensions of spleen cells from â€œcuredâ€•ani Lines of L1210Transfer . effect tion chromo mals. A!! survived until 40 days, when they were challenged VLB5L1210 of with P-1534 leukemia cells indicating their susceptibility, if numberSubtelosome. these cells had been present in the spleens of VCR.S04 â€œcuredâ€•animals. V, parent line L1210/MP ifi Present 1 L1210/A IV Absent 0 ment rather than by therapy initiated soon after L1210/FU Xlii Present 3 implantation. It should be pointed out that Noble L1210/AMPFUXYI2 Present 4 and his associates have also reported resistance in 79 Absent 1 â€œcuredâ€•animals (17). They reported resistance in BDF, hybrid mice â€œcuredâ€• of P-i534 leukemia L1210/AMPFU XYI2, frozen 38 Present 4 and subsequently re-challenged with P1534 cells. 73 Absent 4 We previously reported (39) our inability to repeat L1210/AMPFU XVI 64 Present 4 this phenomenon but suggested the probability of 97 Present 4 tissue incompatibility. Goldin (@5) has reported L1210/AMPFU XYI4, frozen49 41 Present 4 a similar phenomenon with halogenated deriva 60Present Present1 4 tives of amethoptenin in the case of L1@i0 leu kemia, but again this was in an F1 hybrid and did S New code designations: 0 = inactive; 1 = slight activi. ty; 2 = active; 3 = <6 â€œcuresâ€•;4â€”>5 â€œcures.â€•Forcorn not occur in the inbred DBA/@ animal. Noble also parison, the old code designations were: â€”, <24% increase reported â€œcuresâ€•andsubsequent resistance in a in survival time; Â±, 25â€”59% increase in survival time; +, transplantable AKR leukemia in AKR mice. The <60% increase in survival time. curing and subsequent resistance to re-challenge of a strain-specific tumor in the inbred host is lines are characterized by the presence or absence unique in experimental chemotherapy though not of a subte!ocentric chromosome. The presence or unusual in nonstnain-specific tumors in random absence of the chromosome appears to have little bred animals. to do with sensitivity to vinblastine but in general Studies in other laboratories have revealed in it does appear that the more compounds the van teresting data on resistant lines of mouse neo ous lines are resistant to, the greater the sensitivity plasms. Burchenal16 has found vincnistine to be to vinbiastine. Similar studies with vincnistine are effective by several routes in the P-388, P-815, and in progress in Dr. Hutchinson's laboratory.

â€˜SPersonal communication, Dr. Joseph Burchenal, Sloan 10 Personal communication, Dr. Dorris Hutchinson, Sloan Kettering Institute. Kettering Institute.

ToxIcoLoGY Toxicity studies with vincnistine have been con Pharmacology and toxicological studies of these ducted in rats, rabbits, cats, dogs, and monkeys alkaloids in laboratory animals have not been pub with the following results: lished in detail. Vinblastine and vincristine have Rats.â€”Intravenous doses administered 5 times been the most extensively studied by Anderson weekly caused leukopenia, weight loss, and re and his associates.'7 Acute intravenous LD50's for duced food intake in direct relationship to the yinblastine and vincristine in mice are approxi doses, which were 0.5 and 0.@5 mg/kg. Rats re mately 17.0 and @.0mg/kg, respectively. Multiple ceiving 0.@ mg/kg daily by the intraperitoneal doses of vinblastine and vincnistine caused marked route survived 10â€”@1doses. They developed leukopenia in rats and dogs at 0.50 and 0.1 mg/kg, leukopenia, weight loss, diarrhea, alopecia, and respectively, causing death in some instances. blanching of tails, ears, and feet. Clonic extensor Dogs have tolerated daily doses of @.0mg/kg of convulsions and catalepsy occurred before death vinleurosine with slight reversible leukopenia but in one animal. few other toxic effects. A daily dose of 5.0 mg/kg, Rabbits.â€”Intnavenous doses given S times week however, was eventually lethal. Neither vincnistine ly caused leukopenia and death within three to nor vinleurosine is as potent a producer of leuko five doses at levels of 0.1 and 0.@ mg/kg. Before penia in animals as vinblastine. death, signs of muscular weakness (head drop, An interesting phenomenon with vinblastine is kneeling and sprawling positions) appeared. Ani the markedly different LD50 by intravenous and mals receiving 0.0@5 and 0.05 mg/kg appeared intrapenitoneal administration in mice (Table 13). normal after @8doses. The mouse tolerated larger doses of vinblastine TABLE 13 than did the rat or the guinea pig. Subacute toxici LDHOFVINBLASTINEINMICE ty studies in rats given various doses of vinblastine BY DIFFERENT ROUTES revealed loss of weight, with reduction in food intake. The degree of leukopenia produced bone a Intraperitoneal Â±0.02 mg/kg direct relationship to the size of the dosage, and, Intravenous 17 .47 Â±1.57 mg/kg with sublethal doses, the leukocyte count returned Oral3.2 33.12Â±6.03mg/kg to normal after dosage was discontinued. Thirty rats that survived repeated !eukopenia-pnoducing doses of vinbiastine were found to be normal at Cats.â€”The animals received the drug by the necropsy. intravenous route 3 times weekly. Those given 0.@ Given intravenously in leukopenia-producing mg/kg were dead after two doses. A cat adminis but sublethal doses to dogs, vinblastine was found tered 0.1 mg/kg was killed after two doses because to cause a reversible reduction of the myeloid it was moribund. When 0.05 mg/kg was given to components of the bone marrow. one animal, it died after eight doses. A cat receiv The main pathological finding in dogs given ing 0.0@5 mg/kg was killed after thirteen doses because of its extremely poor condition. Weight purposely lethal doses was hypoplasia of the bone loss, general debilitation, clonic convulsions, and marrow. Of lesser prominence and consistency were scattered petechial hemorrhages in other or leukopenia were observed. gans, with slight hepatitis and entenitis. Hypen Dogs.â€”Vincnistine was administered by the in plasia of the marrow and extnamedullany hema travenous route 5 times weekly. The dogs given topoiesis were found at necropsy in those dogs that 0.05 mg/kg were dead or had been killed after the were given courses of vinbiastine in sublethal doses fifth dose. One animal receiving 0.O@5mg/kg was and that were not sacrificed until after recovery killed after seven doses because it was in poor con from the !eukopenic effects of the drug. dition. Three animals survived twenty doses at this level. Leukopenia, tremors, vomiting, weak The cause of death in animals following lethal ness, and bloody stools were observed in these ani doses of vinb!astine was bacterial disease second mals. any to leukopenia. Death did not occur immedi Monkeys.â€”Vincnistine has been administered ately after the dosage but was delayed until infec orally in solution (1 mg/mi) to two male monkeys tion had occurred. Potentiation of phenobarbital @ at dosage levels of and 4 mg/kg, respectively. anesthesia in dogs and prolongation of hexobarbi Hematological and blood chemistry studies on tal â€œsleepingtimeâ€•in mice with the use of vinblas each monkey were essentially unchanged through tine have been observed. out a @9-dayobservation period. Vomiting was

17 Personal communication, Dr. R. C. Anderson, Lilly Re observed in the animal that received 4 mg/kg. search Laboratories. A single intravenous dose of 4 mg/kg killed one

male monkey in 30 hours. Constricted pupils, ano These effects, as well as mild to moderate degen nexia, and diarrhea were observed prior to death. erative changes in the liver and kidney, were re Reduced hematocrit reading, leukocytosis, and lated to dosage. An unusual reaction noted was elevated blood sugar, blood urea nitrogen, and proliferation of the epithelium of the renal collect serum transaminase were also observed. The major ing system. pathological finding was a severe acute cytotoxic A recent observation has made been in this effect on the granulocytic and erthnocytic series in laboratory in reference to host protection of mice the bone marrow. to lethal doses of vincnistine (Table 14). In normal Two male monkeys survived two doses of yin mice folinic acid has a protective effect, whereas @ cristine, mg/kg each, administered 36 days apart. folic does not. Neither folic nor folinic appears to Vomiting, weakness, tremors, weight loss, leuko be involved in the anti-P-1534 activity of vincnis penia, and thrombocytopenia were observed in tine. Indeed, rather than reversing the antitumor both animals. Decrease in blood sugar was noted effect both compounds given with vincristine ap pear to give somewhat better antitumor effects TABLE 14 than vincnistine alone (Table 15). EFFECT OFFOLIC ANDFOLINIC ACIDS ON YCR ToxICITY In view of this host protection phenomenon, we have investigated the effect of vincristine and yin Dosaoz (un/au) blastine on both folic acid reductase and fonmyl tetrahydrofolate synthetase in vitro. The enzymes survival were assayed by either a crude homogenate or par time and VincristineFolic acidFolinic acidAv. days2.5X1 range at SO TABLE 15 (days)Mortality EFFECT OF F0LIc AND FOLINIC ACIDS ON I.Y. (2â€”26) VCR THERAPYOFP.1534 I_P.14OX1 7 (2â€”19) 16/18 (5 mice/group) a52X1X10 X10 I.P.11 7 (4â€”10)17/194/16 Dosaux(uo/aoX1X 10)

in one of the animals. Pathological studies after su@ cent vival sacrifice revealed hypoplastic marrow infiltrated VincristineFolic prolonga . survivors0.2 acidFolinic acidAv. time by lymphocytes. The acinan cells of the pancreas tioiiIndefinite (days)Per were unevenly depleted of secretion granules. Widespread atrophy of the mucosa of the large 0.2 ? ? 5 intestine occurred in one monkey. There were mild 0.2 2.3 77 4 toxic manifestations in the liven and kidney and 13* 0 interstitial edema and adipose infiltration in the 0.2 21 38 4 myocardium of the left ventricle of one monkey. 0.250 140 6 0 1 Eight monkeys (one male and one female per 50140 ? ? 5 140 22 41 3 dosage level) were given repeated doses of vincris 5085 ? ?0 5 tine intravenously at levels of 0.1 mg/kg/day, 0.@ 15*172 0 mg/kg/day, 1 mg/kg twice weekly, and 1 mg/kg weekly. Genera! body weakness, anorexia, tremors, S Saline controls. diarrhea, and ataxia were noted in all groups. No deaths were observed at the 0.1 mg/kg/day dosage tially purified fraction from mouse liver. Folic acid level, but death did occur after seven on twelve reductase activity was measured by the procedure doses of 0.@ mg. vincnistine per kg. daily, two or of Werkheimer et a!. (88) and formyl tetrahydro three doses of 1 mg. vincnistine pen kg. twice week folate by the method of Rabinowitz and Pricer ly, and five doses of 1 mg. vincnistine per kg. (64). Neither alkaloid has any influence on either weekly. of these enzymatic activities in vitro at concentra Leukopenia was generally observed. No major tions of drug up to 100 @g/ml. blood biochemical changes were noted except ten minal blood values from moribund animals and an BIOCHEMICAL AND MECHANISM STUDIES occasional elevation of serum transaminase. Since the alkaloids of Vinca rosea comprise a Autopsy of these monkeys revealed no charac new class of antitumon agents, it was of consider tenistic gross findings. Histological studies showed able interest to us to study the effect of these com reduced celluanity of myeloid and lymphoid tissue. pounds on various biochemical reaction sequences

with the hope of uncovering information concern In intact ascites cells (Table 16), either 8-180 ing their mode of action. or Freund, concentrations of less than 50 ,@g/ml Our initial experiments were devoted to a study vincnistine appear to have no effect on respiration. of the effects of vinb!astine and vincristine on the However, when the concentration of drug is raised rate of respiration and glycolysis of tumor cells to very high levels some inhibition appears. Al under various conditions. S-180 ascites cells were though data are not presented for vinbiastine, es chiefly employed ; however, some studies were per sentially the same results were obtained (10â€”iS formed with Freund ascites cells and also spleen per cent inhibition with 100 @&g/mi).In Table 17 tissue from normal mice and mice carrying P-1534 leukemia. The results of experiments in this area TABLE 18 are summarized in Tables 16 to 19. Each table GLYCOLYSIS OF S-180 AND FREUND ASCITES represents the results of at least two separate ex Cr@LLsIN PRESENCE OF YIN peniments. CRISTINE (50 @iG/ML) TABLE 16 PRODUCTION @ RESPIRATION OF S-18O AND CONTROL)Aerobic- FREUND ASCITES CELLs CaLL TYPETiua (MR.)LACTATE AnaerobicS-180 uptake control)S.180Cell typeAddition of VCR (% (pg/mI)0, 2â€”4 98 101 4â€”8 96 98

10 100 Freund0â€”2 0â€”2 99 97 50 98 2-4 98 99 200 85 4â€”8100 102100 96

Freund0 0 100 10 101 Glycolysis measured as lactate production. 50 99 Krebs-Ringer bicarbonate buffer containing 200100 89 0.2% glucose was employed. Incubation was at 370 C. with 5% CO@-95% 02 or 5% CO@-95% N3 as gas phase. Respiration measured by standard Warburg technic over 4-hour period. TABLE 19 Krebs-Ringer phosphate buffer contain ing 0.1% glucose used with air as atmos ANAEROBIC GLYCOLYSISH0M0G- phere. ENATE OF S-iSO ASCITES CELLs TABLE 17 Addition RESPIRATION OF S-iSO HOMOGENATE cent of VLB control15 AND SPLEEN MINCE (jig/mi)Per

uptake 150103 94 control)NormalCellAddition of VLB (% (jig/mI)@â€˜ Anaerobic glycolysis measured spleen as lactate production. Medium em 20.5 98 ployed was essentially that of La 41.0 102 Page (48).

Leukemic spleen5 0 100 (P.1534) 20.5 99 it can be seen that vinblastine was without effect 41.0 95 on the respiration of spleen mince from normal or leukemic animals. In addition, the complete lack S-iso Ascites cellst0 0 100 100100 99 of influence of vinblastine, at a relatively high con centration, on homogenates of S-iSO ascites cells is noted. This latter result is in contrast to the re S Spleen from DBA/2 mice. Respiration measured with mince in Krebs-Ringer phosphate suits obtainedwith intactcells, since corresponding buffer by standard technic over period of 30 ly high levels of vinblastine do give rise to a iOâ€”i5 minutes. t S-180 homogenate prepared in 0.25 M su per cent inhibition of respiration. In Tables 18 and crose. Medium employed was essentially that of i9, the inability of vinblastine and vincnistine to Aisenberg (1). Results are similar whether suc affect the rate of glycolysis as measured by lactate cinate or pyruvate and fumarate are used as sub strate. 02 uptake measured at intervals over a production is shown. 1-hour period. To summarize our results, it can be said that we

observe neither inhibition nor stimulation of nes The results of an experiment in which animals pination or glycolysis in the presence of these alka were pretreated in vivo with drug is given in Table bids at levels approaching physiological concen @-Twenty-four hours after treatment the ascites trations. At very high concentration values, we cells were removed and incubated in vitro with begin to obtain inhibition. However, we would not consider this particularly meaningful. Our results TABLE 21 agree with those of Beer (6), who measured the GLYCINE-2-C'4 INCORPORATION respiratory quotient of liver from animals treated IN S-180 ASCITES with yinblastine, but differ from those of Hunter,'8 who reported that vinbiastine stimulates aerobic (DPM/uu)RNAACTIVITY acid production and inhibits nespiration. However, ADDITIONTIME (uz@.)Spac. Hunter employed different cell lines so that a di @DNA0 rect comparison with our data is not possible. The pronounced effect of vinblastine on mitosis 90 39,600 68,400 led us to studies on the biosynthesis of nucleic 270 40,000 72,400 acids. We have measured the incorporation of 10@gVCR/ml 30 15,900 24,600 fonmate-C'4 and glycine-@-C'4 into the nucleic acids 90 37,200 69,100 of 5-180 ascites cells in vitro. In Table @0theresults 270 38,700 73,300 of a typical experiment of this type are shown. In 20 @gYLB/ml30 30 14,600 26,000 general, washed 6-day ascites cells were employed 90 36,300 66,100 27016,500 38,60027,400 61,600 TABLE 20

FORMATE-Câ€•INCORPORATION Incubation conditions: 1 ml packed S.180 ascit.es/15 ml IN S-180 AScITES Krebs-Ringer phosphate buffer containing 0.2% glucose; 0.2 @sc(12.5 ,.@g)glycine-2-Câ€•/ml.

(DPM/MG)RNADNA0 ACTIVITY TABLE 22 ADDITIONSpac. GLYCINE-2-C'4 INCORPORATION IN 8-180 AsCITES

ACTIVITY 1 zg YCR/ml 14,600Â±780 4,610 Â±280 (DPM/uo)RNADNA50@gYLB/ml5 1OOj@gVCR/ml 18,300Â±670 4,560Â±230 ADDITIONINCUBATION TIME 20 j@gVLB/ml14,800Â±74018,800Â±6904,520Â±2804,470 Â±280 (MIN.)SPECIFIC

Final concentration: 0.2 @il.packed in 2.5 ml. total volume Krebs-Ringer bicarbonate buffer containing 3.5 gig. (0.3 iic.) 90 1,470 3,290 C'4-formate/ml; 5% CO,-95% N3 gas phase. 25 @gYCR/mlt 30 1,380 2,570 with Krebs-Ringer bicarbonate on Krebs-Ringen 90 1,500 3,020 phosphate buffer containing Odâ€”0.4pen cent glu 10@sgMTX/ml@ 30 1,010 2.090 cose. 90 1,280 2,640 After 60 minutes incubation at 37Â°C., proteins Saline30 30 1,270 2,570 and nucleic acids were precipitated with perchlonic 901,270 1,5902,6603,120 acid and determined after separation by the meth od of Scott et a]. (68). The results shown in Table a Cells from group of six mice treated 24 hours earlier with @0indicate that neither vincristine nor vinblastine 2.5 mg/kg VCR. has any measurable effect on the incorporation of t Cellsfromgroupofsixmicetreated24hoursearlierwith 5.0 mg/kg VLB. formate into nucleic acid in 5-180 ascites. @ Cells from group of six mice treated 24 hours earlier with Table @ipresents the results of a similar expeni 1.0 mg/kg methotrexate. ment in which incorporation of glycine-@-C'4 was Incubation: 0.3 ml. cells (packed) in 2.5 ml. Krebs-Ringer used as a measure of nucleic acid formation. Again phosphate buffer containing 0.1 mmole amino acids (all except glycine), 1 rnmole glucose, and 0.9 @c.(22.5 gig.) glycine-2-C'4. vinblastine and vincristine demonstrate no appre Incubated at 37Â°C. in 02 atmosphere. ciable effects. In this particular experiment, aeno bic conditions were employed. However, anaerobic glycine-@-C'4 under aerobic conditions. As in the experiments with glycine-@-C'4 as substrate gave previous experiment, vinblastine and vincnistine similar results. have no effect, whereas methotrexate treatment

18 Personal communication, Dr. J. C. Hunter, National depresses the incorporation to a significant extent. Cancer Institute. Very recently, however, some preliminary ne

sults have been obtained with HeLa cells in tissue dence of cross-resistance between them (3, 11, 14). culture experiments, lasting over periods of 1 week, The dosage requirements of vinblastine and yin which suggest that vinblastine and possibly also cnistine differ markedly (3). Whereas the weekly vincristine may act as inhibitors of de novo syn intravenous dose for the average solid-tumor pa thesis of nucleic acids. These studies, if confirmed, tient lies between 0.10 and 0.@0 mg/kg of vinbias will be reported elsewhere in detail. tine, the dose of vincnistine is only one-tenth to The capacity of 8-180 cells to incorporate gly one-quarter of this (i.e., 0.01â€”0.03mg/kg). cine-1-C'4 into protein is presented in Table @3.At Both vinblastine and vincristine are alike in relatively low concentrations of vinblastine, no ap sparing hemoglobin and platelet production (3, 4, preciable effect on protein synthesis is noted. At ii, is, 33, 44, 69, 81, 84, 89). The limiting factor a higher concentration, however, some inhibition to dosage with vinblastine is leukopenia (31, SO). becomes apparent. In the second half of Table @3, With vincnistine, on the other hand, leukopenia is the influence of vinblastine on the capacity of a uncommon; and when it has occurred its degree cell-free microsomal system to synthesize protein and duration have generally not constituted a seni was tested. Again no large differences are to be ous problem (3, ii, 13, 44, 45, 66, 69). found between the treated and untreated samples. TABLE 28 In agreement with the results reported by Beer (6)fornucleicacidsynthesisinregeneratingliver INCORPORATION OF GLYCINE-1-Câ€• and Ehnlich ascites, we conclude from our data INTO PROTEIN in Vitro that neither vinblastine nor vincnistine influences the rate of nucleic acid formation of purine syn Exper.pg VLB/miliiliterCounts/mm/mg thesis in S-180 ascites. Furthermore, we can find proteinA* no effect by these agents on protein biosynthesis at relatively low concentrations of drug. The ne 50 150Â±6 150 124Â±5 sults of all of our experiments tend to follow the 0 168Â±8 pattern on little of no activity in vitro at concen tnations approaching physiological levels; whereas Bt10 10 176Â±6 100 166Â±5 we begin to observe inhibition at very high con 0160Â±7 172Â±6 centrations. It should be pointed out that our cx

periments reported in this paper were carried out S Intact S-18O ascites cells incubated in with chiefly a single line of ascites tumor cells in Krebs-Ringer bicarbonate buffer at 37Â°C. for 30 minutes with varying concentrations of VLB vitro. One must consider the possibility that in and glycine-1-C'4. vitro activity cannot be equated with in vivo activi t Microsomal system derived from S-iSO ty. That is, the injected alkaloid may be modified ascites cells. Glycine-l-C'4 incorporation meas in some way by the host to provide an â€œactive ured essentially according to the system of Littlefield and Keller (49). component.â€• With very high doses of vinblastine, manifesta CLINICAL STUDIES WITH THE tions interpreted as central nervous system toxici VINCA ALKALOIDS ty has been encountered (@0,31, 43) ; but this This clinical discussion of the Vinca alkaloids is occurs only rarely, and then only if dosage is in confined to vinblastine sulfate and vincnistine sul creased above the leukopenia-producing magni fate, which are the only members of this family of tude. In contrast, at least some neuromuscular drugs which at this time have undergone extensive abnormality has been encountered in most patients clinical trial. Before a detailed discussion of yin who have been treated with vincnistine, and it is blastine and vincnistine individually, we will brief this problem rather than leukopenia which limits ly compare and contrast the more salient clinical the magnitude of vincnistine dosage (3). Neuno features of both. muscular toxicity apparently may also involve the Both vinblastine and vincnistine have been autonomic system. Indeed, severe and even ob found to be useful in the treatment of some types structive constipation may develop with chronic of human cancer. There is no clinical evidence of vincnistine dosage if stool-softening agents and cross-resistance with radiation or with other pres lubricants are not given to prevent this. Constipa ently used oncolytic agents (4, 9, ii, 13, 14, @0, tion so rarely follows vinbiastine dosage that there 3@,44, 45, 80, 81, 85). Despite only a minor differ is no need to medicate prophylactically on its ac ence in their chemical structures, the clinical ef count. fects of vinblastine and vincristine differ consid In our experience, epilation occurs less frequent erably ; and, surprisingly, there is no clinical cvi ly with vinblastine than it does with vincnistine

approximately 10 per cent of patients being af Indeed, some centers now use vincnistine first fected by the former and perhaps twice this num in the series of drugs they employ for the treatment ber by the latter drug. of acute childhood leukemia because vincnistine Both vinblastine and vincnistine have proved is unlikely to exacerbate the neutropenia, anemia, clinically useful in the treatment of Hodgkin's and thrombocytopenia which may be present in disease and the other lymphomas including lym acute leukemia even before treatment is begun.2Â° phosarcoma (3, 4, 9, ii, 14, @0).Effectiveness has Overlap in the spectra of activity of vinblastine been shown not only when given as de novo therapy and vincnistine has been reported to involve a van but also following development of refractoriness to ety of neoplasms including carcinomas of the alkylating agents and/or radiation. Although it breast2' (4, 43, 84), ovary (4, @0,69,79, 80), kidney remains to be determined which of the two drugs (ii, 33, 79), and cervix (4, 13, 14), chonionepithe will prove more effective in these situations, it is lioma (13, 14, 3@), nhabdomyosarcoma (3, 79), the early impression of the Lilly Clinic group that reticulum-cell sarcoma (14, 33, 84, 89), and neuro vinblastine may be better than vincnistine for blastoma (ii, 69, 79). As is the case with Hodg Hodgkin's disease, whereas vincristine may be kin's disease, comparison between the effectiveness more useful in lymphosarcoma (3, 4). However, of vinblastine and vincnistine against these tumors clinical studies with comparable groups of patients must await further trials as well as a better under in the same stages of their illness will have to be standing of administration and dosage. carried out to determine the correctness of these Vinblastine has been reported to produce tumor impressions. regression in patients with metastatic adenocanci The leukemias constitute another area in which noma of stomach (4, @0,84), colon (34), and rec @ it is of interest to note apparent differences in ne tum (35). Vincristine has thus far not produced sponses to the two drugs. Although published pa significant benefit in any patient with adenocar pens report remissions with vinblastine in the leu cinoma of the gastrointestinal tract. Vincristine kemias as relatively few in number and often has been reported to have some useful effect in a incomplete, in some patients the drug has been single patient with carcinoma of the prostate (13, reported of definite benefit following development 14). Similarly, benefit has been reported with yin of resistance to other agents. Warwick et al. (84) blastine in isolated instances of carcinoma of the reported four of nine patients with acute stem-cell prostate.'9 Obviously more patients with carci leukemia responding to vinblastine. Whitelaw and noma of the prostate should be included in trials Teasdale (89) report eighteen cases of acute blast of these drugs. cell leukemias treated with vinblastine. Of these, three of eight patients without previous therapy, VINBLASTINE SULFATE and four of ten resistant to previous therapy, mani Vinblastine is the official generic name for the fested favorable response for varying periods of alkaloid formerly known as vincaleukoblastine. time. (One of the latter group of four patients re This contraction was necessitated by the U.S. Phan sponding had simultaneous 6-mencaptopunine then macopeia regulation that a generic name should apy.) Hill and Loch (33) included reports on the not imply a therapeutic use. The earlier name was use of vinblastine (often combined with other felt to suggest usefulness in leukemia. agents) in the leukemias and concluded there was Vinblastine was made generally available in sufficient benefit to warrant further clinical trial. 1961, with recommendations for its use in Hodg The Acute Leukemia Cooperative Group B (44, kin's disease (4, 14, @0,@i,84, 85, 89) and chonio 45) obtained remissions in only one of 2.9 children carcinoma (3@,85). Since then it has been reported with acute leukemia. Details of this study have not to be active against many other malignancies in as yet been published. cluding carcinomas of the lung (4, 33, 50, 81, 90), On the other hand, vincnistine has frequently breast (4, 43, 84), cervix (4), uterus (35), ovary been found useful in the leukemias, especially the (4, 80, @0,81, 90), stomach (4, @0,81, 84), colon acute leukemias of children. Karonet al. (45), using (33, 34, 81, 90), rectum (35), and kidney (33, 8i, vincnistine, obtained a complete remission rate in 90), squamous-cell carcinomas@ (33, 81), nhab children with acute leukemia at least as good domyosarcoma (89), melanoma (37, 90), seminoma as with any of the presently accepted standard 20 Personal communication, Dr. Sidney Farber, Children's agents (6-mencaptopurine, steroids, on methotnex Hospital and Dr. Emil J. Freireich, National Cancer Institute. ate). Other investigators have verified the useful 31 Personal communication, Dr. James F. Holland, Roswell ness of this agent in acute leukemia in children.'9 Park Memorial Institute.

19 Data received at the Lilly Laboratories for clinical re 13 Personal communication, Dr. Donald Rochlin, University search. of California, Med. Ctr., Los Angeles.

(84), tenatoma (84), and embryonal-cell carcinoma a new oncolytic drug cannot be properly evaluated of the testis (37), reticulum-cell sarcoma (33, 35, until the optimal magnitude and duration of dos 36, 51, 84, 89) monocytic leukemia (34, 36, 37, age are ascertained. Needless to say this requires 81), erythroleukemia (36), astrocytoma (4, 5@,91), study over a considerable period of time. mycosis fungoides (33, 51). An interesting example of what might be accom Delay in recognition of this broaden spectrum is plished without significant side effects, with use of in part due to the short duration of vinblastine an incremental approach to dosage, is suggested in treatment which was used in earlier trials. In the preliminary report of J. C. W. MacFanlane et many of the early trials, vinblastine therapy was al. (50). This group reported that vinblastine had discontinued if no tumor shrinkage had occurred prolonged the survival of a series of patients with @ after weeks of dosage. It is now evident that in carcinoma of the bronchus as compared with that the case of solid tumors a response to vinblastine of untreated patients or patients treated by radia may occur only after 10â€”if weeks of therapy (4, tion and/or nitrogen mustard. Although some of 34, 43). This is in contrast to the rapid response these patients whose survival was significantly which occurs in cases of Hodgkin's disease and prolonged exhibited tumor shrinkage, others mere choniocancinoma, where benefit usually follows the ly showed an unchanged radiological picture first few doses of vinblastine. throughout their several months of treatment. Not only were some early trials too brief, but This suggests that to require prompt demonstra large and toxic doses were often employed. Pro found leukopenia and other severe side-effects re TABLE 24 sulted from â€œpressingdosage to toxicity.â€• We pre RESPONSE OF CARCINOMA OF BRONCHUS TO YINBLASTINE fer a careful incremental approach to dosage. We believe a patient's dosage with vinblastine may be TumorObjective improve improve established as follows: 1st dose, 0.10 mg/kg; @d responseBronchus: dose, 0.15 mg/kg; 3d dose, 0.@0 mg/kg; 4th dose, mentSubjectivementNo 0.@5 mg/kg; and 5th dose, 0.30 mg/kg, etc. Squamous-cell One intravenous dose every 7 days is given ac Adenocarcinoma 1 1 1 Undifferentiated 1 cording to this schedule until the leukocyte count Oat-cell 11 is reduced to approximately 3,000 cells per cu. Not specified2 17 5 mm. The amount of vinbiastine which will produce this degree of leukopenia varies from patient to patient. Once the leukopenia-producing dose is es tion of gross tumor shrinkage in order to decide tablished, doses 1 increment smaller than those that a drug is effective may result in our missing may be administered at weekly intervals for main more subtle long-term tumor-inhibiting and host tenance. It should be emphasized that, even though supporting effects. We may not always render 7 days have elapsed, the next dose of vinblastine a service to the cancer patient when we try as should not be given until the white cell count has quickly as possible to produce tumor shrinkage returned to at least 4,000 cells per cubic millimeter. by pressing the dose of an oncolytic drug to Should benefit occur with a dose below that which toxicity. This is not meant to imply that it is causes leukopenia, there is no need to increase the unnecessary to demonstrate objective evidence size of subsequent doses. In this way many pa of tumor shrinkage with a new drug, but rather tients may be treated with doses well below the to suggest that a new drug might be found to leukopenia-producing level (4, 84). Our experience prolong survival and inhibit tumor growth if has shown that the maintenance dose for most pa administered in subtoxic doeses for long periods. tients is 0.15 on 0.@0 mg/kg of body weight. Obviously such trials must be conducted in full nec We believe it best to continue with vinblastine ognition of the natural variation in tumor growth dosage at regular 7-day or 14-day intervals for as which can occur spontaneously from time to time. long as a patient will respond (4, 43, 50). Where To avoid drawing falselypositive conclusions, such successful therapy has been discontinued, patients trials must be statistically controlled and should have usually relapsed about 3 weeks after the last include adequate numbers of patients for a suffi dose. Since long-continued dosage at regular 7- to cient length of time. 14-day intervals was only rarely used in the early Carcinoma of the bronchus has also been re trials, it now seems probable that the lack of main ported as responsive to vinbiastine by the Midwest tenance dosage may explain the short duration of Cooperative Chemotherapy Group23 (Table @4).

many of the early responses reported. 23 Paper presented at the Nov., 1961, meeting of the Nation It is apparent that the spectrum of activity of al Cancer Chemotherapy Service Center in Washington, D.C.

To quote from their papen on this topic : â€œThirty thelialized. This remission was maintained for 6 one patients were studied, of whom @1wereevalu weeks.â€• The dosage used for this patient was a ated. All but 4 patients had received prior therapy single course of 0.15 mg/kg of vinblastine, intra with x-ray, alkylating agents, or 5-fluonouracil. venously, daily for 4 consecutive days. No further The priming dose of vinblastine ranged from 0.15 doses were given. In the light of subsequent expe to 0.6 (mean 0.33) mg/kg. Twenty patients re niences this dosage was excessive and lacked the ceived maintenance therapy varying from 0.15 to maintenance treatment necessary for prolonged @.37(mean .7@) mg/kg over 7 to @6(mean 33) benefit. days. Five patients showed objective improvement In 1961 Barbara Johnston et a!. (43) of St of short duration (@i to 48 days), but one patient Luke's Hospital, New York, published on eight with undifferentiated carcinoma showed marked patients with carcinoma of the breast treated with regression of a right hilan mass which, on main vinblastine. Six patients were evaluable; and of tenance therapy, was sustained for @i5days. It is these, three were improved both objectively and of interest that four histologic types of carcinoma, subjectively. Dr. Johnston24 has stated that some squamous, oat cell, adenocarcinoma, and undif of these remissions have now been maintained for ferentiated, were included in those with objective more than 1 year and that her series, now grown improvement. Three patients obtained some de larger, continues to show the same order of respon gree of pain relief lasting 7, 8, and 67 days.â€• siveness. In this series the duration of the trial of vinblas The dosage schedule employed by Johnston et @ tine therapy varied markedly from patient to al. (43) was : â€œ.. . 0.15 mg/kg/day for days fol patient and i4 days' trial was considered adequate lowed by 3 days of rest. If no leukopenia was for evaluation. In the light of other experiences, it noted, one or two additional doses were given until would appear that this duration of trial was too leukopenia occurred. Weekly doses were given brief for the demonstration of a maximal response thereafter depending on the w.b.c.â€• Johnston oh rate. served that vinblastine treatment for at least 6@ In the first clinical trial of vinblastine by the weeks to 3 months was necessary to demonstrate Eastern Cooperative Group in Solid Tumor Chem objective evidence of tumor regression. This is in otherapy (@O),nine patients with carcinoma of the agreement with the experiences of Armstrong et al. bronchus were considered evaluable. None of these (4) (i96@2) in which three of seven patients with was reported to have shown tumor regression. In carcinoma of the breast showed both objective and this series an inadequate duration of trial was de subjective improvement, in one case only after 3 fined : â€œ... when no toxicity occurred and when months of vinblastine treatment. Armstrong et al. the cumulated duration of drug administration and reported less toxicity than did Johnston et al., toxicity was less than @1days.â€•Again the duration probably because they avoided initial heavy load of trial, though it produced a high percentage of ing doses. They followed the incremental approach responses in Hodgkin's disease, was brief for solid to dosage and so avoided leukopenia as much as tumors. possible. The patients of both Johnston et al. and Warwick et al. (84) have stated: â€œTherewas Armstrong et at. were treated for at least 1@ weeks no definite evidence of a correlation between the and, if they responded, were thereafter given degree of leukopenia and the beneficial effect. maintenance dosage indefinitely once every 7 days. Remissions in Hodgkin's disease have been ob For various reasons not related to drug toxicity, served in the absence of leukopenia following vinblastine dosage was discontinued in two of the therapy.â€• The toxicity which can follow the use patients of Armstrong et a!. while the drug was of excessive initial dosage may only deter an still effective. Both patients relapsed during the investigator from continuing the use of a new drug 3d week after their last dose. This again illustrates for an adequate period of trial. the need for continued maintenance dosage for as The earliest reference to the use of vinblastine long as a patient will respond. in the treatment of breast carcinoma was by War In the Midwest Cooperative Chemotherapy wick et a!. (85) in 1960. They wrote: â€œ... on the Group study (1961) of vinblastine, seven patients i@th day the supraclaviculan nodes were no longer with carcinoma of the breast were included, and palpable, and induration and thickening of the none showed any response. Likewise none of eight skin had disappeared. There was less swelling of evaluable patients with carcinoma of the breast the arm. . . The patient was discharged feeling showed any response in the study (1961) of the

well, apart from stiffness in the left shoulder. ... 24 Personal communication, Dr. Barbara Johnston, St. The ulcer on the chest wall had completely epi Luke's Hospital, N.Y.C.

Eastern Cooperative Group in Solid Tumor Chem ployed : â€œ... initial infusions run continuously for otherapy.25 The early criteria employed by many 7@ hours (0.4 mg./Kg.) . . . Re-infusions have been groups for an adequate length of trial of vinblas given at intervals of 6â€”8weeks, or sooner if the pa tine therapy would not have insured the necessary tient's status was deteriorating.â€• While the latter duration of treatment for many patients with car type of regimen would appear to have been more cinoma of the breast. effective, it should be pointed out that neither Noble26 observed that mature female rats can investigator gave maintenance dosage intrave tolerate larger doses of vinblastine than can im nously after intra-artenial infusions. It is conceiva mature female rats. They further demonstrated ble that beneficial effects might be prolonged by that the immature rats could be made equally intravenous maintenance dosage started 7â€”14days tolerant of vinblastine as are mature rats if they after intra-arterial infusion and continued regular were pretreated with stilbestrol for a few days be ly each week for so long as a patient showed im fore dosage with vinblastine. This finding may provement. well have a clinical bearing when we come to treat Hertz et a!. (3@) demonstrated that vinblastine women who have previously undergone ovariec can be effective in patients with methotnexate tomy, as is so often the case with breast carcinoma resistant choniocarcinoma. Responses to vinblas patients. tine in both male and female patients with tropho Armstrong et al. have noted that a woman with blastic tumors have also been reported by others. breast carcinoma was able to tolerate weekly in The observations of Hertz are most interesting in travenous vinbiastine doses no larger than 0.05 that they relate to a magnitude of vinblastine mg/kg without leukopenia while she was receiving dosage considerably larger than has been used by testosterone. After several weeks of this combined others. As might be anticipated, this high dosage therapy, testosterone was discontinued. Within a produced considerable toxicity. Hertz is the only month the patient was found to have increased her investigator to report : â€œSeveremental depression tolerance of vinbiastine so that doses in the range was uniformly noted during the toxic phase and of 0.15 to 0.@0 mg/kg/week could be given with this was more marked than would be accounted safety. Only after the patient had improved objec for by the respective clinical status of each of the tively following the use of vinblastine treatment patients.â€• This followed single total doses of @4 was testosterone discontinued. Had testosterone mg. and 36 mg. The weight of the patients who been discontinued immediately prior to the use of received these doses was not stated ; but, assuming vinblastine, the benefit seen could have been at the weight of the average patient to have been tnibuted to testosterone withdrawal. In this pa about 70 kg., this represented dosage in the range tient, testosterone may have been acting in the of 0.30â€”0.50 mg/kg. In many of his patients, same manner as does the absence of ovarian func Hertz observed leukocyte counts below 1,000 cells tion in immature female rats. per cubic millimeter following vinblastine dosage. Mealey (5@) and Wilson (91) have separately At this dosage he also reported : â€œ... suppression reported on the administration of vinblastine by of deep tendon reflexes associated with each course intra-antenial infusion to patients with brain tu of therapy. Two [of 81 patients complained of mors. Only two of ten patients in Mealey's series numbness and panaesthesias in the fingers. These derived benefit (5@). In the series of Wilson, ten changes proved to be reversible. In addition to of seventeen evaluable patients were improved these apparent neurotoxic effects, there was sug (91).Thisdifferenceinresponsenatemaybere gestive evidence of panasympatholoytic action of lated to the different dosage regimens employed. the drug. All patients, during at least one course Mealey reported that : â€œTenpatients with malig of therapy, experienced marked constipation for S nant cerebral astrocytomas were given post-opera to ii days. . . . There was one episode of urinary tive infusions of vinblastine (0.5 mg./Kg.) into the retention. Six [of 8] patients had bilateral pam carotid or vertebral arteries, usually over a period and tenderness of the panotid glands, usually as of several days.â€• Wilson on the other hand em sociated with marked dryness of the mouth. Two @ Personal communication, Dr. Emil Frei, III, National patients developed sinus tachycardia in the ab Cancer Inst., who as chairman of the Eastern Solid Tumor sence of fever or other apparent cause.â€•Following Group study of YLB reports that this group's continuing study a course of 6 mg. of vinbiastine twice daily for 3 of YLB has now demonstrated responses among patients with consecutive days, Hertz et al. reported â€œtypically carcinoma of the breast in approximately the same order of severe toxicityâ€• with decrease in the platelet count frequency as with 5-PU. 16 Personal communication, Dr. R. L. Noble, University of by 30â€”60per cent, white blood cell count below British Columbia. p2,000cells per cubic millimeter, and an absence of

reticulocytes from the peripheral blood for @â€”9administration of glutamic acid in capsules pro days. He went on to state that : â€œCompleterecov duced severe nausea. However, by giving glutamic eny of all of the bone marrow elements, as mani acid as an effervescent solution prepared by mixing fested by the peripheral blood findings, occurred it with an equal volume of sodium bicarbonate, it within 3 weeks and there has been neither evidence was well tolerated. Four hundred mg/kg of irglu of cumulative toxicity with successive courses of tamic acid were given daily in this way. In some treatment nor decreasing effects in the bone man of their patients the tolerated vinblastine dose was row.â€• increased by 30 per cent with glutamic acid treat It is interesting to learn from Hertz et a!. that ment, but this produced no demonstrable improve massive dosage with vinblastine can produce con ment in antitumor effect. Accumulation of meta stipation and neurotoxicity somewhat resembling phases was noted in bone-marrow specimens after the toxicity produced by yincnistine at a dosage combined vinblastine and glutamic acid therapy, within the therapeutic range. However, there the but this accumulation was much less than with resemblance ends, because yincnistine does not usu vinblastine therapy alone; and the suppression of ally depress the leukocyte count at neurotoxic 1ev postmetaphase stages was at no time complete. els of dosage. It should also be noted that vinbias No investigation has yet been made of the possi tine does not usually depress the platelet on red bility that glutamic acid, etc., might be useful in blood cell counts at dosage just sufficient to pro preventing systemic toxicity from large doses of duce moderate leukopenia; nor does it commonly vinblastine infused intra-artenially into tumor depress the white blood cell count for longer than bearing areas of the body. 7â€”10days. Toxicological studies at the Lilly Research Lab Johnson et al. (4%) observed that the inhibiting oratories have revealed that vinblastine potenti effect of vinblastine on a tissue culture of human ated phenobarbital anesthesia in dogs and pro monocytic leukemia cells could be blocked by glu longed EvipalÂ® sleeping time in mice. It is there tamic acid, aspartic acid, arginine, citrulline, and fore conceivable that should vinbiastine be given ornithine and observed reversal of anti-P-1534 ac to a patient undergoing barbiturate anesthesia re tivity in vivo (39). Based on these observations, covery of consciousness might be delayed. A very two clinical groups investigated the possibility that large number of cancer patients have now been some of these substances might be useful in revers treated with vinblastine during the daytime and ing the leukopenic effect of vinblastine. Armstrong have taken barbiturates the same night to facili et al. (4) gave capsules of glutamic acid orally in tate sleep. In spite of this there have been no ne doses up to 3 gm. S times daily to several patients ports of prolonged or augmented sedation in such at various times in relation to vinbiastine dosage. patients. However, since vinblastine may be rapid Monosodium monopotassiuxn glutamate solution ly fixed and metabolized, the clinical situation was was given intravenously to other patients. Still not completely analogous to the administration of others were given capsules of fraspartic acid by vinblastine and barbiturates simultaneously. It mouth in doses up to 3 gm. 3 times daily. The only would be interesting to compare the leukopenic consistent effects noted with all three forms of and oncolytic effects of vinblastine administered treatment, regardless of the size and duration of alone with those of vinblastine given at the time dosage, were anorexia, nausea, abdominal colicky of barbiturate anesthesia. pain, and weight loss. The data did suggest that Tumor shrinkage with significant benefit has fol ir.glutamic acid in oral doses of 3 gm. 3 times daily lowed the administration of uncoated compressed may have prevented the leukopenic effect of mod tablets of vinblastine by mouth (4, 36). However, erately sized but not of large doses of vinblastine. the leukopenic response following dosage was un Only when the glutamic acid had been given for predictably variable from week to week even some days before treatment and continued past though the same dose was used each week. The the period during which leukopenia might be cx incidence of gastrointestinal side-effects with these pected to appear (5 days after dosage) was any tablets was high. Some patients who responded to possible leukopenia-sparing effect seen. Patients oral vinblastine later relapsed in spite of its con first treated with glutamic on aspartic acid when tinued weekly use but then responded again after already leukopenic from vinblastine dosage showed intravenous vinblastine was substituted for the no more rapid recovery from leukopenia than pa oral drug. It was concluded that these tablets of tients not given these amino acids. Vaitkevicius vinblastine were not consistently absorbed from (81)notedâ€œsomeapparentameliorationof(yin the gut and were therefore unsuitable for further blastine) toxicityâ€• when ir-glutamic acid was given use (4). More recently the Lilly Clinic group has orally. They, like Armstrong et al., noted that oral begun to investigate a new type of vinblastine

tablet which passes through the stomach before National Cancer Institute29 when vincnistine was beginning to slowly disintegrate in the intestine used as the first drug. Several groups now initiate over a period of several hours. Preliminary obser therapy with vincnistine because it does not usu vations with this new tablet indicate that it is ally depress marrow function. superior to the earlier one in that it less often After remission from acute leukemia has been causes side-effects and is more predictable in its obtained with vincnistine, one has the choice of leukopenic effects. Now that a Hodgkin's disease continuing dosage on a maintenance basis for as patient has obtained complete remission with this long as the patient will respond on of withholding new preparation, its evaluation will be continued treatment until it is needed again. A controlled by a larger number of investigators. study of these alternatives is being carried out by the cooperative groups in an endeavor to establish VINcrn5TINE SULFATE which of these regimens is the most effective in To initiate the trial of vincristine, Armstrong, prolonging survival. Dyke, and Fouts (3) of the Lilly Clinic gave small Vincristine dosage much below 0.10 mg/kg/ weekly intravenous doses of only 0.01 mg/kg to week has not been effective in inducing complete two men. One of these suffered from Hodgkin's remissions from acute childhood leukemia. Arm disease and the other from lymphosarcoma. Even strong et al., influenced by their good results with these first tentative doses produced prompt and @ gratifying responses. Within months it became TABLE 25 obvious that vincnistine was useful for a variety of AGE DISTRIBUTION AND RESPONSES OF 95 LEUKEMIC malignant conditions at doses of 0.01â€”0.03mg/kg/ CHILDREN TREATED WITH VINCRISTINE week. At this time the trial was enlarged to include

several other centers. Kanon, Freireich, and Frei Age of the National Cancer Institute soon discovered (years)Totalresponse1â€” patientsCompleteremissionPartial remissionNo that vincnistine could induce remissions from acute 3 (40%) (27%) (38% leukemia in children when given in doses larger 4â€”6 22 (42%) 15 (29%) 15 (29% than had been employed by the Lilly group (44, 7â€”9 17 7 (41%) 6 (85%) 4 (24% 10â€”15 10 2 (20%) 4 (40%) 4 (40%) 45). Approximately 0.iO mg/kg/week is necessary 16â€”20 19587292916 04 05 for an optimal remission rate from acute childhood leukemia. Totals52 The remission rates obtained with vincristine in acute childhood leukemia by Karon (45) and by lesser dosage when treating adults with lympho the Acute Leukemia Group B27 have been at least mas and solid tumors, failed to attain the dosage as good as can be obtained with the standard necessary for leukemic children. As a result they agentsâ€”6-mercaptopurine, steroids, or a folic acid produced merely partial rather than complete re antagonist. The responses in a series of 95 children missions with weekly doses in the range of 0.0@5- with acute leukemia treated jointly by six cen 0.075 mg/kg. tens28 are seen in Table @5. It is fortunate that leukemic children tolerate There is noted even distribution of responses in large doses of vincnistine better than do adults. age groups up to 10 years. It is also of interest that The weekly dose of 0.10 mg/kg which is necessary some of the leukemias responding to vincristine are for induction of remission in childhood leukemia of the myelogenous type. produces profound toxicity in adults. Fortunately The majority of these patients received vincris adults with lymphomas and solid tumors will often tine as a fourth drug after sequential treatment respond to weekly doses in the range of 0.0@â€”0.05 with the standard drugs. More recently, a higher mg/kg and in some cases to doses as low as 0.01 remission rate has been obtained with vincristine mg/kg. There is no question that the incidence and used as the initial treatment for acute leukemia. severity of central nervous system side-effects are Complete remission was obtained in eight of nine related to the magnitude of vincristine dosage (4, children at the Boston Children's Cancer Research 13, 44, 45). The incidence of epilation, the most Foundation2Â° and in seven of eight children at the frequently occurring side-effect, is not so clearly 27 Personal communication, Drs. M. R. Karon and E. Frei, dose-related. III, NationalCancerInstitute. With single weekly doses, leukopenia, neuritic 28 Data received at the Lilly Laboratories for Clinical Re pain, constipation, and difficulty in walking due search from National Cancer Institute, the Boston Children's Cancer Research Foundation, the Roswell Park Memorial In to muscle weakness have usually been of short stitute, the Bobs Roberts Hospital, the Sloan-Kettering Insti 29 Personal communication, Dr. M. R. Karon, National tute, and the Lilly Clinic. Cancer Institute.

durationâ€”i.e., less than 7 days. When subsequent tients have been continued on their usual weekly dosage was reduced, these side-effects often less vincnistine dosage. To date vincnistine has not ened or disappeared. When the same total amount been administered intrathecally to any human be of vincnistine per week has been given in several ing. This route of administration has been avoided doses over a number of days, these side-effects because Costa3Â° found that vincnistine given have usually persisted indefinitely while therapy intracisternally to anesthetized dogs produced was continued. To avoid such prolongation it ascending paralysis and death. Since it is known seems advisable to administer vincnistine no more that the Vinca alkaloids are capable of potentiat often than once weekly. ing barbiturate anesthesia, it is conceivable that Other side-effects, such as epilation, sensory the above events which followed intrathecal ad loss, paresthesias, slapping gait, loss of deep ten ministration of vincnistine reflected potentiation don reflexes, hoarseness, and muscle weakness of the barbiturate anesthesia rather than an effect have not reversed so rapidly and have persisted for of vincnistine itself. at least as long as therapy has been continued. In After it was found necessary to employ a weekly most instances these latter side-effects have even dosage of vincnistine as high as 0.10 mg/kg to in tually disappeared by about the 6th week after duce an optimal number of remissions in acute discontinuance of treatment. Muscle weakness has childhood leukemia, similar doses were then used been reported most often to involve the hands, in treating both children and adults with other quadriceps, dorsiflexors of the foot, and less often malignant conditions. In retrospect, it became ap the larynx and the extrinsic muscles of the eye. parent that this high dosage did not improve the Frequently there has been a sequence in the response rate among patients with vincristine development of side-effects. Initially, most pa sensitive tumors but did increase the toxicity. tients have reported only sensory impairment and Holland2' et at. of the Eastern Cooperative Group paresthesias. With continued treatment neunitic in Solid Tumor Chemotherapy have returned to pain has sometimes appeared and later still, in using weekly doses in the neighborhood of 0.0@25 some cases, motor difficulties. mg/kg and believe that this may be no less effec Constipation, taking the form of colicky ab tive for patients with Hodgkin's disease, lympho dominal pain and upper colon impaction, has often sarcoma, and carcinoma of the breast than is high occurred only after several doses of vincnistine. In er dosage. They have found that this lesser dosage such cases the rectum has usually been empty and is noticeably less productive of side-effects than is a flat-plate x-ray of the abdomen has been neces higher dosage. However, they state that further sary to confirm the diagnosis. Because it has been experience will be required before it is proved be difficult to free such high impaction, it would seem yond doubt that 0.0@5 mg/kg dosage is no less advisable to employ a routine prophylactic regi effective than 0.050 mg/kg dosage. men against constipation for all patients receiving Carbone and Bnindley (1 1) of the National Can vincnistine. After encountering some severe cases cer Institute have reported that five of five pa of cecal impaction among the first several patients tients with Hodgkin's disease and three of three in their series, the Lilly Clinic group subsequently patients with lymphosarcoma responded to vim have given all further patients, from the beginning cnistine. They wrote : â€œFiveof these 8 patients of therapy, a feces-wetting agent (such as Dox were resistant to on had not responded to vinblas anTM) and a methylcellulose bulk laxative (such tine and alkylating agents. This order of respon as Cologel TM) 3 times daily by mouth. On any siveness in the absence of bone marrow toxicity is day should a patient, in spite of these medications, a unique experience in lymphoma chemotherapy. not have a bowel movement by noon, he is immedi Less striking evidence of antitumor activity has ately given magnesium sulfate ; and if that does been observed in patients with solid tumors, par not produce a stool by evening, an enema is given. ticulanly neuroblastoma and renal cell carcinoma.â€• This prophylactic regimen has been found to dim Costa et a!. (13) of Roswell Park Memorial In mate the problem of constipation. stitute also reported : â€œ... rapid regressions of Central nervous system leukemia was reported four of five lymphomas in patients refractory to in eleven of 66 children undergoing otherwise bene standard treatments. . . Unequivocal partial re ficial therapy with vincnistine.'Â° This suggests that gressions of single cases of metastatic choniocan vincristine, like 6-mercaptopunine and folic acid cinoma, testicular teratocancinoma, carcinomas of antagonists, may not penetrate well into the cere cervix and of bladder are in progress. Real effects,

brospinal fluid. Intrathecal amethopterin has been 30 Personal communication, Dr. G. Costa, Roswell Park successfully used for this problem while the pa Memorial Institute.

indicating some drug activity, have been seen in Selawny et al. (69) of Roswell Park Memorial single cases of myeloma, prostatic carcinoma and Institute gave vincnistine in weekly doses of 1.5â€”5 chronic lymphocytic leukemia.â€• mg/sq meter, averaging 3 mg/sq meter of body It should be mentioned that Dr. Costa,3Â°in his surface, to fourteen children. â€œObjective tumor capacity as chairman of the Eastern Solid Tumor response was noted in 4/5 patients with acute lym Group's myeloma study group, has reported sub phoblastic leukemia . . . @/4patients with neuro sequently that vincristine was not significantly blastoma . . . and 1/1 patient with ovarian dys useful with any consistency in a large series of genminoma. . . . Noteworthy was the absence of multiple myeloma patients. thrombocytopenia. Weight loss of 13â€”@5per cent Bohannon et al. (9) have reported that â€œ... 4 of during 5â€”10weeks of drug administration consti 11 [adult] patients with Hodgkin's disease and 1 tuted a serious problem in 5 patients and appeared patient with chronic myelocytic leukemia have to be unrelated to G.I. toxicity. This drug seems had a temporary subjective and/or objective clini to have a wide spectrum of activity in childhood cal response.â€• This group employed â€œdosesof0.03 tumors. . . .â€œ @ mg/kg to 0.06 mg/kg, I.V. repeated at to 14 day Weight loss of this magnitude has also been re intervals depending on the leukocyte response. ... ported by the National Cancer Institute group but Treatment was continued until leukopenia, throm not by others. It may be significant that both the bocytopenia or other toxicity occurred.â€• The un Roswell Park Memorial Institute and the National usually low response rate reported by this group Cancer Institute groups employed weekly doses with lymphoma patients is possibly due to the for some patients in excess of 3 mg/sq meter of irregular spacing of doses. body surface. Further, the National Cancer Insti Tan et at. (79) also followed a dosage schedule tute workers have examined their data for any different from that of other investigators. In a influence which dosage magnitude might have on @ series of children, they reported : â€œThedosage weight change. They found that patients who re was 0.05 mg. per kilogram of body weight given ceived weekly doses in excess of 0.10 mg/kg lost 1 to 3 times weekly to an average total dose of weight, whereas those who received weekly doses 0.5 mg. per kilogram of body weight. The drug smaller than 0.09 mg/kg gained weight. From this was discontinued when toxicity or an adequate there would seem to be merit in giving a weekly therapeutic response occurred. Toxic effects con - vincnistine dose no larger than 0.10 mg/kg to leu sisted of leukopenia, thnombocytopenia, alopecia, kemic children. Also, to avoid excessive neurotox and cellulitis following extravasation of the drug. icity, it seems advisable not to increase the weekly Transient urinary symptoms such as dysunia, fre dose, if possible, above 0.0@5 mg/kg for patients quency, urgency and polyunia without microscopic with solid tumors. It remains to be established abnormalities occurred in 5 patients. Ptosis of the whether some lymphoma and solid-tumor patients lids with no other meningeal signs occurred in 5 might benefit from higher weekly dosage than patients. . - . In 1 of 4 children with Wilm's Tumon 0.0@5 mg/kg if no response is obtained with this an abdominal mass disappeared without subjec dose. @ tive benefit; 1 child with Hodgkin's disease, of Costa et al. of Roswell Park Memorial Institute 3 children with embryonal Rhabdomyosarcoma, 1 have reported that patchy liven necrosis was seen @ of with neuroblastoma and 1 child with embryo at autopsy in some of their vincristine-treated nal adenocarcinoma had objective regression with patients (14). However, they also noted that: @ clinical benefits continuing for 1 to months.â€• â€œTheliver function tests performed in these pa@ Only in these two reports (9, 79) from the Sloan tients had not been altered by drug administna Kettering Institute have leukopenia and throm tion.â€• bocytopenia been classed as prominent side-effects. Costa et a!. (14) reported in addition that: It should be understood that only in these two â€œPartial remissions were observed in single in series were doses of vincnistine often administered stances in carcinoma of the cervix, choriocarcino more frequently than once every 7 days. Other ma, anaplastic carcinoma, testicular teratocar investigators have commented rather on the rela cinoma, and carcinoma of the prostate.â€• They tive freedom from marrow toxicity which vincnis continued : â€œTheactivity in carcinoma of the cer tine affords. Significantly, these others have not vix is worthy of particular attention, since it nepne regularly spaced their doses so closely as did the sents one of the human neoplasms singularly re Sloan-Kettering investigators. From this it ap sistant to chemotherapy. Confirmation of such pears that there is merit in not giving vincnistine activity of vincnistine in carcinoma of the cervix doses more frequently than once every 7 days. has been obtained in a larger series of patients.â€•

The duration of dosage before complete remis either no toxicity or much less toxicity occurred sion occurred in 37 children with acute leukemia than would be expected from such doses given in averaged 88 days (range, 11â€”106days).'9 Patients travenously. This suggests that both vinblastine with Hodgkin's disease or lymphosarcoma usu and vincristine are cleared from the blood and ally begin responding to vincnistme within a day fixed rapidly on malignant tissue when adminis after the first or second dose. For this reason it tered intra-artenially. seems advisable to increase the fluid intake of Beer has shown that, when material derived by patients at the time of the first few doses to avoid titration of vinblastine was injected intravenous excessive elevation of the blood uric acid. It re ly into rats, it was cleared very rapidly from the mains to be established how late one can expect a blood.@ Within minutes of injection much of the response in the various other solid tumors, but radioactivity was detected in the liver; and in less tumor shrinkage has been variously reported to than an hour the blood was clear. The radioactivi occur as soon as a few days after the first dose and ty was subsequently traced from the liver through as late as after the sixth weekly dose of vincnistine the biliany system into the gut. Only 5 per cent of in some patients with various types of carcinoma. the radioactivity appeared in the urine. Since significant benefit and gross tumor shrinkage With vincnistine it has been shown that dosage have occurred in some patients only after 1@weeks is more toxic to rats which have undergone ligation of dosage with the similar alkaloid vinblastine (4, of the common bile ducts than to rats with patent 43), it would seem that a similar period of trial biliary tnacts.@ It has also been reported that pa might be appropriate with vincnistine. tients with obstructive jaundice experienced more As with vinblastine, withdrawal of vincnistine severe side-effects than patients without biliany dosage has in several cases been followed by the obstruction Here again then, as in the case of reappearance of tumor growth within @â€”3weeks vinblastine, we have some evidence that vincnis of the last dose.19 For this reason it would seem tine may be largely and rapidly excreted by the advisable to continue with weekly vincristine main liver. A summary of the differences and similar ac tenance dosage for as long as a patient derives tions of vinblastine and vincnistine in clinical use benefit. It is not known whether smaller weekly as understood in December, 196@, is shown in doses than are necessary to produce tumor shrink Table @6. age can be relied upon to maintain a beneficial response once it has occurred. A cooperative group DISCUSSION study of this problem would be important, because Included in the invitation to present this review it has been observed that downward adjustment was a paragraph indicating that the purpose of of dosage magnitude in some of the patients of this presentation, as well as of the entire symposi Armstrong et al. has been followed by reversal or um, was to document progress; and in addition to reduction in neurotoxicity. point up problems which remain to be solved, to In that vincristine causes little if any depression depict the nature of the obstacles which appear to of bone-marrow function (3, 11, 13, 14, 44, 45, 66, be impeding further progress, and to re-evaluate 69), this drug should be relatively safe for use in existing concepts. Participants were urged not conjunction with radiation therapy. only to be candid and objective, but also to be Like vinbiastine (5Q, 91), vincnistine has been speculative in approach. Recognizing these objec administered intra-artenially with beneficial results tives and the limitations of time and space, we to some patients with brain tumors.2' Vincnistine have attempted to document the present state of has also been infused directly into the hepatic an our knowledge concerning the production, chemis tery of a patient with primary carcinoma of the try, toxicology, experimental activity, mechanism liver, a procedure which was followed by tumor of action, and clinical efficacy of these new agents, shrinkage and clinical improvement.2' A few other emphasizing what we view as areas requiring fur patients with primary liver carcinoma have been then exploration. treated with vincnistine administered intravenous ly rather than into the hepatic artery. None of the SOME ASPECTS OF STRUCTURE AND MODIFICATION latter patients have shown any response. The structures of vinblastine, vincnistine, and The doses of vinbiastine or vincnistine infused to a lesser extent, vinleunosine, are reasonably well intra-arterially into tumor-bearing pants of the established. Modification of these large alkaloidal body have been several times larger than can be molecules is difficult. One such modification which @ given intravenously@' (37, 5@, 91) . In all cases experimentally appeared to be useful was not con

31 Unpublished data, Dr. James G. Armstrong, Lilly Lab 32 Personal communication, Dr. C. T. Beer, University of oratories for Clinical Research. British Columbia.

COMPARISON OF THE CLINICAL CHARACTERISTICS OF VINBLASTINE SULFATE (VLB) AND VINCRISTINE SULFATE (YC R) AS UNDERSTOOD IN DECEMBER, 1962

CharacteristicVLBVCR

Weekly intravenous dose for solid 0.10â€”0.30mg/kg; average, 0.15 mg/kg 0.01â€”0.05 mg/kg; average, 0.025 mgI tumors kg

Weekly intravenous dose for acute Benefit reported in some cases Approx. 0.10 mg/kg childhood leukemia

Weekly oral dosage Possibly 2â€”3Xthe intravenous dose; Not studied remains to be firmly established Leukopenia Limiting factor to dosage Uncommon; rarely limits dosage

Anemia and thrombocytopenia Rarely occur Rarely occur

Neuromuscular manifestations Very rarely occur; rapidly reversible Limiting factor to dosage; slowly re versible

Constipation Only rarely occurs Almost invariable if preventive medi cation not used

Acute leukemia in children Reported useful in some situations Complete remissions very frequent

Acute monocytic leukemia Can induce complete remissions Remains to be determined

Hodgkin's disease Complete remissions frequent Complete remissions frequent

Lymphosarcoma Usually only partial remissions Complete remissions frequent

Carcinoma of breast Tumor shrinkage and clinical benefit Rapid useful effect in a (?) higher per after 8â€”12weeks' dosage in about centage of patients 30 per cent of patients

Carcinoma of bronchus As above Remains to be determined

Carcinomas of G.I. tract As above Remains to be determined

Carcinoma of ovary Some reports of useful tumor shrink- Not known age; more evaluation needed

Carcinoma of cervix As above Benefit reported; more evaluation needed

Carcinoma of prostate Benefit reported; more evaluation Benefit reported; more evaluation needed needed

Malignant melanoma Some useful remissions reported (?) Not significantly useful

Chorionepithelioma and other tro- Frequently beneficial Frequently beneficial phoblastic tumors

Carcinoma of kidney Benefit reported; more evaluation Benefit reported; more evaluation needed needed

Primary brain tumors (intra-arteri- Reported as frequently clinically Reported as frequently clinically al dosage) beneficial beneficial Mycosis fungoides Some complete remissions reported Not known Hand SchUllerChristian Disease Usefulremissionsreported Not known and Letterer-Siwe Disease

Neuroblastoma Benefit reported Benefit reported

Rhabdomyosarcoma Benefit reported; more evaluation Benefit reported; more evaluation needed needed

firmed in the clinic, but does illustrate some inter To add to our problems, many of these compounds esting features of even slight modification of these are obtained from the Vinca plant in low yield. compounds. Problems of yield are recurrent and chronic. Be Preliminary clinical evaluation of vinleurosine fore rejection of a compound, we must study it had not yet been initiated when it was observed for any evidence of activity to determine if at experimentally that the methiodide of vinleurosine tempts to increase the yield are worthwhile. It gave a striking increase in activity over the parent would be gratifying if some of the more abun compound when tested in the P-1534 system. Pro dantly available, but less active or inactive, com longations of 70â€”160per cent were obtained oven pounds could be converted to active materials. On a dose range of 30â€”60mg/kg, and in addition paper a number of alterations seem feasible-for marked prolongation and survival were obtained example, the oxidation of an N-methyl group of in near-terminal animals. In contrast to the in vinleurosine. However, with any method proposed crease in P-1534 activity, there was a loss of ne to accomplish a useful change, many reactive cen sponse in all other tumors carried in our expeni tens on these complex molecules are readily af mental spectrum. This was interpreted to indicate fected, as for example, in the oxidation of the basic an increase in specificity, and for this reason yin nitrogen in the catharanthine moiety to an amide, leurosine methiodide was selected for preliminary all biological activity is lost. clinical evaluation. An additional pharmacological difference of some interest was the effect on pe PHARMACOLOGY ripheral white blood cell counts. As has long been A number of problems which basically have a recognized, vinblastine and, to a lesser extent, yin bearing on toxicology of these compounds deserve leurosine cause leukopenia as a result of bone-man full discussion and some ne-evaluation. All the ac row depression. The methiodide of vinleurosine, in tive antitumon alkaloids appear to have some capa contrast, was not observed to produce leukopenia bility for arresting mitosis of the mammalian cell under the conditions of our investigation. On the in vitro and in vivo (1@,16, 39, 4@,6@,81). A num contrary, there was noted an induction of leukocy ben of amino acids including glutamic, ornithine, tosis in nonleukemic cancer patients. Although citrulline, arginine, aspartic, and tryptophan have some evidence of minimal tumor shrinkage was been reported from this laboratory to reverse or noted clinically, vinleunosine methiodide has not block the TCD50 dose of vinblastine in 1-week been pursued further in humans because of the growth curves in tissue culture (4@). severity of side-effects not encountered with the These observations were extended in vivo in the other clinically active Vinca alkaloids. P-1534 system and were found to block either the These marked differences in activity and side antitumon effect or toxicity in nonleukemic ani effects after only minor changes in the molecule mals given an ED,0 of vinblastine. Cutts (16) emphasize the importance of even minute changes found a reduced percentage of cells in mitosis, in configuration of these compounds. Continued under certain conditions of administration, in an structure analysis, both of the parent compounds in i@ivosystem following treatment with vinblas and products produced due to their being metabo tine, if glutamic acid and tryptophan were also lized, is essential for the understanding of the administered. The effects observed by Cutts were mechanism of their activity. The striking differ similar to those observed clinically by Vaitkevicius ence in clinical activity of vinblastine and vincnis (81) with glutamic acid. tine in the acute leukemias of childhood must be Two points need to be reconsidered in the rela related to the presence of an N-formyl group in tionship of these amino acids to the biological vincnistine instead of the N-methyl group found action of vinbiastine. First, in this laboratory, in vinblastine. effects of these amino acids in nitro were observed Observations on molecular modifications some only at 50 per cent end-point levels of vinblastine, times give rise to additional problems. In the cited and this was also true of in vivo work. The fact case of vinleurosine and its methiodide, vinleuno that optimal doses of vinblastine were unaffected sine was temporarily withheld from a clinical eval suggests that the amino acids play only an ancil uation, and the methiodide given priority, because lary role on vinblastine action. While the sugges the quatennary compound appeared superior in tion was made by one of the authors (39, 4@) that terms of P-1534 activity. However, our subsequent the antituinon mechanism of vinblastine might be experiences, just described, serve to emphasize by interference with metabolic pathways involving that an enormity of effort can come to nought these amino acids, these observations obviously when the clinical trial fails to substantiate leads do not present a complete picture. suggested by experimental evaluation in animals. Secondly, however, as far as we are aware, there

has been little effort by clinical investigators, other ing to note that it has previously been shown that than the limited work of Vaitkevicius and Arm both vinblastineand vincnistine potentiate barbitu strong to study or take advantage of the possible rate anesthesia in animals, and Noble@ has re use of these amino acids in host-protection studies ported increased toxicity and mortality in rats with vinbiastine. In parallel studies with P-1534- treated with vinblastine when they were under beaning and tumor-free mice, it was observed that barbiturate anesthesia. From these considerations, glutamic acid offered host protection and blocked it would seem appropriate to determine whether antitumor effect, whereas tryptophan blocked an the increased toxicity observed by Costa in dogs titumor effect but offered no host protection. might be associated with the fact that the animals Aspartic acid, however, did not reverse antitumor receiving vincnistine intracistennally were also un effects and did offer host protection (39). It would der barbiturate anesthesia. In addition, there have seem that a greater clinical effort to explore these been interesting differences reported in LD50's of relationships in terms of host protection is war vinblastine given by different routes. This com ranted. pound is approximately 6 times more toxic in rats Clinically the use of vincnistine is associated by the intrapenitoneal route than when adminis with neuromuscular manifestations which have tered intravenously. Thus, the question of toxicity not as yet been fully studied experimentally. Par of these compounds by different routes in the pres enthetically, it is interesting that similar neuno muscular manifestations have been reported in TABLE 27 some malignant conditions without treatment of EFFECT OF VINCRISTINE THERAPY ON any kind. Whether or not the neuromuscular mani INTRACRANIAL P-1534 LEUKEMIA festations following vincristine therapy are indeed Ten mice per group related to the parent compound or to its meta bolic products which subsequently may pass the Dose cent of life (mg Xkg prolonga survivors so-called blood-brain barrier; or whether the com (days)Per pound exerts some more fundamental effect of days)1x I)RouteAv. tion*Indefinite (73 triggering a common, unrecognized mechanism .3 sometimes found in certain malignant conditions 1.3 Subcutaneous 23 46 2 1.3 Intravenous 26 534 5 without therapy, remains to be clarified. A diffi SalineIntraperitonealAll S routes16 160 0 culty has been the lack of a convenient expeni

mental system in which these same features can * Based on animals which died only. be studied. The muscular weakness and head-drop observed in rabbits following administration of ence and absence of anesthesia and in different vincristine might be such a system and deserve species, would seem to require more extensiye in further exploration. Recent observations in this vestigation, and in addition it would appear ap laboratory of a host-protection effect of folinic acid propriate to observe whether on not intrathecal without reversal of the anti-P-1534 activity of use of folinic acid with vincnistine might not de vincristine would also appear to deserve clinical crease toxicity observed by the use of this route. investigation. Leukemia of the central nervous system has POSSIBLE ADDITIONAL CLINICAL APPLICATIONS been observed in patients in hematological remis There are four other areas in which a more ex sion following vincristine therapy. Thus, it has tensive clinical investigation of these compounds been suggested that vincnistine might not pass the seems warranted, although in some of these situa blood-brain barrier. The results of recent investi tions it has been difficult to find useful experimen gation of vincnistine therapy in intracranially im tILl systems to provide a basis for direct extension planted P-1534 mice demonstrate that vincnistine of these clinical studies. This would not seem to can be â€œcurativeâ€•inthis experimental CNS leu be the case, however, in Noble's finding that rats kemia (Table @7).From the work of Costa it might of different sex and different age groups, in the be implied that the neuromuscular effects of this case of females, differ in tolerance to yinblastine. compound are due to direct action of vincnistine This work and his additional finding of the effects on the CNS. This implication could perhaps be of estrogens on vinblastine tolerance has already suggested by his comparison of toxicity of vincnis been alluded to, and is an example of a problem tine administered by intravenous, intrapenitoneal, which deserves a more intensive clinical investiga and intracisternal routes in dogs. In this study it tion. This finding is of obvious importance in

â€˜was found that there was a marked increase in evaluating the effect of vinblastine, and perhaps toxicity by the intracisternal route. It is interest vincristine, in carcinoma of the breast and perhaps

other tumors as well. Evaluation of vinblastine in cristine should be of obvious interest, but vim the treatment of carcinoma of the breast serves to blastine should probably also be considered. illustrate an additional obstacle to clinical prog Lastly are the effects of prolonged administra ness in determining the extent of usefulness of tion of vinbiastine at low dosage levels. We have these new agents. It would seem desirable to study commented on the importance of maintenance a much larger number of breast carcinoma patients therapy with these compounds and, in addition, treated for atleast 1@ weeks with vinblastine. How the duration of therapy required for a useful effect ever, the Breast Carcinoma Cooperative Chemo of vinblastine in carcinoma of the breast and per therapy Groups tend to focus their attention on haps of the bronchus. In the latter case, it was steroidal compounds leaving few uncommitted pa stated that it appeared possible to obtain a signifi tients or investigators to investigate nonsteroidal cant increase in survival in the absence of tumor but obviously active compounds such as vinblas shrinkage. Recently, Walker and Wright (8@)have tine. made an observation in tissue culture of primary A second inadequately studied area is the pos explants from a variety of human neoplasms which sible use of the Vinca alkaloids in combination may also lend support to the concept of prolonged with or as an adjunct to radiation therapy. Since nontoxic therapy with vinblastine At subtoxic vincristine is not a severe depressor of the bone levels of vinblastine, these workers observed mor marrow, it would seem to be a particularly good phological alteration of these cultures from pleo candidate, but it would seem equally appropriate morphic multipolan elongated cells to an epithelial to investigate low dosage levels of vinblastine as type. These workers took note of the fact that, in well. Some drugs have been demonstrated clini general, normal cell lines have polygonal or epi cally to render radiation-resistant tumors sensitive thelial-like cells which might change to a fusiform, to radiation therapy. The usefulness of the Vinca fibroblast-like morphology, and that the effect of alkaloids in this area has not been adequately in vinblastine in these cultures appeared to be the vestigated. Various modalities in terms of time of reversal of this type of transformation which is radiation in reference to states of metaphase an also frequently observed in virus-induced tnans nest should be borne in mind during any such in formation. One could interpret their observations vestigation. as a â€œre-differentiationâ€•from a state of â€œde-differ There has been no effort to investigate these entiation.â€• In any event, the clinical evaluation of compounds as surgical adjuncts. Noble (17) has prolongedlow-level therapy with vinblastine where reported the clearing of leukemic cells from the clinically feasible seems warranted. peniphenal circulation of rats by vinblastine. The complete obliteration of P-1534 leukemias in ani ACKNOWLEDGMENTS mals in which vincnistine therapy was delayed The authors would like to thank the following individuals for technical assistance: Miss Louise Wood, Mr. H. F. Wright, until the animals were in a near-terminal state of Mr. G. A. Poore, Mr. C. P. Dorsey, and Mr. L. A. Baker. We generalized disease has already been described in would like to thank Mrs. P. Steuernagel and Mr. L. T. Willis this repont. Recently, Schabel33 has investigated for assistance with the manuscript, and C. J. Jansen, M.D., the effect of vinblastine in a series of hamster tu for critically reviewing certain portions of it. In addition, the mors which are characterized by wide-spread me isolation and chemical work described herein were carried out in cooperation with Drs. Gordon Svoboda, Norbert Neuss, tastases. Vinblastine decreased the incidence of H. E. Boaz, and Mr. A. J. Barnesof the Lilly Laboratories, metastases of some of these tumors at times in the and a special acknowledgment is given to Prof. K. Biemann absence of any effect on the growth of the primary of M.I.T. for recording and interpretation of mass spectral tumor. Tumors in which such effects have been data. We would like to thank P. J. Fouts, M.D., and R. 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Irving S. Johnson, James G. Armstrong, Marvin Gorman, et al.

Cancer Res 1963;23:1390-1427.

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