the vinca alkaloids : a new class of oncolytic agents...the vinca alkaloids : a new class of...

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 Vinca ro@eaLinn. has demonstrated thata number of alkaloidal substances can be obtained with antitumon activity. Over 30alkaloids have been obtained, of which fourâ€”vinbiastine, vinleunosine, vincnistine,and vinrosidineâ€”are known definitely to be active. Chemically these compounds areclosely related to one another and to two monomeric alkaloids, vindoline and catharanthine. The structure of these latter two compounds has been determined, and partialstructures for the biologically active alkaloids have been proposed. They represent anew class of large complex dimenic alkaloids containing both indole and dihydnoindolemoieties.

Experimentally, a strain-specific, transplantable, acute, lymphocytic leukemia(P-1534) carried in DBA/@mice served as a bioassay for obtaining these compoundsand for predicting their clinical activity. Vinblastine, vincnistine, and vinrosidine arecapable of prolonging and/on â€œcuringâ€•mice of the P-1534 leukemia even when therapyis delayed until a near-terminal state of generalized disease. Resistance to an additionalchallenge of leukemic cells has been observed in these â€œcuredâ€•animals. Parenteral administration of vincnistine has been demonstrated to â€œcureâ€•mice given intracranialimplants of the P-1534. The experimental tumor spectrum and toxicological studiesare presented and discussed.

Biochemical studies performed to date do not reveal any effect on cellular nespiration, glycolysis, protein or nucleic acid synthesis. The mechanisms of action of thesecompounds, which may differ within the group as well as from those of other knownagents, remain to be determined.

Only two of these compounds, vinbiastine and vincnistine, have received extensiveclinical evaluation. In spite of their close similarity, chemically, a somewhat differentgroup of human neoplasms responds to these compounds, and there has been a singularlack of cross-resistance between these two drugs and any other oncolytic drug now inwide use. Vinblastine has proved effective in chonioepithelioma, Hodgkin's disease,and other lymphomas, and a number of beneficial results have been obtained in carcinoma of the breast and bronchus. In addition, there have been smaller numbers of avariety of other neoplasias reported as responding to this compound.

Vincnistine has been striking in its ability to induce complete hematological remissionof the acute leukemias of childhood, both lymphocytic and myelogenous in type. Responses have also been reported in a number of other malignancies.

The problems and obstacles encountered in obtaining a full realization of the clinicalefficacy of these new types of oncolytic compounds are discussed in addition to areasof clinical application other than those previously reported.

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

1390

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JOHNSON et al.â€”Vinca Alkaloids . 1391

phytochemical research on this plant, Svoboda haspointed out that the probable correct botanicalname for this plant is Catharanthus roseus G. Don,but, owing to the frequent and prevalent use ofVinca ro8ea, the latter name will be used synonymously.

This plant has enjoyed a popular reputation inindigenous medicine in various parts of the world.Peckholt (63) in 1910 described the use in Brazilof an infusion of the leaves to control hemorrhageand scurvy, as a mouthwash for toothaches, andfor the healing and cleaning of chronic wounds. Inthe British West Indies it has been used to treatdiabetic ulcer (86) and in the Philippines has beenreported as being an effective oral hypoglycemicagent (fl). The plant is also used in South Africaas a hypoglycemic, and, in fact, a preparation under the name of â€œCovincaâ€•is marketed for thispurpose. In England the plant has been sold fordiabetic treatment under the name â€œVin-q-lin.â€•

The folklore reputation which this plant enjoyedas an oral hypoglycemic agent independently stimulated its phytochemical investigation in two different laboratories, unknown to each other. One ofthese groups included Noble, Beer, and Cutts, thenat the Collip Laboratories, University of WesternOntario, and the other a group in the Lilly Research Laboratories including Svoboda, Johnson,

Neuss, and Gonman. Although neither group couldsubstantiate the hypoglycemic activity, the Canadian group observed a peripheral granulocytopenia and bone marrow depression in rats associated with certain fractions (15, 18). Continuedinvestigation led to their preparation of vincaleukoblastine (VLB) sulfate, an alkoloid capable ofproducing severe leukopenia in rats (17, 60, 61).During this period the Lilly group had demonstrated that certain alkaloidal fractions gave a noproducible prolongation oflife of DBA/@ mice givenimplants of the acute lymphocytic neoplasm, theP-1534 leukemia (41).

The detection of activity against the P-1534 leukemia was considered particularly significant, owing to the fact that this tumor system has detectedother clinically useful antitumor agents in our laboratory (40) and had been sensitive enough tostudy structure-activity relationships of activecompounds which correlated with the clinical activity (@3,46, 53). An intensive phytochemical investigation resulted in the obtaining by Svoboda(7@) of leunosine, a new dimeric alkaloid closely

related chemically to VLB, as well as VLB sulfate.The effectiveness of both of these alkaloids againstthe P-1534 leukemia was first demonstrated in theLilly Laboratories.

Early in this investigation it became apparent

that â€œindefiniteâ€•survival of animals implantedwith the P-1534 leukemia was being obtained bytneatment with crude fractions of the plant whichwere chemically free of both leurosine and VLB

(38). Further investigation of those fractions nesulted in Svoboda's obtaining still two other newactive alkaloids, leurocnistine and leurosidine (74).The A.M.A. Council on Drugs has approved yinblastine' (VLB), vinleurosine (VLR), vincnistine2(VCR), and vinnosidine (VRD) as generic namesfor these alkaloids; and they will be referred to bythese names in the balance of this report.

The purpose of this report is to summarize ourcurrent knowledge of this new class of antineoplastic agents, including the chemistry, pharmacology,possible mechanisms of action, and experimentaland clinical activity.

ISOLATION AND CHEMISTRYFollowing the initial observation that extracts

of Vinca rosea Linn. produced prolongation of lifein mice with P-1534 leukemia, a detailed fractionation of the plant was undertaken. It was shown(41) that the activity was found entirely in thealkaloidal constituents of this material, and thatthe alkaloids of the leaves were far more activethan those contained in either the stems or theroots (73). The leaf material was therefore used forthe preparation of the compounds described heroin, and a procedure of differential extractions wasdeveloped (78) which separated the alkaloids intoseveral groups, as shown in Chart 1, according totheir varying basicities This procedure, which involves the conversion of the bases to their tartratesfollowed by extraction into organic solvents, wasfirst used by Svoboda (71) to prepare alkaloids fromanother plant of the same family, ALitonia constricta F. Muell. It was found that the antitumor activity was primarily located in fraction A(Chart 1).

Fraction A was partially purified by chromatography on alumina, deactivated by treatment with10 per cent acetic acid. This yielded, in additionto a number of inactive alkaloids (the results ofpurification of fraction A are shown in Chart @),two pure compounds possessing antitumor activity(7@), vinleurosine and VLB (isolated as sulfate).

When the noncnystalline residues obtained fromthis chromatography were tested for antitumor activity, certain of the post-VLB fractions appearedto be more active than either vinleurosine or VLB(74). These materials were subjected to a gradientpH technic involving partition of the mixturesbetween benzene and buffers from pH @.8to 7.5

1 VelbanÂ® (Vinblastine, Lilly).

2 OncovinTM (Vincristine, Lilly).



â€œAlkalineBenzeneExtract

1) 2% Tartaric Acid

2) EtCI2

GroundPlant

I SkellyB

Extract

1) HCI(2N)

2) NH4OH.CHCI3

Drug

1) NH3

2) Benzene

1) NH4OH

Alkaline

EtOH

Extract

PhenolicAlkaloids(C,D)

Benzene Benz-CHCI,(1:1) CHCI,I I@ 1

I@ @[email protected] [CATHARICINEJ

IVINDOLININE(.2HCl)@ISOLEUROSINBenzBenz(deactivated)FVIROSINE11CHCI,residuesI CATHARANTHINE LEUROSINE Moth Llq. Al,03 LOCHNERIDINE Combined 1

IA.JMALICINE@j VLB(-H,S04) chromatography@ chromatographyI Ai,O,

. CHCI3 Benz CHCI3 CHCI3I I

________________________________________ Benz.CHCI, CHCI3OSINEJ (1:3)@ Gradient

@ GradIent@ pH

____________I2.1.3.4Benz@CHCI3

IVINDOUDINEI(1:3)

ICAROSID1N.!JICombinedI1:3ResIdues1

â€˜Gradient

IpH(4.4.5.4) Ichromatog.[VINCARODINEI

CHCI3. CH3OH(99:1)

, [NE0LEuR0sIDINE]

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

Phase

1) NaOH (pH 11)

2) EtCI,

2)chromatography

GradientpHofmoth.Iiq. (3.4)

[itEUROCRI@@j

DetailedDrug

1) 2% Tartarlc Acid

2) Benzene

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

[Frac@on@

Ai,03

Chromatography

@ii@iy-SolubleI@lkalolds(E)]



.L@ I IBenz-CHCI3Benz@CHCl, CHCI,(3:1) (1:1)

I ILAjmaucinel[LOIHNERI1@iE]___________râ€”1-PERIVINEI

@ Moth.Liq.

BenzeneI (3:1)

IS1TSIRIKINE( Y2H2S04)@ Catharanthinetlne@1@ndolinej

H,S04

NameFormulaAjmalicine

TetrahydroalstonineSerpentineLochnerineAkuammine*Reserpine*C,1H,4N@O@

C,1H34N,O,C21H,2N20,C20H24N,02C,@[email protected]@O4C@H@N2O,

JOHNSON et al.â€”Vinca Alkaloids 1393

in 0.5 pH unit increments. The resulting materialswere then crystallized as the bases or sulfate saltsas shown in Chart @.By this procedure two additional biologically active alkaloids, vincnistine andvinrosidine, were obtained pure (74) . These compounds differed in their activity from the previoustwo in that they routinely gave survivors (38),rather than just limited prolongation of life inP-1534 leukemia. The remaining fractions (Chart1) were purified as described above for fractionA, and the results obtained are outlined in Chart3 (75, 76). In addition to the alkaloids listed inChant 3 from fraction E, Moza and Trojanek (54)

At the time of our entry into this area a numberof well-known alkaloids had been reported fromthis plant (78) ; these are listed in Table 1. Theyare all found in other genera and species of thefamily Apocynaceae, and their structures are welldocumented. No direct chemical relationship couldbe seen between the alkaloids in Table 1 and thefour active alkaloids. The remaining alkaloidswhich are listed in Table@ with their tentativeempincal formulae, melting points, rotations, andpK'@ values were all of unknown structure butwere, by and large, indole and dihydroindole innature (77). A comparison of physical properties

[FractionB1I

Al,O,Chromatography

Benz@CHCl3(3:1)

(Ajmalicine]

I Fraction

Al,03Chromatography

Benz.CHCI3(3:1)

IVindolineIITETRAHYDROALSTONINEI

Benzene

[FractionB

IA120,Chromatography

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

have recently reported two additional compoundstentatively named vindolidine3 and locherinine.

The biological comparison of the four activealkaloids from this plant (38) indicated considerable differences in the nature and scope of theiractivities, and it was felt that an understanding ofthe chemistry of this apparently new class of oncolytic agents was important to the eventual understanding of their mode of action as well as toenable one to study chemical modifications of thesecompounds which might show enhanced activities.

3 Since the name vindolidine had previously been given to

another alkaloid (Table 2) Prof. Trojanek has suggested thealternate name vindorosine for his compound.

TABLE 1

ALKALOIDS FROM Vinca roses LINN.

a Akuammine (vincamajoridine) and reserpine have not been encountered in our investigation, whereas the presence of the other four hasbeen confirmed.

IFractionEIAl,03IChromatography

F IBenzene Benz-CHCI,

I (3:1)I LOCHNERICINEI[Tetrahydroalstonine@ [VlndOJifld

IFractionF]IA120,

@J@hromatography

CHC1,

ISERPENTINE( HJ1O,I

Benz@CHCl3(2:1)

I-CATHARINEIVINDOLICINE



NameFormulaM.P., Â°C.*(a)@ CHCIspK'@ in DMFEtOH U.v.)@max.,m@Vinleurosine

VinblastineVirosinePerivineCatharanthineLochnericineVindolinine-2HC1VindolineIsoleurosineLochnenidineSitsirikine' 1/2 H@SO4VincamicineCatharineVindolicine

VinrosidineVincristineC4H@N4O,t

C4H@N4O,C@H,.N@O4fCHH@N,O$C@H24N,O,C,iHUN@O@C@H@N@O,â€¢2HClC,@HI,N,OC4@HoN4O,tC,OHUN,OS@

@ 1/2 H@SO4(Dimeric)[email protected]@N4O,.CU,OH([email protected])

(Dimeric)[email protected]@N4O@0202â€”205

(decompn.211â€”216(decompn.258â€”264(decompn.180â€”181126â€”128190â€”193(decompn.)210-212 (decompn.)154â€”155202â€”206(decompn.211â€”214(decompn.289-241 (decompn.224â€”228(decompn.271â€”275(decompn.248-251 (melts, recryst.)

265â€”267(decompn.208â€”211(decompn.218â€”220(decompn.+

72+ 42@â€”160 .5â€”121.4+ 29 .8â€”482â€” 9 (H,O)â€”18+ 61 .2â€”607.5+ 23 (Base)+418â€”54 .2â€”48 .4

+ 55.8+ 17 .0 (EtCh)5

.5, 7 .5 (H20)S .4, 7 .4 (H,O)5 .85 (66%)7.5(66%)6 .8 (66%)4 .2 (66%)3 .8, 7 . 1 (66%)5 .5 (66%)4 .8, 7.3 (66%)5 .5 (66%)7 .6 (66%)4 .80, 5 .85 (66%)5 .34 (66%)5 .4 (66%)

5 .0, 8 .8 (33%)5 .0, 7 .4 (38%)214,

259214, 259226, 270226,314226, 284, 292226, 297, 827245, 300212, 250, 304214,261,287230, 298, 328224,282,288214, 264, 815, 341222, 265, 292212,257,308

214, 265220, 255,296Carosidine

CarosinePleurosineNeoleurosidineVincarodineCatharicineVindolidineNeoleurocristineVindolidi@ie#(Dimeric)

C@H@N4O@0fC4H,sN4OiotC@4@H@N4Oi@t

@ C441L,N4O@otC@H,,N4O,otC4,H.@N4O@otC4,H,N4O@,tC,,H,0N@O5263-278,

288 (decompn.)

214-218191â€”194 (decompn.219â€”225(decompn.258â€”256(decompn.281â€”284(decompn.)244â€”250(decompn.)188â€”196(decompn.)167â€”

89 .8

+ 6 .0+ 61 .0+ 41 .6â€”197.4+ 34 .8â€”113.2â€”57 .87â€”814

.4, 5 .5 (88%)4 .4, 5 .55 (33%)5 . 1 (88%)5 .8 (66%)5 .8, 6 .3 (88%)5 .3 (83%)4 .68 (38%)212,254,308

255, 294267,308214, 268280, 272, 298214, 268, 293, 315261, 311220, 257, 298

250,302Lochneridinine#C@H,sN@O4163-169424247,326

Cancer Research Vol. @3,September 19631394

such as titration, infrared and ultraviolet spectra,and nuclear magnetic resonance spectra of the fouractive alkaloids indicated first of all that theyare closely related chemically, and secondly thatthey are nonsymmetrical â€œdimericâ€•alkaloids (30).Thus, for vinleurosine and VLB titration showedthe presence of two basic nitnogens, pK'a 5.5 and7.5, one of which would quatennize when treatedwith methyl iodide or similar reagents. Analyses of

Comparison of these spectra with those of several other Vinca rosea alkaloids, notably catharanthine and vindoline (31) (Table @),clearly mdicated a close interrelationship between these compounds. When the infrared spectrum of a solutioncontaining equimolar quantities of catharanthineand vindoline was compared with those of vinleurosine and VLB, it was virtually supenimposablefrom@ to 8@ and quite similar up to 16@ (Chant

TABLE 2

NEW ALKALOIDS FROM Vinca rosea LINN.

a The melting points were determined on a Kofler microstage. The ultraviolet absorption spectra were obtained with a Carymodel 14 spectrophotometer.

t Whilethesemolecularformulasagreewellwiththeanalyticalresultsforeachparticularalkaloid,it shouldbenotedthattheyare 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. Wilsonof Stanford,wenowpreferthe

C,@,formulationratherthan the C1,firstreported(54).#SeeRef.58.

the free bases, sulfates, dihydrochlonides, and quaternary salts indicated that they are C@H@_58N4O9compounds (57). A study of the infrared spectra(Chart 4, Aâ€”D)of the bases further supported thiscontention (thus the intensity of the indole N-Hwas approximately one-half that expected for atypical indolic C21 alkaloid) and indicated thatVLB contains a hydroxyl group (X CHC13 @.8,1O.0@) that is not present in vinleunosine. Asidefrom this difference, the infrared spectra are verysimilar (Chart 4, Aâ€”D)(30).

5). It was assumed that the double molecules yinleurosine and VLB were composed of catharanthine and vindoline moieties, with minor molecularmodification, bonded together in some uniquemanner (30) . It was therefore possible to investigate the structures of the more plentiful smallermolecules and then relate this informatioa to thedimenic compounds.

Cathananthine (31, 56) (I)@ was found to be aC21H2@N@O2compound and to form a methiodide

4 Catharanthine and derivatives are shown in Chart 8.



FREQUENCY(CM@')

WAVELENGTH (MICRONS)

I


CHART 4.â€”Infrared spectra of biologically active Vines alkaloids

A. Vinleurosine C.B. Vinblastine D.



1396 Cancer Research Vol. p23, September 1963

readily. The physical characteristics of the quaternary salt showed that the basic nitrogen (pK'a 7.0)was analogous to the one forming this salt in thedimeric compounds (77). Spectral data indicatedthat the alkaloid was a simple @,3-substituted pentacyclic indole containing an isolated double bond,

ester group, and closely related to the isoquinuclidine alkaloid coronanidine5 (II), C21H26O2N2(@7).The N.M.R. spectrum (Chart 6B) confirmed theabove assignments and indicated that the doublebond contained one vinyl proton at 5.SÃ¶,and thata C-ethyl group characterized by a methyl tripleta 1.1Ã¶and an allylic methylene quartet at 1.9Ã¶waspresent (56). These data clearly suggested structure(I) for cathananthine (Chant 8). Supporting cvidence for the position of the double bond was ob

double bond at C3-C4â€”viz., voacangine (III), dihydrocatharanthine (IV) (vide infra)â€”the Ci-C18bond is broken to afford 3-methyl-S-ethyl pynidine(83).

Mild hydrogenation of I afforded only one isomen, dihydrocatharanthine (IV). Its infrared spectrum was strikingly similar to that of coronar

idine. The differences in these compounds couldbe explained by examining Dreiding models whichshowed that the hydrogens could only have entered the molecule from the side nearest to Nb togive the axial ethyl group in dihydnocatharanthine.The ethyl group in the Iboga alkaloids has beenshown to be equatorial (@).7

The reduction of catharanthine and dihydrocatharanthine with LiA1H4 afforded corresponding

FREQUENCY (CM1)

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

z0


alcohols. The formation of the tetrahydro-1,3-oxazine derivative (70) from cathananthinol gave

additional evidence for the position of canbomethoxyl at C18.

The Iboga alkaloids possessing a carbomethoxyfunction at C13are known to decarboxylate smooth

7 The following evidence can also be correlated with this

assignment:a) The pK'a values of the axial series are higher than those

in the equatorial series; dihydrocatharanthine pK'@ 6.4,coronaridine pK'. 6.1 (38% DMF).

b) The rate of methiodide formation has been found to bea sensitive indicator of the configuration of the C-ethyl group,the rate being much faster in the axial series (private communication, Prof. M. Shamma, Penn. State Univ., State College,Pennsylvania).

c) The Re values of these compounds vary when they arerun by thin-layer chromatography on silica gel plates. (RT ofdihydrocatharanthine 0.2, coronaridine 0.8, solvent ethyl acetate-chloroform, 1 : 1.) This difference may be ascribed to theacidity of the silica.

tamed recently from decoupling experiments performed on the vinyl proton of the alkaloid.6

Saturation of this proton led to changes in theshape of the C-@ proton at 2.6@6, and, in addition,small decoupling effects were seen on the protonsat 4.18@(C-5 proton) and 1.96 (methylene of ethylgroup). As expected, dehydrogenation of catharanthine with Pd on carbon yielded 3-ethyl pyridineby the cleavage of the allylic C1-C2 bond. In isoquinuclidine Iboga alkaloids which do not have a

6 The correlation of isoquinucidine alkaloids (with no aro

matic enethexyl groups) is readily found by the observance ofa triplet centered at 6.8@ in the infrared spectrum. N. Neuss,â€œLillyCollection of Physical Data on Indole and Dihydroindole Alkaloids,â€• Eli Lilly and Company, Indianapolis, Indiana(1961).

6 The decoupling experiments were carried out by Mr. PaulLandis, Eli Lilly and Company, on a proton-proton decouplerpatterned after that described by Mr. L. F. Johnson, VarianAssociates, Palo Alto, California (private communication).




ly on saponification (@9), followed by mild acidtreatment, or alternately after prolonged refiuxingof the ester with hydrazine (65) in ethanol. Analogously, dihydrocatharanthine afforded the conresponding descarbomethoxy base epi-ibogamine(V), differing from the known ibogamine (VI) only

in the orientation of the C-4 ethyl group as previously mentioned. The final confirmation of thenature of the ring system was given by the isolation of 4-ethyl-@,6-dimethyl-11-H-indolo (@, 3-C)quinoline from Se-dehydrogenation of epi-ibogamine. The identical compound has been obtained

B

@@@ @u

â€˜ â€˜ â€˜ I I

400 300 260 100 [email protected].

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



1398 Cancer Research Vol. @3,September 1963

earlier by the same treatment of ibogamine (57).Catharanthine (I) must therefore be &-dehydnocoronanidine.

The nature of the second half of the dimericcompounds could best be understood by a studyof the dihydroindole alkaloid vindoline (VII) (@6,31), a C@[email protected] basic nitrogen didnot form a methiodide in analogy to the nitrogenwith PK'a 5.4 in the dimenic alkaloids. The natureof the six oxygens was determined by the formation of suitable derivatives (31). Thus, brief treatment of vindoline with acid afforded desacetylvindoline, C2@H30N2O5,corresponding to the loss of an

a Chart 9 shows vindoline and related compounds.

acetyl group. This was further corroborated in theinfrared spectrum by the appearance of a hydnoxylband (@.7@i)and corresponding reduction of intensity of the canbonyl band.

The presence of the methyl ester was shown bylithium aluminum hydride reduction of the basewith simultaneous removal of the acetyl to yieldvindolinol, C@H30N2O4. The infrared spectrum ofvindoline, in which a broad band at 3.5â€”4.0@twasconspicuous, showed that the fifth oxygen was ahydrogen-bonded hydroxyl. Formation of the diacetate C27H3@N2O7(acetic anhydnide in pynidine)was accompanied by the disappearance of thisbroad 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; Dihydrocat 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, thesixth oxygen was shown to be present as an anomatic methoxyl on a dihydroindole chromophore.The confirmation of the assignment of the oxygenfunctional groups was obtained from the N.M.R.spectrum (Chart 6A).

Vindoline consumed 1 mole of hydrogen at atinosphenic pressure, showing it to be a pentacycliccompound and afforded dihydrovindoline (VIII)which could be converted to an amorphous hygroscopic hydrochloride (Chart 9). Pyrolysis of

this salt at 195Â°â€”@00Â°C. in vacuum gave a distillate from which a C21H2@N@O2compound (IX) wasobtained in a 15 per cent over-all yield by directcrystallization from hexane. The methoxydihydno

(8). Thecleavagecanberationalizedasoccurringin the manner shown in Chant 10.@

The position of the carbonyl in the ketone (IX)was assigned on the basis of the presence of thepeak at m/e @98(M-4@ is equivalent with loss ofketene in IX or ethylene in XI) which suggestedthat this group involved either C-3 or C-4. Position4 was selected for two reasons; the typical ABXpattern at low field (3â€”3.58)in the N.M.R. spectrum which was consistent with structure VIII indicated three protons between the canbonyl groupand nitrogen; and equilibration with CH3OD/methoxide resulted in the introduction of only twodeuterium atoms per molecule (M = 34@); whereas if the carbonyl was at C-3 there would be three

I DELTA VALUES

2.65 3.45.u2.1â€”3.0

6.91 5.236.30

5.433.75@ __ 900

3.802.68

VII

CH3O R px R:CH3C@-XI R=CH3

H

CH)O@@@J1@HCH3

XIIVIII

H COOCH3

XIV

CH3d''211@

CH3

XIII

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

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

Vindoline (VII) was found to possess the samering system as the alkaloid aspidospermine (X)(7), obtained from plants of the Aspidospermagenus which is in the same family as Vinca. Thisobservation was made by comparing the massspectral fragmentation patterns of the pyrolysisketone and dihydnovindoline with that of the aspidospermine (X) derivative desacetyl-N-methyl aspidospermine (XI). In each compound the identicalintense peaks were found at rn/c 1@4,174, 188, and

@98(@8). A study by Biemann and co-workers hasshown that these four peaks are uniquely formedby cleavage of this type of a pentacyclic system

B

@N@YDCH3Q IC

CH3

IxXI

CLEAVAGE AT A,C rn/c â€¢174B.C rn/c =188C.D rn/c =298

(HEAVY LINES) A,D rn/c :124

CHART 10.â€”Mass spectral fragmentation of the vindoline

pyrolysis ketone (IX, Y =C= 0) and desacetyl-Nmethyl

aspidospermine (Y



1400 Cancer Research Vol. â€˜23,September 1963

exchangeable hydrogens. The other fragmentationpeaks occurring in these vindoline derivatives (p28)are consistent with the proposed skeletal structureand are due to more complex fragmentation whichwill be discussed elsewhere (7) . The similarities inthe mass spectra of dihydrovindoline and the C21-ketone indicate that no rearrangements of the ringsystem occurred during the pyrolysis. The correlation of the mass spectrum of vindoline with dihydrovindoline is rendered more complex because ofthe double bond in the former which alters thetypical fragmentation patterns somewhat; however, all other physical measurements are consistent with the simple hydrogenation of an iso

lated double bond in the conversion of VII to VIII.There remained then the problem of the place

ment of functional groups and the stereochemistry

still be consistent with the remainder of the spectrum). Since in the N.M.R. spectrum of vindoline(Chart 6A) there is a single unsplit proton peak atS.43Ã´ which shifts on removal of the 0-acetylmoiety to 4.07Ã´ the carbon bearing the acetoxylmust contain a single proton and those carbons oneither side of it must contain no protons. Likewise,acetylation of the 3-hydroxyl in vindoline does notresult in an analogous shift of any protons to alower field, and it must therefore be tertiary. Theonly arrangement consistent with these data isthat shown for vindoline (VII) with the hydroxyland carbomethoxyl groups attached to carbon 3and the acetoxyl on carbon 4.

The double bond was placed as shown also onthe basis of the N.M.R. spectrum which shows twocis 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 [email protected]. Since vindoline is stable to both borohydnide reduction and zinc and acid reduction, thedouble bond is not present as an enamine (7, Sposition) and therefore can only be in the 6, 7position as shown. It is interesting to note that a.related dihydroindole alkaloid, vindolinine (19)(Tablep2),foundinthesameplant,hasbeenshownto have structure XIV and possess an ester anddouble bond in the same position as those of yindoline.

The stereochemistry of the ring system of vindoline (VII) is assumed to be as found in aspidospermine (X), and this conclusion is supported by

the observation that the methyl (of the ethylgroup) in vindoline yields a 3 proton triplet in theN.M.R. at abnormally high field (0.48Ã¶).One canexplain this shift on the basis of increased shieldingdue to ring currents from the aromatic ring. Observation of models suggests that, with the aspidospermine stereochemistry, the methyl is in theproper position to exhibit such enhanced shielding.The stereochemistry of the functional groups atpositions S and 4 is, as yet, not certain, and a.discussion of this problem will be published elsewhere.

As mentioned above, spectral evidence stronglysuggested that the dimeric alkaloids were composed of a molecular combination of catharanthine(I) and vindoline (VII) (59). This assumptioncould be further substantiated by the productsof acid cleavage (concentrated hydrochloric acid,stannous chloride, tin, refiux) carried out on thefour active dimenic alkaloids. In each case therewas obtained upon chromatography of the reaction mixture an indole compound (vide infra) , followed by vindoline derivatives (Chart 11). Vin

XVII XVIII

COOCH3 CH3O@ â€¢OOCH3

XV R=CH3XVI R=CHO

CHART 11.â€”Structures of dimeric alkaloids and derivatives

of the molecule. The position of the aromaticmethoxyl was shown to be at either C-is or C-16by examination of the typical 1,@,4 aromatic proton pattern (ortho splitting J = 8 c.p.s., meta splitting J = @.Sc.p.s.). The final selection of C-16 isbased on the comparison of the infrared and ultraviolet spectra of vindoline with 6-methyoxy-N-methyldihydroindole (XII) and by the isolation ofind-N-methylnorhanmine (XIII), from a soda limedistillation of vindoline at 3@50â€¢This derivativealso indicates the position of the N-CH3 as beingon the anilino nitrogen. A second product (IX)which was identical to the C21-ketone obtainedabove was also isolated from the soda lime reaction.

The formation of the four major fragmentationpeaks in the mass spectrum of dihydrovindoline(vide supra) not only indicated the ring system ofthe alkaloid but showed that all the oxygen functions other than the aromatic methoxyl must belocated at carbons 3 and 4 (i.e., the peak m/e @98could not contain the ester, acetyl, or hydroxyl and




blastine (XV),9 vinleurosine, and vinnosidine afforded desacetylvindoline, thus proving the identity of the dihydroindole portion of these alkaloids.The corresponding fraction from the cleavage ofvincristine (XVI) yielded des-N(a@-methyldesacetylvindoline, M = 400, C@[email protected] massspectrum, as well as ultraviolet and infrared spectra, demonstrates the relationship of this compound to desacetylvindoline. Both VLB and yincnistine yield the same tetracyclic indole derivativevelbanamine (XVII), C19H2@N2O, the infraredspectrum of which clearly shows the oxygen to bepresent as a hydnoxyl (57).

A related tetracyclic indole derivative, cleavamine (XVIII), C19H24N2, was obtained from thecleavage of vinleurosine (the correct formula ofvinleurosine is still in doubt owing to difficulty ofpreparing a solvent-free sample) . Isolation of cleavamine (XVIII) demonstrates the relationship ofthe indole portion of C46alkaloids to catharanthine(I), since it was also obtained by a similar acidtreatment of the latter (@6, @7,@9).Mass spectralevidence indicates that cleavamine has structureXVIII and that velbanamine is its dihydrohydroxy analog XVIII. Recently, the structure ofcleavamine has been unambiguously substantiatedby an x-ray structure determination (47). Thestructure of the tetracyclic indole derived fromvinrosidine has not, as yet, been determined.

Thus, vinleurosine, vinrosidine, and VLB differfrom one another only with respect to the catharanthine portion of the molecule, whereas VLB(XV) and vincnistine (XVI) differ only with respect to the vindoline portion. The nature of thislatter difference was demonstrated in the followingway:

A comparison of the empirical formulae of VLB(XV) and vincnistine (XVI) (Table @)shows thatthe former has one less oxygen and two more hydrogens than the latter. The U.V. spectrum of yincnistine is quite different from those of the otherthree dimeric compounds (Table @),and this difference is indicative of a different substitution on

N(a) of the dihydroindole moiety. The infraredspectra of VLB and vincristine (Chart 4) are similar, with the exception of the presence of a strongadditional band,@ S.94j@ in the spectrum ofthe latter. The N.M.R. spectra differ in that theN-CH3 proton resonance at @.73Ã´present in VLB(Chart7)ismissinginvincnistine;and,conversely,in place of only one low field proton 9.85 in VLB,there are two in vincnistine at 9.5 and 8.9Ã¶.

Lithium aluminum hydride reduction of the twoalkaloids afforded good yields of the same penta

a Chart 11 contains structures of dimeric compounds andderivatives.

hydnoxy derivative, C42H5,N406 (XIX). This formulation is consistent with the reduction of twomethyl esters, one acetate and an N-fonmyl groupin the case of vincnistine. The N.M.R. spectrumof XIX shows, accordingly, only two methyl signals, aromatic OCH3 at 3.88 and N-CH3 at @.73Ã¶.The N-CHO of vincristine is, therefore, at theanilino nitrogen in place of the N-CH3 in VLB, andtherefore vincnistine is des-N(a)-methyl N(a)formylVLB (57).

The mode of attachment of the indole portionof the dimenic alkaloids to the dihydroindole fragment must be through the C-is aromatic carbon,since the N.M.R. spectrum shows only two protonsin a 1 :4 relationship in the aromatic portion of themolecule rather than the 1,@,4 three-proton pattern present in vindoline (Charts 6 and 7).

From the data presented thus fan one can drawa fairly complete picture of the structures of thedimenic alkaloids. Vincnistine and vinblastine arebest represented by structures XV and XVI, nespectively.'Â° The point of attachment of the vindoline portion as well as the location of the hydroxylin the catharanthine moiety is, as yet, obscure;however, on the basis of the biogenetic considerations discussed below, we prefer position 4' for thehydroxyl group. An N.M.R. study of the fullyacetylated VLB shows that this hydroxyl is tentiary. The point of attachment of vindoline in theother two biologically active alkaloids is probablythe same as for VLB, but the nature of their oxygenation in this region of the molecule is, as yet,unknown and under investigation.

The remaining alkaloids in Table@ can be classifled into three general groups on the basis of thechemical data available to date. Eleven of thealkaloids (Table @)are definitely of the dimenicindole-indoline class and closely related to the fouralready discussed. These are listed in Table 3 Theresemblance is seen in their infrared, ultravioletand N.M.R. spectra as well as the results of acidcleavage of several of them.

The second group (Table 3) which includes cathananthine and vindoline consists of ten monomenic alkaloids ranging in size from C18to C25cornpounds. The known alkaloids of Table 1 are alsoall in this category. In addition to vindoline (@8),catharanthine (56), and vindolinine (19) the structune of lochnenidine (47) and vindonosine (54)(desmethoxyl vindoline) have been described, andpartial structures of penivine (78), lochnenicine

10 The possibility of the indole portion being in the open

tetracycic form as in velbanamine (XVII) cannot be entirelyexcluded on the basis of analytical (57) or infrared data (58).

Note Added in Proof: Recent mass spectral studies haveshown that the formula of VLB should be CuH58O9N4, andthus the indole moiety must be tetracycic as in velbanamine.



ActivityAlkyl

halides(% prolongation)Leurosine:Methyl

iodideMethyl chloride67-14464Methylbromide41Methyl

tosylateDimethyl sulfateEthyl iodide45

44128N-Propyliodide33Ally!iodide71Bencyl

bromideCarbethoxymethyl bromide@9-Hydroxyethyl bromideInactive

Inactive45VLB:Methyl

iodideEthyl iodide2625Propy!

iodideDimethyl sulfate73 60

Cancer Research Vol. @3,September 1963140@

(31), sitsirikine (76), and lochneninine (54) havebeen proposed.

The third group (Table 3) consists of dimenicalkaloids which are not of the indole-indoline type.Vindolicine (76) and vindolidine (75) appear to bedimers of vindoline, and vincanodine (75) seems tobe related to the alkaloid vincine from V. minorLinn.

With this background of knowledge concerningthe chemical nature of the dimenic alkaloids our

TABLE 3

CLASSIFICATION OF THE ALKALOIDSFROM Vines rosea LINN.

@1Band Leurosine-like Alkaloids:1. Vincaleukoblastine (Vinblastine)2. Leurosine (Vinleurosine)3. Leurocristine (Vincristine)4. Leurosidine (Vinrosidine)5. Isoleurosine6.7. Carosine8.9. Catharicine

10. Neoleurocristine11. Neoleurosidine

Monomeric Alkaloids:1.2. Virosine8. Catharanthine4. Vindoline5. Lochnericine6. Vindolinine7. Lochneridine8. Sitsirildne9.

10.Miscellaneous Dimeric Alkaloids:

1. Vindolicine2. Vindolidine8. Vincamicine4. Vincarodine5. Carosidine

attention was turned toward the effect of structuna! changes on biological activity. One can consider the four active alkaloids as minor modifications of one another, and, as will be shown below,each of these compounds has a somewhat differentantitumor spectrum; as far as is known, they alsoexert a varying clinical spectrum and possess dif

ferent toxic manifestations (vide infra). A numberof the dimenic alkaloids of Table 3 such as neoleurosidine, neoleurocnistine, and cathanine possessonly one pK'. value (Table @),indicating that thebasic nitrogen in the catharanthine portion of themolecule has been changed, most probably byamide formation. None of these derivatives hasany activity.

It is apparent that all the alkaloids possess anacetyl group at C4 (vindoline portion). Removal of

this group destroyed antileukemic activity in VLB.VLB contains two hydroxyls, whereas vinleurosinehas only one (58). Complete acetylation of thesehydroxyls yields the respective acetates devoid of

antileukemic properties. LiA1H4 reduction of yinleurosine and VLB, yielding the correspondingcarbinols (58), likewise destroyed activity. The hydrogenation of the double bond in these compoundsalso reduces activity.â€•

An interesting change in the spectrum of biological activity of the active alkaloids was observedupon quatennization of the basic nitrogen in thecatharanthine moiety of these compounds withvarious alkyl halides (Table 4). Catharanthine(pK'a 7.5) readily forms a series of quaternary saltsfrom the appropriate alkyl halide in benzene atroom temperature, whereas the basicity of thebasic N (pK'a 5.5) in vindoline is such that it doesnot react under these conditions. In the case ofvinleurosine the methiodide and ethiodides were

TABLE 4

PER CENT PROLONGATION OF DIFFERENTALKYL HALIDES OF LEUROSINE AND

VBL IN P.1534LEUKEMIA

much more active than the parent base. WithVLB, vinrosidine, and vincristine, the quaternarysalts were considerably less active than the parentcompounds.'2 These results will be described inmore detail below. It therefore appears that therequirements for activity in the vindoline portionof the molecule are much more rigid than in thecatharanthine portion. A number of other denivatives are currently under investigation.

The nature of the biogenesis of these interestingnew dimeric compounds is of great interest. Wenkert and others have outlined detailed schemes forthe biosynthesis of many monomeric alkaloids of

1@Private communication, Dr. N. Neuss, Lilly Research

Laboratories.

12 Personal communication, Dr. G. H. Svoboda, Lilly Or

ganic Development Laboratories.



MaterialDosage(mg/kg/day)Av.

wt. change(gm) T/CAv.

survivaltime (days) TICPer

centincrease in

survivaltimePer

centindefinite

.survivorsDefatted

whole plantFractionAFractionBTotalalkaloids120

.00.530.0

6.07.515.015.075(Oral)+0

1/+2 7â€”0.6/+1.2â€”1.2/+1.2+0.8/+2.9+0.2/+0.6â€”2.3/+0.6+0.3/+0.5â€”1.5/+0.626/15

27/1924/1928/1730/1320/1330/1321/1373

38256112251130530

20402006000

JOHNSON el al.â€”Vinca Alkaloids 1403

the Iboga (catharanthine) on Aspidosperirta (yindoline) types. These are formed by the interactionof an appropriately oriented unit with tryptophan,and it has been shown that tryptophan is an excellent precursor for the Vinca alkaloids.'3 The nontryptophan portion is thought to be derived fromeither shikimic and pyruvic acids or, alternately,from acetate via mevalonate to cyclopentane monoterpenoid intermediates. The details of these

schemes can be found in the paper by Wenkert(87), and we shall discuss only the method of at

tachment. This is most probably photochemicalsince, as mentioned above, the major portion ofthe dimenic alkaloids are found in the leaves of theplant. The first step can be visualized as an oxidation of vindoline to a free radical by a peroxidasetype of enzyme followed by attack at the doublebond of catharanthine by this radical. Alternately,

A possibly analogous chemical reaction to thebiochemical addition of an aromatic radical to adouble bond is the Meerwein anylation reaction(67), in which the aromatic radical formed by decomposition of a diazonium salt adds to the leastsubstituted position of a double bond according tothe reaction in Chart 1@. Further investigationsinto the nature of this biosynthetic mechanism areplanned.

EXPERIMENTAL BIOLOGICALPROPERTIES

A@rITtmzoR STUDIES

The most striking experimental biological effectof the four Vinca alkaloids under discussion aretheir effectiveness in prolonging life or, in somecases, â€œcuringâ€•DBA/@ mice given implants of theP-1534 leukemia. The procedures for antitumor

TABLE 5

AcTIvITY OF ORIGINAL EXTRACT OF WHOLE PLANT AND CRUDE

FRACTIONS AGAINST P.1534 LEUKEMIA

the vindoline may react in the phenolic state before methylation, and the cathananthine may bealready oxygenated at the double bond (hydnoxy!or epoxide).'4 The resulting intermediate couldthen be reduced to a neutral molecule in variousways (such as reaction with water) to yield thewide variety of dimeric products found in theplant. The large number of dimeric products speaksfor the addition step of the free radical not beingenzymatical!y controlled. The formation of theN-CHO derivative of VLB (i.e., vincristine) isanalogous to the oxidation of various dimethylanilino compounds by horseradish penoxidase enzymeand hydrogen peroxide where oxidative demethylation occurs (p24). The peroxidase-catalyzed di

menization of aniline derivatives is known to yieldcompounds similar to vindolicine (a vindolinedimer).

13 Unpublished data, Drs. R. E. McMahon and M. Gorman,

Lilly Research Laboratories.

â€˜4This scheme resulted from a discussion with Prof. E.

Wenkert, Indiana University.

AR-N2C(@ [AR.] H2-C=CR@

AR@CH2@CR2@X [AR-CHf CR2]x

CHART 12.â€”Meerwein arylation reaction

testing in this laboratory have been previously de

scribed (4@) but essentially consist of intrapenitoneal inoculation of leukemic on ascites cells andsubcutaneous trocar implantation of solid tumors,with therapy being initiated the day following implantation and continuing for ten daily injections.Effectiveness is described in terms of per cent prolongation over saline-treated control groups in thecase of leukemic and ascites-beaning animals andper cent inhibition in terms of comparison of meantumor diameters of treated groups to those ofsaline-treated controls in the case of solid tumors.

The results obtained with the first extracts ofVinca rosea in December of 1957 against the P1534 leukemia are seen in Table S. From extracts



MaterialDosage(mg/kg/day)ToxicdeathsPer

centprolonga

tionJndef

inite

survivorsVinleurosine7

.510.0112520.0

150.0(Oral)0

00100

324176460

001

0Vinblastine0.05

0.10.30.450.450.61.5(Oral)0

00001041

537098131150700

00000

0Vincristine0

350.300.250.200.150.120.090.002

201000030

?226110

554932241

SS

200

0Vinrosidine10

.07.55.04.03.02.01

00000?

?127

7530214

50000

Dosage(mg/kg/

day)ToxicdeathsPer

centprolongationIndefinitesurvivors3.0

3.03.00.618.04.59.09.06.020.030.00

0222010000?

??

1811471511471741231482385

5300121414

TUMORHOSTALKALOIDS*ABCDLilly

mammarySarcoma 180Adenocarcinoma 755C-1498 leukemiaP-l534leukemiaL1210 leukemiaRidgeway osteogenic sarcomaMecca lymphosarcomaAKR leukemiaEhrlich ascitesFreund ascitesS-180 ascitesB82A leukemiaWalker carcinosarcoma 256Lilly mammaryGardner lymphosarcomaS-91melanomaX-5568 myelomaHigh malignancy cloneLilly rhabdomyosarcomaDBA/1

CAP'C57BL/6C57BL/6DBA/2DBA/2AKRAKRAKRCox std.CAl?'CAF@C58RatC3HCSHDBA/1C3HCSHRat+

â€”

â€”â€”

+++â€”

â€”

â€”

â€”

+++++++++

+++

â€”â€”

N.D.N.D.

+â€”

â€”

â€”â€”

++â€”

â€”

â€”

+++++++

++

â€”â€”

N.D.N.D.

â€”â€”

â€”

â€”

â€”

+++â€”

++++-

â€”

+++

+++

+â€”

+â€”

â€”+

â€”

+â€”

+++â€”

++++++

â€”

++++++++

â€”

â€”

â€”

+â€”

â€”

Cancer Research1404

with this type of potency and with the P-1534 asthe bioassay, Svoboda obtained vinleurosine andvinblastine. Results obtained with crude extractschemically free of vinleurosine and vinblastine areseen in Table 6. From extracts of this type ofpotency Svoboda obtained vincnistine and vinrosidine. A comparison of the anti-P-1534 activity ofthe four compounds under discussion is seen inTable 7. In this connection, it must be stated,however, that the response of the P-1534 leukemia

TABLE 6

ANTI-P-1534 ACTIVITY OF AMORPHOUSFRACTIONS FREE OF VINBLASTINE

AND VINLEUROSINE

(5 mice/group)

TABLE 7

ANTI-P-1534 ACTIVITY OF VINCA ALKALOIDS

(5 mice/group)

TABLE 8

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

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 prolongationof leukemias; +++ = 100% inhibition of solid tumors of > 100% prolongation of leukemias; N.D. = notdone.

Vol. @23,September 1963



CompoundDosage(mg/kgX 1 X 10)Av.

wt.change(gm)Av.

surv.(days)Per

cent

prolongationIndefinite

.survivorsVinrosidine

(VRS)Vinleurosine (VIAl)Vinblastine (VLB)Vincristine (VCR)VRS+VLRVR@S+VLBVRS+VCRSaline3

75015 .0000.2000 125

Asaboveâ€˜4 a

U ii+0

9+1 .2+1 .5+0 .4â€”0.8â€”0.6

â€”0.219

1822243123291437

296174

12869

1090

0010330

Treatmentstarted

(days afterimplantation)Mg/kg

Xfreq.Av.

surv.

(days) T/CToxic deathsPercent pro

longationIndefinitesurvivors1

36

1136100.2SX1X1O

025X1X100.25X1X10

0.25X1X102.5X12.5X12.5X12.5X1?/14

28/1433/14

30/1526/1523/1531/1533/153/5

3/50/5

0/103/103/100/100/10?

108146

937081981122/5

0/54/5

2/104/100/100/101/10


as maintained in our laboratories has changed inresponse to vinleurosine and vinblastine since 1958â€”

196@2,but the response to vincristine and vinrosidine during the period 1960 to the present hasremained unchanged. Vinb!astine currently givesa lower order of activity (50 per cent prolongation)at a maximum tolerated dose, whereas vinleurosinegives a somewhat greater and certainly more uniform response than previously. Unfortunately aâ€œtumorbankâ€• was not available to us during this

greater therapeutic effect in the combination thenapy. Combinations of the various active Vincaalkaloids themselves gave no evidence of anygreater activity in this tumor system either, withthe possible exception of vinrosidine (Table 9),which seems to be additive with both vinleurosineand vincristine but not vinblastine.

In a different tumor system, the B8@A leukemia,there was a suggestion of additive effect with yinleurosine and vincristine under certain regimens of

TABLE 9

COMBINATION THERAPY OF THE P-1534 LEUKEMIA WITH VINIt0SIDINE

AND OTHER VINCA ALKALOIDS

(5 mice/group)

TABLE 10

EFFECTS OF DELAYED TREATMENT WITH VINCRISTINE SULFATE ON P-1534

period, and we are unable to check sensitivity ret

roactively.The four â€œactiveâ€•alkaloids have similar expeni

mental tumor spectra but with some significantdifferences (Table 8). The most striking differencein spectrum is the activity of vinnosidine and yincristine against several AKR tumors.

A number of studies have been made expenimentally of combining vinbiastinc or vincnistinetherapy with therapy with other â€œstandard,â€•useful antitumor compounds against the P-i534 icukemia. These studies will be described in somedetail elsewhere, but in all cases there was a noticeable lack of any evidence for potentiation or

therapy. The two regimens in which this suggestion might be made was by giving one compounddaily for a week followed by a week's therapy withthe other compound, and secondly by giving onecompound in the A.M. and the other in the P.M.In the same system vinblastine and vincristinewere not additive.

Vinbiastine has previously been shown to beeffective against the P-1534 leukemia when therapy was delayed for several days after implantation(4@). Vincnistine is also effective in this manner(Table 10), More remarkable, perhaps, was thefact that â€œcuresâ€•or indefinite survivors could beobtained when therapy was initiated late in the



GroupNo. in groupNo.surviving

at 40 daysAv.surv.

time(days)A

BCD4)Challenged

with4@, spleensfrom

@i â€œcuredâ€•4) P-lS34animals@)ChaIIe@@@ed

with4@, P-l534leuke44J miacellS.2

[email protected]

.Lines of L1210Transfer

genera.tion

numberSubtelo

.centric

chromosome.

Relativeeffect

ofVLB5L1210

V, parent lineL1210/MP ifiL1210/A IVL1210/FU Xlii

L1210/AMPFUXYI2

L1210/AMPFU XYI2, frozen

L1210/AMPFU XVI

L1210/AMPFU XYI4, frozen49

79

3873

6497

4160Present

PresentAbsentPresent

PresentAbsent

PresentAbsent

PresentPresent

PresentPresent1

103

41

44

44

44

1406 Cancer Research Vol. @3,September 1963

disease. These animals were proved to be free ofleukemia by passage of sp!eens of such â€œcuredâ€•animals to normal DBA/@ mice. These animalsremained free of disease but were not resistant toa challenge with P-1534 cells. A typical experiment is seen in Table 11.

Resistance to a new challenge to P-1534 cellsafter â€œcuresâ€•has been seen, however, and deserves some comment in reference to the Vincaalkaloids in general. This resistance, in our hands,has not been a regularly predictable phenomenon.It is our impression that this occurs most frequently in â€œsurvivorsâ€• obtained by delayed treat

TABLE 11

PASSAGE EVIDENCE FOR â€œCURESâ€•OF YINCRISTINE-S04-TREATED ANIMALS

B-8174 leukemias and to a lessen extent in theB8@. He has also observed a peculiar relationshipin terms of cross-resistance between vincnistineand certain compounds in sensitive and resistantlines of these leukemias (10) . Vincnistine was mactive in P-815 and P-388 leukemias resistant toNSC 38@80 but is not additive to this compoundin a sensitive line. NSC 38@80 is potentiated inthese systems by stilbamidine, tniparanol, andamphotericin, whereas vincnistine is not. Crossresistance of these compounds is difficult to interpret in view of the obvious differences in chemotherapeutic behavior. Burchenal has also observedcross-resistance of vincnistine and vinblastine in aline of P-815 made resistant to vinblastine.

Dr. Dornis J. Hutchinson'6 has observed amarked effect of vinb!astine on several lines ofL1@10 leukemia resistant to amethopterin, 6-MP,5-FU, and all three compounds (Table 1@). These

TABLE 12

EFFECT OF VINBLASTINE ON RESISTANTLINES OF L1210

Four groups of normal DBA/2 mice were given intraperitoneat injections of suspensions of spleen cells from â€œcuredâ€•animals. A!! survived until 40 days, when they were challengedwith P-1534 leukemia cells indicating their susceptibility, ifthese cells had been present in the spleens of VCR.S04â€œcuredâ€•animals.

ment rather than by therapy initiated soon afterimplantation. It should be pointed out that Noble

and his associates have also reported resistance inâ€œcuredâ€•animals (17). They reported resistance inBDF, hybrid mice â€œcuredâ€• of P-i534 leukemia

and subsequently re-challenged with P1534 cells.We previously reported (39) our inability to repeatthis phenomenon but suggested the probability oftissue incompatibility. Goldin (@5) has reporteda similar phenomenon with halogenated derivatives of amethoptenin in the case of L1@i0 leukemia, but again this was in an F1 hybrid and didnot occur in the inbred DBA/@ animal. Noble alsoreported â€œcuresâ€•and subsequent resistance in atransplantable AKR leukemia in AKR mice. Thecuring and subsequent resistance to re-challengeof a strain-specific tumor in the inbred host isunique in experimental chemotherapy though notunusual in nonstnain-specific tumors in randombred animals.

Studies in other laboratories have revealed interesting data on resistant lines of mouse neoplasms. Burchenal16 has found vincnistine to beeffective by several routes in the P-388, P-815, and

S New code designations: 0 = inactive; 1 = slight activi.

ty; 2 = active; 3 = <6 â€œcuresâ€•;4 â€”>5 â€œcures.â€•For cornparison, the old code designations were: â€”, <24% increasein survival time; Â±, 25â€”59% increase in survival time; +,<60% increase in survival time.

lines are characterized by the presence or absenceof a subte!ocentric chromosome. The presence orabsence of the chromosome appears to have little

to do with sensitivity to vinblastine but in generalit does appear that the more compounds the vanous lines are resistant to, the greater the sensitivityto vinbiastine. Similar studies with vincnistine arein progress in Dr. Hutchinson's laboratory.

â€˜SPersonal communication, Dr. Joseph Burchenal, Sloan

Kettering Institute.10 Personal communication, Dr. Dorris Hutchinson, Sloan

Kettering Institute.



IntraperitonealIntravenousOral3.2

Â±0.02 mg/kg17 .47 Â±1 .57 mg/kg33.12Â±6.03mg/kg


ToxIcoLoGY

Pharmacology and toxicological studies of thesealkaloids in laboratory animals have not been published in detail. Vinblastine and vincristine havebeen the most extensively studied by Andersonand his associates.'7 Acute intravenous LD50's foryinblastine and vincristine in mice are approximately 17.0 and @.0mg/kg, respectively. Multipledoses of vinblastine and vincnistine caused markedleukopenia in rats and dogs at 0.50 and 0.1 mg/kg,respectively, causing death in some instances.Dogs have tolerated daily doses of @.0mg/kg ofvinleurosine with slight reversible leukopenia butfew other toxic effects. A daily dose of 5.0 mg/kg,however, was eventually lethal. Neither vincnistinenor vinleurosine is as potent a producer of leukopenia in animals as vinblastine.

An interesting phenomenon with vinblastine isthe markedly different LD50 by intravenous andintrapenitoneal administration in mice (Table 13).The mouse tolerated larger doses of vinblastinethan did the rat or the guinea pig. Subacute toxicity studies in rats given various doses of vinblastinerevealed loss of weight, with reduction in foodintake. The degree of leukopenia produced bone adirect relationship to the size of the dosage, and,with sublethal doses, the leukocyte count returnedto normal after dosage was discontinued. Thirtyrats that survived repeated !eukopenia-pnoducingdoses of vinbiastine were found to be normal atnecropsy.

Given intravenously in leukopenia-producingbut sublethal doses to dogs, vinblastine was foundto cause a reversible reduction of the myeloidcomponents of the bone marrow.

The main pathological finding in dogs givenpurposely lethal doses was hypoplasia of the bone

marrow. Of lesser prominence and consistency

were scattered petechial hemorrhages in other organs, with slight hepatitis and entenitis. Hypenplasia of the marrow and extnamedullany hematopoiesis were found at necropsy in those dogs thatwere given courses of vinbiastine in sublethal dosesand that were not sacrificed until after recoveryfrom the !eukopenic effects of the drug.

The cause of death in animals following lethaldoses of vinb!astine was bacterial disease secondany to leukopenia. Death did not occur immediately after the dosage but was delayed until infection had occurred. Potentiation of phenobarbitalanesthesia in dogs and prolongation of hexobarbital â€œsleepingtimeâ€•in mice with the use of vinblastine have been observed.

17 Personal communication, Dr. R. C. Anderson, Lilly Re

search Laboratories.

Toxicity studies with vincnistine have been conducted in rats, rabbits, cats, dogs, and monkeyswith the following results:

Rats.â€”Intravenous doses administered 5 timesweekly caused leukopenia, weight loss, and reduced food intake in direct relationship to thedoses, which were 0.5 and 0.@5 mg/kg. Rats receiving 0.@ mg/kg daily by the intraperitonealroute survived 10â€”@1doses. They developedleukopenia, weight loss, diarrhea, alopecia, andblanching of tails, ears, and feet. Clonic extensorconvulsions and catalepsy occurred before deathin one animal.

Rabbits.â€”Intnavenous doses given S times weekly caused leukopenia and death within three tofive doses at levels of 0.1 and 0.@ mg/kg. Beforedeath, signs of muscular weakness (head drop,kneeling and sprawling positions) appeared. Animals receiving 0.0@5 and 0.05 mg/kg appearednormal after @8doses.

TABLE 13

LDHOFVINBLASTINEIN MICEBY DIFFERENT ROUTES

Cats.â€”The animals received the drug by theintravenous route 3 times weekly. Those given 0.@mg/kg were dead after two doses. A cat administered 0.1 mg/kg was killed after two doses becauseit was moribund. When 0.05 mg/kg was given toone animal, it died after eight doses. A cat receiving 0.0@5 mg/kg was killed after thirteen dosesbecause of its extremely poor condition. Weightloss, general debilitation, clonic convulsions, andleukopenia were observed.

Dogs.â€”Vincnistine was administered by the intravenous route 5 times weekly. The dogs given0.05 mg/kg were dead or had been killed after thefifth dose. One animal receiving 0.O@5mg/kg waskilled after seven doses because it was in poor condition. Three animals survived twenty doses atthis level. Leukopenia, tremors, vomiting, weakness, and bloody stools were observed in these animals.

Monkeys.â€”Vincnistine has been administeredorally in solution (1 mg/mi) to two male monkeysat dosage levels of@ and 4 mg/kg, respectively.Hematological and blood chemistry studies oneach monkey were essentially unchanged throughout a @9-dayobservation period. Vomiting wasobserved in the animal that received 4 mg/kg.

A single intravenous dose of 4 mg/kg killed one



VincristineFolic acidFolinic acidAv.

survivaltime and

range(days)Mortality

at SOdays2.5X1

I.Y.

a52X1X10I_P.14OX1

X10I.P.11

(2â€”26)

7 (2â€”19)

7 (4â€”10)17/19

16/18

4/16

VincristineFolicacidFolinic acidAv.

su@vival

time

(days)Per

centprolonga

tioiiIndefinite

.survivors0.2

0.20.2

0.20.250 140

501401405085

?2.313*

216?

22?

15*172

?77

380?

41?0

540

415350

Cancer Research Vol. p23, September 19631408

male monkey in 30 hours. Constricted pupils, anonexia, and diarrhea were observed prior to death.Reduced hematocrit reading, leukocytosis, andelevated blood sugar, blood urea nitrogen, andserum transaminase were also observed. The majorpathological finding was a severe acute cytotoxiceffect on the granulocytic and erthnocytic series inthe bone marrow.

Two male monkeys survived two doses of yincristine,@ mg/kg each, administered 36 days apart.Vomiting, weakness, tremors, weight loss, leukopenia, and thrombocytopenia were observed inboth animals. Decrease in blood sugar was noted

TABLE 14

EFFECT OFFOLIC ANDFOLINIC ACIDS ON YCR ToxICITY

Dosaoz (un/au)

These effects, as well as mild to moderate degenerative changes in the liver and kidney, were related to dosage. An unusual reaction noted wasproliferation of the epithelium of the renal collecting system.

A recent observation has made been in thislaboratory in reference to host protection of miceto lethal doses of vincnistine (Table 14). In normalmice folinic acid has a protective effect, whereasfolic does not. Neither folic nor folinic appears tobe involved in the anti-P-1534 activity of vincnistine. Indeed, rather than reversing the antitumoreffect both compounds given with vincristine appear to give somewhat better antitumor effectsthan vincnistine alone (Table 15).

In view of this host protection phenomenon, wehave investigated the effect of vincristine and yinblastine on both folic acid reductase and fonmyltetrahydrofolate synthetase in vitro. The enzymeswere assayed by either a crude homogenate or par

TABLE 15

EFFECT OF F0LIc AND FOLINIC ACIDS ON

VCR THERAPYOFP.1534(5 mice/group)

Dosaux(uo/aoX 1X 10)

in one of the animals. Pathological studies aftersacrifice revealed hypoplastic marrow infiltrated

by lymphocytes. The acinan cells of the pancreaswere unevenly depleted of secretion granules.Widespread atrophy of the mucosa of the largeintestine occurred in one monkey. There were mildtoxic manifestations in the liven and kidney and

interstitial edema and adipose infiltration in themyocardium of the left ventricle of one monkey.

Eight monkeys (one male and one female perdosage level) were given repeated doses of vincristine intravenously at levels of 0.1 mg/kg/day, 0.@mg/kg/day, 1 mg/kg twice weekly, and 1 mg/kgweekly. Genera! body weakness, anorexia, tremors,diarrhea, and ataxia were noted in all groups. Nodeaths were observed at the 0.1 mg/kg/day dosagelevel, but death did occur after seven on twelvedoses of 0.@ mg. vincnistine per kg. daily, two orthree doses of 1 mg. vincnistine pen kg. twice weekly, and five doses of 1 mg. vincnistine per kg.weekly.

Leukopenia was generally observed. No majorblood biochemical changes were noted except tenminal blood values from moribund animals and anoccasional elevation of serum transaminase.

Autopsy of these monkeys revealed no charac

tenistic gross findings. Histological studies showedreduced celluanity of myeloid and lymphoid tissue.

S Saline controls.

tially purified fraction from mouse liver. Folic acidreductase activity was measured by the procedure

of Werkheimer et a!. (88) and formyl tetrahydrofolate by the method of Rabinowitz and Pricer(64). Neither alkaloid has any influence on eitherof these enzymatic activities in vitro at concentrations of drug up to 100 @g/ml.

BIOCHEMICAL AND MECHANISM STUDIES

Since the alkaloids of Vinca rosea comprise anew class of antitumon agents, it was of considerable interest to us to study the effect of these compounds on various biochemical reaction sequences



CaLL TYPETiua(MR.)LACTATE

PRODUCTION

@ CONTROL)Aerobic-

AnaerobicS-180

Freund0â€”2

2â€”44â€”8

0â€”22-44â€”8100

9896

9998

102100

10198

979996

Cell typeAddition of VCR(pg/mI)0,

uptake(%control)S.180

Freund0

1050

200

01050

200100

1009885

100101

9989

Additionof VLB(jig/mi)Per

centcontrol15

150103 94CellAddition of VLB

(jig/mI)@â€˜

uptake

(%control)Normal

spleen

Leukemic spleen5(P.1534)

S-iso Ascites cellst0

20.541.0

020.541.0

0100100

98102

1009995

10099


with the hope of uncovering information concerning their mode of action.

Our initial experiments were devoted to a studyof the effects of vinb!astine and vincristine on therate of respiration and glycolysis of tumor cellsunder various conditions. S-180 ascites cells werechiefly employed ; however, some studies were performed with Freund ascites cells and also spleentissue from normal mice and mice carrying P-1534leukemia. The results of experiments in this areaare summarized in Tables 16 to 19. Each tablerepresents the results of at least two separate expeniments.

TABLE 16

RESPIRATION OF S-18O ANDFREUND ASCITES CELLs

In intact ascites cells (Table 16), either 8-180or Freund, concentrations of less than 50 ,@g/mlvincnistine appear to have no effect on respiration.However, when the concentration of drug is raisedto very high levels some inhibition appears. Although data are not presented for vinbiastine, essentially the same results were obtained (10â€”iSper cent inhibition with 100 @&g/mi).In Table 17

TABLE 18

GLYCOLYSIS OF S-180 AND FREUND ASCITESCr@LLsIN PRESENCE OF YIN

CRISTINE (50 @iG/ML)

Glycolysis measured as lactate production.Krebs-Ringer bicarbonate buffer containing0.2% glucose was employed. Incubation was at370 C. with 5% CO@-95% 02 or 5% CO@-95% N3

as gas phase.

TABLE 19

ANAEROBIC GLYCOLYSISH0M0G-ENATE OF S-iSO ASCITES CELLs

Respiration measured by standardWarburg technic over 4-hour period.Krebs-Ringer phosphate buffer containing 0.1% glucose used with air as atmosphere.

TABLE 17

RESPIRATION OF S-iSO HOMOGENATEAND SPLEEN MINCE

Anaerobic glycolysis measuredas lactate production. Medium employed was essentially that of LaPage (48).

it can be seen that vinblastine was without effecton the respiration of spleen mince from normal orleukemic animals. In addition, the complete lackof influence of vinblastine, at a relatively high concentration, on homogenates of S-iSO ascites cellsis noted. This latter result is in contrast to the resuits obtainedwith intactcells, since correspondingly high levels of vinblastine do give rise to a iOâ€”i5per cent inhibition of respiration. In Tables 18 andi9, the inability of vinblastine and vincnistine toaffect the rate of glycolysis as measured by lactateproduction is shown.

To summarize our results, it can be said that we

S Spleen from DBA/2 mice. Respiration

measured with mince in Krebs-Ringer phosphatebuffer by standard technic over period of 30minutes.

t S-180 homogenate prepared in 0.25 M sucrose. Medium employed was essentially that ofAisenberg (1). Results are similar whether succinate or pyruvate and fumarate are used as substrate. 02 uptake measured at intervals over a1-hour period.



ADDITIONTIME(uz@.)Spac.

ACTIVITY(DPM/uu)RNA

@DNA0

10@gVCR/ml

20 @gYLB/ml30

90270

3090

270

3090

27016,500

39,60040,000

15,90037,20038,700

14,60036,30038,60027,400

68,40072,400

24,60069,10073,300

26,00066,10061,600

ADDITIONSpac.

ACTIVITY(DPM/MG)RNADNA0

1 zg YCR/ml1OOj@gVCR/ml20 j@gVLB/ml14,800Â±740

14,600Â±78018,300Â±67018,800Â±6904,520Â±280

4,610 Â±2804,560Â±2304,470 Â±280

ADDITIONINCUBATION TIME

(MIN.)SPECIFIC

ACTIVITY(DPM/uo)RNADNA50@gYLB/ml5

25 @gYCR/mlt

10@sgMTX/ml@

Saline30

90

3090

3090

30901,270

1,470

1,3801,500

1,0101,280

1,2701,5902,660

3,290

2,5703,020

2.0902,640

2,5703,120

Cancer Research Vol. 23, September 19631410

observe neither inhibition nor stimulation of nespination or glycolysis in the presence of these alkabids at levels approaching physiological concentrations. At very high concentration values, webegin to obtain inhibition. However, we would notconsider this particularly meaningful. Our resultsagree with those of Beer (6), who measured therespiratory quotient of liver from animals treatedwith yinblastine, but differ from those of Hunter,'8who reported that vinbiastine stimulates aerobicacid production and inhibits nespiration. However,Hunter employed different cell lines so that a di

rect comparison with our data is not possible.The pronounced effect of vinblastine on mitosis

led us to studies on the biosynthesis of nucleicacids. We have measured the incorporation offonmate-C'4 and glycine-@-C'4 into the nucleic acidsof 5-180 ascites cells in vitro. In Table @0the resultsof a typical experiment of this type are shown. Ingeneral, washed 6-day ascites cells were employed

TABLE 20

FORMATE-Câ€•INCORPORATIONIN S-180 AScITES

The results of an experiment in which animalswere pretreated in vivo with drug is given in Table

@-Twenty-four hours after treatment the ascites

cells were removed and incubated in vitro with

TABLE 21

GLYCINE-2-C'4 INCORPORATIONIN S-180 ASCITES

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

TABLE 22

GLYCINE-2-C'4 INCORPORATION IN 8-180 AsCITES

Final concentration: 0.2 @il.packed in 2.5 ml. total volumeKrebs-Ringer bicarbonate buffer containing 3.5 gig. (0.3 iic.)C'4-formate/ml; 5% CO,-95% N3 gas phase.

with Krebs-Ringer bicarbonate on Krebs-Ringenphosphate buffer containing Odâ€”0.4pen cent glucose.

After 60 minutes incubation at 37Â°C., proteinsand nucleic acids were precipitated with perchlonicacid and determined after separation by the method of Scott et a]. (68). The results shown in Table

@0indicate that neither vincristine nor vinblastinehas any measurable effect on the incorporation offormate into nucleic acid in 5-180 ascites.

Table @ipresents the results of a similar expeniment in which incorporation of glycine-@-C'4 wasused as a measure of nucleic acid formation. Againvinblastine and vincristine demonstrate no appreciable effects. In this particular experiment, aenobic conditions were employed. However, anaerobicexperiments with glycine-@-C'4 as substrate gavesimilar results.

18 Personal communication, Dr. J. C. Hunter, National

Cancer Institute.

a Cells from group of six mice treated 24 hours earlier with2.5 mg/kg VCR.

t Cellsfromgroupofsixmicetreated24hoursearlierwith5.0 mg/kg VLB.

@ Cells from group of six mice treated 24 hours earlier with1.0 mg/kg methotrexate.

Incubation: 0.3 ml. cells (packed) in 2.5 ml. Krebs-Ringerphosphate buffer containing 0.1 mmole amino acids (all exceptglycine), 1 rnmole glucose, and 0.9 @c.(22.5 gig.) glycine-2-C'4.Incubated at 37Â°C. in 02 atmosphere.

glycine-@-C'4 under aerobic conditions. As in theprevious experiment, vinblastine and vincnistinehave no effect, whereas methotrexate treatmentdepresses the incorporation to a significant extent.

Very recently, however, some preliminary ne



Exper.pg VLB/miliiliterCounts/mm/mgproteinA*

Bt10

501500

101000160Â±7

150Â±6124Â±5168Â±8

176Â±6166Â±5172Â±6


sults have been obtained with HeLa cells in tissueculture experiments, lasting over periods of 1 week,which suggest that vinblastine and possibly alsovincristine may act as inhibitors of de novo synthesis of nucleic acids. These studies, if confirmed,will be reported elsewhere in detail.

The capacity of 8-180 cells to incorporate glycine-1-C'4 into protein is presented in Table @3.Atrelatively low concentrations of vinblastine, no appreciable effect on protein synthesis is noted. Ata higher concentration, however, some inhibitionbecomes apparent. In the second half of Table @3,

the influence of vinblastine on the capacity of acell-free microsomal system to synthesize proteinwas tested. Again no large differences are to befound between the treated and untreated samples.

In agreement with the results reported by Beer(6)fornucleicacidsynthesisin regeneratingliverand Ehnlich ascites, we conclude from our datathat neither vinblastine nor vincnistine influencesthe rate of nucleic acid formation of purine synthesis in S-180 ascites. Furthermore, we can findno effect by these agents on protein biosynthesisat relatively low concentrations of drug. The nesults of all of our experiments tend to follow thepattern on little of no activity in vitro at concentnations approaching physiological levels; whereaswe begin to observe inhibition at very high concentrations. It should be pointed out that our cxperiments reported in this paper were carried outwith chiefly a single line of ascites tumor cells invitro. One must consider the possibility that in

vitro activity cannot be equated with in vivo activity. That is, the injected alkaloid may be modifiedin some way by the host to provide an â€œactivecomponent.â€•

CLINICAL STUDIES WITH THEVINCA ALKALOIDS

This clinical discussion of the Vinca alkaloids isconfined to vinblastine sulfate and vincnistine sulfate, which are the only members of this family ofdrugs which at this time have undergone extensiveclinical trial. Before a detailed discussion of yinblastine and vincnistine individually, we will briefly compare and contrast the more salient clinicalfeatures of both.

Both vinblastine and vincnistine have beenfound to be useful in the treatment of some typesof human cancer. There is no clinical evidence ofcross-resistance with radiation or with other presently used oncolytic agents (4, 9, ii, 13, 14, @0,3@,44, 45, 80, 81, 85). Despite only a minor difference in their chemical structures, the clinical effects of vinblastine and vincristine differ considerably ; and, surprisingly, there is no clinical cvi

dence of cross-resistance between them (3, 11, 14).The dosage requirements of vinblastine and yin

cnistine differ markedly (3). Whereas the weekly

intravenous dose for the average solid-tumor patient lies between 0.10 and 0.@0 mg/kg of vinbiastine, the dose of vincnistine is only one-tenth toone-quarter of this (i.e., 0.01â€”0.03mg/kg).

Both vinblastine and vincristine are alike insparing hemoglobin and platelet production (3, 4,ii, is, 33, 44, 69, 81, 84, 89). The limiting factorto dosage with vinblastine is leukopenia (31, SO).With vincnistine, on the other hand, leukopenia isuncommon; and when it has occurred its degree

and duration have generally not constituted a senious problem (3, ii, 13, 44, 45, 66, 69).

TABLE 28

INCORPORATION OF GLYCINE-1-Câ€•INTO PROTEIN in Vitro

S Intact S-18O ascites cells incubated in

Krebs-Ringer bicarbonate buffer at 37Â°C. for30 minutes with varying concentrations of VLBand glycine-1-C'4.

t Microsomal system derived from S-iSOascites cells. Glycine-l-C'4 incorporation measured essentially according to the system ofLittlefield and Keller (49).

With very high doses of vinblastine, manifestations interpreted as central nervous system toxicity has been encountered (@0,31, 43) ; but thisoccurs only rarely, and then only if dosage is increased above the leukopenia-producing magnitude. In contrast, at least some neuromuscularabnormality has been encountered in most patientswho have been treated with vincnistine, and it isthis problem rather than leukopenia which limitsthe magnitude of vincnistine dosage (3). Neunomuscular toxicity apparently may also involve theautonomic system. Indeed, severe and even obstructive constipation may develop with chronicvincnistine dosage if stool-softening agents andlubricants are not given to prevent this. Constipation so rarely follows vinbiastine dosage that thereis no need to medicate prophylactically on its account.

In our experience, epilation occurs less frequently with vinblastine than it does with vincnistine



Cancer Research Vol. @23,September 1963141@

approximately 10 per cent of patients being af

fected by the former and perhaps twice this number by the latter drug.

Both vinblastine and vincnistine have provedclinically useful in the treatment of Hodgkin'sdisease and the other lymphomas including lymphosarcoma (3, 4, 9, ii, 14, @0).Effectiveness hasbeen shown not only when given as de novo therapybut also following development of refractoriness toalkylating agents and/or radiation. Although itremains to be determined which of the two drugswill prove more effective in these situations, it isthe early impression of the Lilly Clinic group thatvinblastine may be better than vincnistine forHodgkin's disease, whereas vincristine may bemore useful in lymphosarcoma (3, 4). However,clinical studies with comparable groups of patientsin the same stages of their illness will have to becarried out to determine the correctness of theseimpressions.

The leukemias constitute another area in whichit is of interest to note apparent differences@ in nesponses to the two drugs. Although published papens report remissions with vinblastine in the leu

kemias as relatively few in number and often

incomplete, in some patients the drug has beenreported of definite benefit following developmentof resistance to other agents. Warwick et al. (84)reported four of nine patients with acute stem-cellleukemia responding to vinblastine. Whitelaw andTeasdale (89) report eighteen cases of acute blastcell leukemias treated with vinblastine. Of these,three of eight patients without previous therapy,and four of ten resistant to previous therapy, manifested favorable response for varying periods oftime. (One of the latter group of four patients responding had simultaneous 6-mencaptopunine thenapy.) Hill and Loch (33) included reports on theuse of vinblastine (often combined with otheragents) in the leukemias and concluded there wassufficient benefit to warrant further clinical trial.

The Acute Leukemia Cooperative Group B (44,45) obtained remissions in only one of 2.9 childrenwith acute leukemia. Details of this study have notas yet been published.

On the other hand, vincnistine has frequentlybeen found useful in the leukemias, especially theacute leukemias of children. Karonet al. (45), using

vincnistine, obtained a complete remission rate inchildren with acute leukemia at least as goodas with any of the presently accepted standardagents (6-mencaptopurine, steroids, on methotnex

ate). Other investigators have verified the useful

ness of this agent in acute leukemia in children.'9

Indeed, some centers now use vincnistine firstin the series of drugs they employ for the treatmentof acute childhood leukemia because vincnistineis unlikely to exacerbate the neutropenia, anemia,and thrombocytopenia which may be present inacute leukemia even before treatment is begun.2Â°

Overlap in the spectra of activity of vinblastineand vincnistine has been reported to involve a vanety of neoplasms including carcinomas of thebreast2' (4, 43, 84), ovary (4, @0,69, 79, 80), kidney(ii, 33, 79), and cervix (4, 13, 14), chonionepithelioma (13, 14, 3@), nhabdomyosarcoma (3, 79),reticulum-cell sarcoma (14, 33, 84, 89), and neuroblastoma (ii, 69, 79). As is the case with Hodgkin's disease, comparison between the effectivenessof vinblastine and vincnistine against these tumorsmust await further trials as well as a better understanding of administration and dosage.

Vinblastine has been reported to produce tumorregression in patients with metastatic adenocancinoma of stomach (4, @0,84), colon (34), and rectum (35). Vincristine has thus far not producedsignificant benefit in any patient with adenocar

cinoma of the gastrointestinal tract. Vincristinehas been reported to have some useful effect in asingle patient with carcinoma of the prostate (13,14). Similarly, benefit has been reported with yinblastine in isolated instances of carcinoma of theprostate.'9 Obviously more patients with carcinoma of the prostate should be included in trialsof these drugs.

VINBLASTINE SULFATE

Vinblastine is the official generic name for thealkaloid formerly known as vincaleukoblastine.This contraction was necessitated by the U.S. Phanmacopeia regulation that a generic name shouldnot imply a therapeutic use. The earlier name wasfelt to suggest usefulness in leukemia.

Vinblastine was made generally available in1961, with recommendations for its use in Hodgkin's disease (4, 14, @0,@i,84, 85, 89) and choniocarcinoma (3@,85). Since then it has been reportedto be active against many other malignancies including carcinomas of the lung (4, 33, 50, 81, 90),breast (4, 43, 84), cervix (4), uterus (35), ovary(4, 80, @0,81, 90), stomach (4, @0,81, 84), colon(33, 34, 81, 90), rectum (35), and kidney (33, 8i,90), squamous-cell carcinomas@ (33, 81), nhabdomyosarcoma (89), melanoma (37, 90), seminoma

20 Personal communication, Dr. Sidney Farber, Children's

Hospital and Dr. Emil J. Freireich, National Cancer Institute.

31 Personal communication, Dr. James F. Holland, Roswell

Park Memorial Institute.

13 Personal communication, Dr. Donald Rochlin, University

of California, Med. Ctr., Los Angeles.19 Data received at the Lilly Laboratories for clinical re

search.



TumorObjective improvementSubjective

improvementNo

responseBronchus:

Squamous-cellAdenocarcinomaUndifferentiatedOat-cellNot specified2

1111

1

17

1

5


(84), tenatoma (84), and embryonal-cell carcinomaof the testis (37), reticulum-cell sarcoma (33, 35,36, 51, 84, 89) monocytic leukemia (34, 36, 37,

81), erythroleukemia (36), astrocytoma (4, 5@,91),mycosis fungoides (33, 51).

Delay in recognition of this broaden spectrum isin part due to the short duration of vinblastinetreatment which was used in earlier trials. Inmany of the early trials, vinblastine therapy wasdiscontinued if no tumor shrinkage had occurredafter@ weeks of dosage. It is now evident that inthe case of solid tumors a response to vinblastinemay occur only after 10â€”if weeks of therapy (4,34, 43). This is in contrast to the rapid responsewhich occurs in cases of Hodgkin's disease andchoniocancinoma, where benefit usually follows thefirst few doses of vinblastine.

Not only were some early trials too brief, butlarge and toxic doses were often employed. Profound leukopenia and other severe side-effects resulted from â€œpressingdosage to toxicity.â€• We prefer a careful incremental approach to dosage. Webelieve a patient's dosage with vinblastine may beestablished as follows: 1st dose, 0.10 mg/kg; @ddose, 0.15 mg/kg; 3d dose, 0.@0 mg/kg; 4th dose,0.@5 mg/kg; and 5th dose, 0.30 mg/kg, etc.

One intravenous dose every 7 days is given according to this schedule until the leukocyte countis reduced to approximately 3,000 cells per cu.mm. The amount of vinbiastine which will producethis degree of leukopenia varies from patient topatient. Once the leukopenia-producing dose is established, doses 1 increment smaller than thosemay be administered at weekly intervals for maintenance. It should be emphasized that, even though7 days have elapsed, the next dose of vinblastineshould not be given until the white cell count hasreturned to at least 4,000 cells per cubic millimeter.Should benefit occur with a dose below that whichcauses leukopenia, there is no need to increase thesize of subsequent doses. In this way many pa

tients may be treated with doses well below theleukopenia-producing level (4, 84). Our experiencehas shown that the maintenance dose for most patients is 0.15 on 0.@0 mg/kg of body weight.

We believe it best to continue with vinblastinedosage at regular 7-day or 14-day intervals for aslong as a patient will respond (4, 43, 50). Wheresuccessful therapy has been discontinued, patientshave usually relapsed about 3 weeks after the lastdose. Since long-continued dosage at regular 7- to14-day intervals was only rarely used in the earlytrials, it now seems probable that the lack of maintenance dosage may explain the short duration ofmany of the early responses reported.

It is apparent that the spectrum of activity of

a new oncolytic drug cannot be properly evaluateduntil the optimal magnitude and duration of dosage are ascertained. Needless to say this requiresstudy over a considerable period of time.

An interesting example of what might be accomplished without significant side effects, with use ofan incremental approach to dosage, is suggested inthe preliminary report of J. C. W. MacFanlane etal. (50). This group reported that vinblastine hadprolonged the survival of a series of patients withcarcinoma of the bronchus as compared with thatof untreated patients or patients treated by radiation and/or nitrogen mustard. Although some ofthese patients whose survival was significantlyprolonged exhibited tumor shrinkage, others merely showed an unchanged radiological picturethroughout their several months of treatment.This suggests that to require prompt demonstra

TABLE 24

RESPONSE OF CARCINOMA OF BRONCHUS TO YINBLASTINE

tion of gross tumor shrinkage in order to decidethat a drug is effective may result in our missingmore subtle long-term tumor-inhibiting and hostsupporting effects. We may not always rendera service to the cancer patient when we try asquickly as possible to produce tumor shrinkageby pressing the dose of an oncolytic drug totoxicity. This is not meant to imply that it isunnecessary to demonstrate objective evidenceof tumor shrinkage with a new drug, but ratherto suggest that a new drug might be found toprolong survival and inhibit tumor growth ifadministered in subtoxic doeses for long periods.Obviously such trials must be conducted in full necognition of the natural variation in tumor growthwhich can occur spontaneously from time to time.To avoid drawing falselypositive conclusions, suchtrials must be statistically controlled and shouldinclude adequate numbers of patients for a sufficient length of time.

Carcinoma of the bronchus has also been reported as responsive to vinbiastine by the MidwestCooperative Chemotherapy Group23 (Table @4).

23 Paper presented at the Nov., 1961, meeting of the Nation

al Cancer Chemotherapy Service Center in Washington, D.C.



Cancer Research Vol. @23,September 19631414

To quote from their papen on this topic : â€œThirtyone patients were studied, of whom @1were evaluated. All but 4 patients had received prior therapywith x-ray, alkylating agents, or 5-fluonouracil.The priming dose of vinblastine ranged from 0.15to 0.6 (mean 0.33) mg/kg. Twenty patients received maintenance therapy varying from 0.15 to

@.37(mean .7@) mg/kg over 7 to @6(mean 33)days. Five patients showed objective improvementof short duration (@i to 48 days), but one patientwith undifferentiated carcinoma showed markedregression of a right hilan mass which, on maintenance therapy, was sustained for @i5days. It is

of interest that four histologic types of carcinoma,squamous, oat cell, adenocarcinoma, and undifferentiated, were included in those with objective

improvement. Three patients obtained some degree of pain relief lasting 7, 8, and 67 days.â€•

In this series the duration of the trial of vinblastine therapy varied markedly from patient topatient and i4 days' trial was considered adequatefor evaluation. In the light of other experiences, itwould appear that this duration of trial was toobrief for the demonstration of a maximal responserate.

In the first clinical trial of vinblastine by theEastern Cooperative Group in Solid Tumor Chemotherapy (@O),nine patients with carcinoma of thebronchus were considered evaluable. None of thesewas reported to have shown tumor regression. Inthis series an inadequate duration of trial was defined : â€œ.. . when no toxicity occurred and whenthe cumulated duration of drug administration andtoxicity was less than @1days.â€•Again the durationof trial, though it produced a high percentage ofresponses in Hodgkin's disease, was brief for solidtumors.

Warwick et al. (84) have stated: â€œTherewasno definite evidence of a correlation between thedegree of leukopenia and the beneficial effect.Remissions in Hodgkin's disease have been ob

served in the absence of leukopenia followingtherapy.â€• The toxicity which can follow the useof excessive initial dosage may only deter aninvestigator from continuing the use of a new drugfor an adequate period of trial.

The earliest reference to the use of vinblastinein the treatment of breast carcinoma was by Warwick et a!. (85) in 1960. They wrote: â€œ.. . on thei@th day the supraclaviculan nodes were no longerpalpable, and induration and thickening of theskin had disappeared. There was less swelling ofthe arm. . . The patient was discharged feelingwell, apart from stiffness in the left shoulder. ...The ulcer on the chest wall had completely epi

thelialized. This remission was maintained for 6weeks.â€• The dosage used for this patient was asingle course of 0.15 mg/kg of vinblastine, intravenously, daily for 4 consecutive days. No furtherdoses were given. In the light of subsequent expeniences this dosage was excessive and lacked themaintenance treatment necessary for prolongedbenefit.

In 1961 Barbara Johnston et a!. (43) of StLuke's Hospital, New York, published on eightpatients with carcinoma of the breast treated withvinblastine. Six patients were evaluable; and ofthese, three were improved both objectively andsubjectively. Dr. Johnston24 has stated that someof these remissions have now been maintained formore than 1 year and that her series, now grownlarger, continues to show the same order of responsiveness.

The dosage schedule employed by Johnston etal. (43) was : â€œ.. . 0.15 mg/kg/day for@ days fol

lowed by 3 days of rest. If no leukopenia wasnoted, one or two additional doses were given untilleukopenia occurred. Weekly doses were giventhereafter depending on the w.b.c.â€• Johnston ohserved that vinblastine treatment for at least 6@weeks to 3 months was necessary to demonstrateobjective evidence of tumor regression. This is inagreement with the experiences of Armstrong et al.(4) (i96@2) in which three of seven patients withcarcinoma of the breast showed both objective andsubjective improvement, in one case only after 3months of vinblastine treatment. Armstrong et al.reported less toxicity than did Johnston et al.,probably because they avoided initial heavy loading doses. They followed the incremental approachto dosage and so avoided leukopenia as much aspossible. The patients of both Johnston et al. andArmstrong et at. were treated for at least 1@ weeks

and, if they responded, were thereafter givenmaintenance dosage indefinitely once every 7 days.For various reasons not related to drug toxicity,vinblastine dosage was discontinued in two of thepatients of Armstrong et a!. while the drug wasstill effective. Both patients relapsed during the3d week after their last dose. This again illustratesthe need for continued maintenance dosage for aslong as a patient will respond.

In the Midwest Cooperative ChemotherapyGroup study (1961) of vinblastine, seven patientswith carcinoma of the breast were included, andnone showed any response. Likewise none of eightevaluable patients with carcinoma of the breastshowed any response in the study (1961) of the

24 Personal communication, Dr. Barbara Johnston, St.

Luke's Hospital, N.Y.C.



JOHNSON el al.â€”Vinca Alkaloids 1415

Eastern Cooperative Group in Solid Tumor Chemotherapy.25 The early criteria employed by manygroups for an adequate length of trial of vinblastine therapy would not have insured the necessaryduration of treatment for many patients with carcinoma of the breast.

Noble26 observed that mature female rats cantolerate larger doses of vinblastine than can immature female rats. They further demonstratedthat the immature rats could be made equallytolerant of vinblastine as are mature rats if theywere pretreated with stilbestrol for a few days before dosage with vinblastine. This finding maywell have a clinical bearing when we come to treatwomen who have previously undergone ovariectomy, as is so often the case with breast carcinomapatients.

Armstrong et al. have noted that a woman withbreast carcinoma was able to tolerate weekly intravenous vinbiastine doses no larger than 0.05mg/kg without leukopenia while she was receivingtestosterone. After several weeks of this combinedtherapy, testosterone was discontinued. Within amonth the patient was found to have increased hertolerance of vinbiastine so that doses in the rangeof 0.15 to 0.@0 mg/kg/week could be given withsafety. Only after the patient had improved objectively following the use of vinblastine treatmentwas testosterone discontinued. Had testosteronebeen discontinued immediately prior to the use ofvinblastine, the benefit seen could have been attnibuted to testosterone withdrawal. In this patient, testosterone may have been acting in thesame manner as does the absence of ovarian function in immature female rats.

Mealey (5@) and Wilson (91) have separatelyreported on the administration of vinblastine byintra-antenial infusion to patients with brain tumors. Only two of ten patients in Mealey's seriesderived benefit (5@). In the series of Wilson, tenof seventeen evaluable patients were improved(91).Thisdifferencein responsenatemayberelated to the different dosage regimens employed.Mealey reported that : â€œTenpatients with malignant cerebral astrocytomas were given post-operative infusions of vinblastine (0.5 mg./Kg.) into thecarotid or vertebral arteries, usually over a periodof several days.â€• Wilson on the other hand em

@ Personal communication, Dr. Emil Frei, III, NationalCancer Inst., who as chairman of the Eastern Solid TumorGroup study of YLB reports that this group's continuing studyof YLB has now demonstrated responses among patients withcarcinoma of the breast in approximately the same order offrequency as with 5-PU.

16 Personal communication, Dr. R. L. Noble, University of

British Columbia.

ployed : â€œ.. . initial infusions run continuously for7@ hours (0.4 mg./Kg.) . . . Re-infusions have been

given at intervals of 6â€”8weeks, or sooner if the patient's status was deteriorating.â€• While the lattertype of regimen would appear to have been moreeffective, it should be pointed out that neitherinvestigator gave maintenance dosage intravenously after intra-artenial infusions. It is conceivable that beneficial effects might be prolonged byintravenous maintenance dosage started 7â€”14daysafter intra-arterial infusion and continued regularly each week for so long as a patient showed improvement.

Hertz et a!. (3@) demonstrated that vinblastinecan be effective in patients with methotnexateresistant choniocarcinoma. Responses to vinblastine in both male and female patients with trophoblastic tumors have also been reported by others.The observations of Hertz are most interesting inthat they relate to a magnitude of vinblastinedosage considerably larger than has been used byothers. As might be anticipated, this high dosageproduced considerable toxicity. Hertz is the onlyinvestigator to report : â€œSeveremental depressionwas uniformly noted during the toxic phase andthis was more marked than would be accountedfor by the respective clinical status of each of thepatients.â€• This followed single total doses of @4mg. and 36 mg. The weight of the patients whoreceived these doses was not stated ; but, assumingthe weight of the average patient to have beenabout 70 kg., this represented dosage in the rangeof 0.30â€”0.50 mg/kg. In many of his patients,Hertz observed leukocyte counts below 1,000 cellsper cubic millimeter following vinblastine dosage.At this dosage he also reported : â€œ.. . suppressionof deep tendon reflexes associated with each courseof therapy. Two [of 81 patients complained ofnumbness and panaesthesias in the fingers. Thesechanges proved to be reversible. In addition tothese apparent neurotoxic effects, there was suggestive evidence of panasympatholoytic action ofthe drug. All patients, during at least one courseof therapy, experienced marked constipation for Sto ii days. . . . There was one episode of urinaryretention. Six [of 8] patients had bilateral pamand tenderness of the panotid glands, usually associated with marked dryness of the mouth. Twopatients developed sinus tachycardia in the absence of fever or other apparent cause.â€•Followinga course of 6 mg. of vinbiastine twice daily for 3consecutive days, Hertz et al. reported â€œtypicallysevere toxicityâ€• with decrease in the platelet count

by 30â€”60per cent, white blood cell count belowp2,000cells per cubic millimeter, and an absence of




reticulocytes from the peripheral blood for @â€”9days. He went on to state that : â€œCompleterecoveny of all of the bone marrow elements, as manifested by the peripheral blood findings, occurredwithin 3 weeks and there has been neither evidenceof cumulative toxicity with successive courses oftreatment nor decreasing effects in the bone manrow.â€•

It is interesting to learn from Hertz et a!. thatmassive dosage with vinblastine can produce constipation and neurotoxicity somewhat resemblingthe toxicity produced by yincnistine at a dosagewithin the therapeutic range. However, there theresemblance ends, because yincnistine does not usually depress the leukocyte count at neurotoxic 1evels of dosage. It should also be noted that vinbiastine does not usually depress the platelet on redblood cell counts at dosage just sufficient to produce moderate leukopenia; nor does it commonlydepress the white blood cell count for longer than7â€”10days.

Johnson et al. (4%) observed that the inhibiting

effect of vinblastine on a tissue culture of humanmonocytic leukemia cells could be blocked by glutamic acid, aspartic acid, arginine, citrulline, andornithine and observed reversal of anti-P-1534 activity in vivo (39). Based on these observations,two clinical groups investigated the possibility thatsome of these substances might be useful in reversing the leukopenic effect of vinblastine. Armstronget al. (4) gave capsules of glutamic acid orally indoses up to 3 gm. S times daily to several patientsat various times in relation to vinbiastine dosage.Monosodium monopotassiuxn glutamate solutionwas given intravenously to other patients. Stillothers were given capsules of fraspartic acid bymouth in doses up to 3 gm. 3 times daily. The onlyconsistent effects noted with all three forms of

treatment, regardless of the size and duration of

dosage, were anorexia, nausea, abdominal colickypain, and weight loss. The data did suggest thatir.glutamic acid in oral doses of 3 gm. 3 times dailymay have prevented the leukopenic effect of moderately sized but not of large doses of vinblastine.Only when the glutamic acid had been given forsome days before treatment and continued pastthe period during which leukopenia might be cxpected to appear (5 days after dosage) was anypossible leukopenia-sparing effect seen. Patientsfirst treated with glutamic on aspartic acid whenalready leukopenic from vinblastine dosage showedno more rapid recovery from leukopenia than patients not given these amino acids. Vaitkevicius(81)notedâ€œsomeapparentameliorationof (yinblastine) toxicityâ€• when ir-glutamic acid was givenorally. They, like Armstrong et al., noted that oral

administration of glutamic acid in capsules produced severe nausea. However, by giving glutamicacid as an effervescent solution prepared by mixingit with an equal volume of sodium bicarbonate, itwas well tolerated. Four hundred mg/kg of irglutamic acid were given daily in this way. In someof their patients the tolerated vinblastine dose wasincreased by 30 per cent with glutamic acid treatment, but this produced no demonstrable improvement in antitumor effect. Accumulation of metaphases was noted in bone-marrow specimens aftercombined vinblastine and glutamic acid therapy,but this accumulation was much less than withvinblastine therapy alone; and the suppression ofpostmetaphase stages was at no time complete.No investigation has yet been made of the possibility that glutamic acid, etc., might be useful inpreventing systemic toxicity from large doses ofvinblastine infused intra-artenially into tumorbearing areas of the body.

Toxicological studies at the Lilly Research Laboratories have revealed that vinblastine potentiated phenobarbital anesthesia in dogs and prolonged EvipalÂ® sleeping time in mice. It is therefore conceivable that should vinbiastine be givento a patient undergoing barbiturate anesthesia recovery of consciousness might be delayed. A very

large number of cancer patients have now beentreated with vinblastine during the daytime andhave taken barbiturates the same night to facilitate sleep. In spite of this there have been no neports of prolonged or augmented sedation in suchpatients. However, since vinblastine may be rapidly fixed and metabolized, the clinical situation wasnot completely analogous to the administration ofvinblastine and barbiturates simultaneously. Itwould be interesting to compare the leukopenicand oncolytic effects of vinblastine administeredalone with those of vinblastine given at the timeof barbiturate anesthesia.

Tumor shrinkage with significant benefit has followed the administration of uncoated compressedtablets of vinblastine by mouth (4, 36). However,the leukopenic response following dosage was unpredictably variable from week to week eventhough the same dose was used each week. Theincidence of gastrointestinal side-effects with thesetablets was high. Some patients who responded tooral vinblastine later relapsed in spite of its continued weekly use but then responded again afterintravenous vinblastine was substituted for theoral drug. It was concluded that these tablets ofvinblastine were not consistently absorbed fromthe gut and were therefore unsuitable for furtheruse (4). More recently the Lilly Clinic group hasbegun to investigate a new type of vinblastine



Age

(years)Total patientsCompleteremissionPartial remissionNoresponse1â€”

34â€”67â€”9

10â€”1516â€”20

Totals52

1710

16

(40%)22 (42%)

7 (41%)2 (20%)04

(27%)15 (29%)6 (85%)4 (40%)05

(38%15 (29%4 (24%4 (40%)

195872929

JOHNSON et aLâ€”Vinca Alkaloids 1417

tablet which passes through the stomach beforebeginning to slowly disintegrate in the intestineover a period of several hours. Preliminary observations with this new tablet indicate that it issuperior to the earlier one in that it less oftencauses side-effects and is more predictable in itsleukopenic effects. Now that a Hodgkin's disease

patient has obtained complete remission with thisnew preparation, its evaluation will be continuedby a larger number of investigators.

VINcrn5TINE SULFATE

To initiate the trial of vincristine, Armstrong,Dyke, and Fouts (3) of the Lilly Clinic gave smallweekly intravenous doses of only 0.01 mg/kg totwo men. One of these suffered from Hodgkin'sdisease and the other from lymphosarcoma. Eventhese first tentative doses produced prompt andgratifying responses. Within@ months it becameobvious that vincnistine was useful for a variety ofmalignant conditions at doses of 0.01â€”0.03mg/kg/week. At this time the trial was enlarged to includeseveral other centers. Kanon, Freireich, and Freiof the National Cancer Institute soon discoveredthat vincnistine could induce remissions from acuteleukemia in children when given in doses largerthan had been employed by the Lilly group (44,45). Approximately 0.iO mg/kg/week is necessaryfor an optimal remission rate from acute childhoodleukemia.

The remission rates obtained with vincristinein acute childhood leukemia by Karon (45) and bythe Acute Leukemia Group B27 have been at leastas good as can be obtained with the standardagentsâ€”6-mercaptopurine, steroids, or a folic acidantagonist. The responses in a series of 95 childrenwith acute leukemia treated jointly by six centens28 are seen in Table @5.

There is noted even distribution of responses inage groups up to 10 years. It is also of interest thatsome of the leukemias responding to vincristine areof the myelogenous type.

The majority of these patients received vincristine as a fourth drug after sequential treatmentwith the standard drugs. More recently, a higherremission rate has been obtained with vincristineused as the initial treatment for acute leukemia.Complete remission was obtained in eight of ninechildren at the Boston Children's Cancer ResearchFoundation2Â° and in seven of eight children at the

27 Personal communication, Drs. M. R. Karon and E. Frei,

III, NationalCancerInstitute.28 Data received at the Lilly Laboratories for Clinical Re

search from National Cancer Institute, the Boston Children'sCancer Research Foundation, the Roswell Park Memorial Institute, the Bobs Roberts Hospital, the Sloan-Kettering Institute, and the Lilly Clinic.

National Cancer Institute29 when vincnistine wasused as the first drug. Several groups now initiatetherapy with vincnistine because it does not usually depress marrow function.

After remission from acute leukemia has beenobtained with vincnistine, one has the choice ofcontinuing dosage on a maintenance basis for aslong as the patient will respond on of withholdingtreatment until it is needed again. A controlledstudy of these alternatives is being carried out bythe cooperative groups in an endeavor to establishwhich of these regimens is the most effective inprolonging survival.

Vincristine dosage much below 0.10 mg/kg/week has not been effective in inducing completeremissions from acute childhood leukemia. Armstrong et al., influenced by their good results with

TABLE 25

AGE DISTRIBUTION AND RESPONSES OF 95 LEUKEMICCHILDREN TREATED WITH VINCRISTINE

lesser dosage when treating adults with lymphomas and solid tumors, failed to attain the dosagenecessary for leukemic children. As a result they

produced merely partial rather than complete remissions with weekly doses in the range of [email protected] mg/kg.

It is fortunate that leukemic children toleratelarge doses of vincnistine better than do adults.The weekly dose of 0.10 mg/kg which is necessaryfor induction of remission in childhood leukemiaproduces profound toxicity in adults. Fortunatelyadults with lymphomas and solid tumors will oftenrespond to weekly doses in the range of 0.0@â€”0.05mg/kg and in some cases to doses as low as 0.01mg/kg. There is no question that the incidence andseverity of central nervous system side-effects arerelated to the magnitude of vincristine dosage (4,13, 44, 45). The incidence of epilation, the most

frequently occurring side-effect, is not so clearly

dose-related.With single weekly doses, leukopenia, neuritic

pain, constipation, and difficulty in walking dueto muscle weakness have usually been of short

29 Personal communication, Dr. M. R. Karon, National

Cancer Institute.




durationâ€”i.e., less than 7 days. When subsequentdosage was reduced, these side-effects often lessened or disappeared. When the same total amountof vincnistine per week has been given in severaldoses over a number of days, these side-effectshave usually persisted indefinitely while therapywas continued. To avoid such prolongation itseems advisable to administer vincnistine no moreoften than once weekly.

Other side-effects, such as epilation, sensoryloss, paresthesias, slapping gait, loss of deep tendon reflexes, hoarseness, and muscle weaknesshave not reversed so rapidly and have persisted forat least as long as therapy has been continued. Inmost instances these latter side-effects have eventually disappeared by about the 6th week afterdiscontinuance of treatment. Muscle weakness hasbeen reported most often to involve the hands,quadriceps, dorsiflexors of the foot, and less oftenthe larynx and the extrinsic muscles of the eye.

Frequently there has been a sequence in thedevelopment of side-effects. Initially, most patients have reported only sensory impairment andparesthesias. With continued treatment neuniticpain has sometimes appeared and later still, insome cases, motor difficulties.

Constipation, taking the form of colicky abdominal pain and upper colon impaction, has oftenoccurred only after several doses of vincnistine. Insuch cases the rectum has usually been empty anda flat-plate x-ray of the abdomen has been necessary to confirm the diagnosis. Because it has been

difficult to free such high impaction, it would seemadvisable to employ a routine prophylactic regimen against constipation for all patients receivingvincnistine. After encountering some severe casesof cecal impaction among the first several patientsin their series, the Lilly Clinic group subsequentlyhave given all further patients, from the beginningof therapy, a feces-wetting agent (such as DoxanTM) and a methylcellulose bulk laxative (such

as Cologel TM) 3 times daily by mouth. On anyday should a patient, in spite of these medications,not have a bowel movement by noon, he is immediately given magnesium sulfate ; and if that doesnot produce a stool by evening, an enema is given.This prophylactic regimen has been found to dimmate the problem of constipation.

Central nervous system leukemia was reportedin eleven of 66 children undergoing otherwise beneficial therapy with vincnistine.'Â° This suggests thatvincristine, like 6-mercaptopunine and folic acidantagonists, may not penetrate well into the cerebrospinal fluid. Intrathecal amethopterin has beensuccessfully used for this problem while the pa

tients have been continued on their usual weeklyvincnistine dosage. To date vincnistine has notbeen administered intrathecally to any human being. This route of administration has been avoidedbecause Costa3Â° found that vincnistine givenintracisternally to anesthetized dogs producedascending paralysis and death. Since it is knownthat the Vinca alkaloids are capable of potentiating barbiturate anesthesia, it is conceivable thatthe above events which followed intrathecal administration of vincnistine reflected potentiationof the barbiturate anesthesia rather than an effectof vincnistine itself.

After it was found necessary to employ a weeklydosage of vincnistine as high as 0.10 mg/kg to induce an optimal number of remissions in acutechildhood leukemia, similar doses were then usedin treating both children and adults with othermalignant conditions. In retrospect, it became apparent that this high dosage did not improve theresponse rate among patients with vincristinesensitive tumors but did increase the toxicity.

Holland2' et at. of the Eastern Cooperative Groupin Solid Tumor Chemotherapy have returned tousing weekly doses in the neighborhood of 0.0@25mg/kg and believe that this may be no less effective for patients with Hodgkin's disease, lymphosarcoma, and carcinoma of the breast than is higher dosage. They have found that this lesser dosageis noticeably less productive of side-effects than ishigher dosage. However, they state that furtherexperience will be required before it is proved beyond doubt that 0.0@5 mg/kg dosage is no lesseffective than 0.050 mg/kg dosage.

Carbone and Bnindley (1 1) of the National Cancer Institute have reported that five of five patients with Hodgkin's disease and three of threepatients with lymphosarcoma responded to vimcnistine. They wrote : â€œFiveof these 8 patientswere resistant to on had not responded to vinblastine and alkylating agents. This order of responsiveness in the absence of bone marrow toxicity isa unique experience in lymphoma chemotherapy.Less striking evidence of antitumor activity hasbeen observed in patients with solid tumors, particulanly neuroblastoma and renal cell carcinoma.â€•

Costa et a!. (13) of Roswell Park Memorial Institute also reported : â€œ.. . rapid regressions offour of five lymphomas in patients refractory tostandard treatments. . . Unequivocal partial regressions of single cases of metastatic choniocan

cinoma, testicular teratocancinoma, carcinomas ofcervix and of bladder are in progress. Real effects,

30 Personal communication, Dr. G. Costa, Roswell Park

Memorial Institute.




indicating some drug activity, have been seen insingle cases of myeloma, prostatic carcinoma andchronic lymphocytic leukemia.â€•

It should be mentioned that Dr. Costa,3Â°in hiscapacity as chairman of the Eastern Solid TumorGroup's myeloma study group, has reported subsequently that vincristine was not significantlyuseful with any consistency in a large series ofmultiple myeloma patients.

Bohannon et al. (9) have reported that â€œ.. . 4 of11 [adult] patients with Hodgkin's disease and 1patient with chronic myelocytic leukemia havehad a temporary subjective and/or objective clinical response.â€• This group employed â€œdosesof 0.03mg/kg to 0.06 mg/kg, I.V. repeated at@ to 14 dayintervals depending on the leukocyte response. ...Treatment was continued until leukopenia, thrombocytopenia or other toxicity occurred.â€• The un

usually low response rate reported by this groupwith lymphoma patients is possibly due to theirregular spacing of doses.

Tan et at. (79) also followed a dosage scheduledifferent from that of other investigators. In aseries of@ children, they reported : â€œThedosagewas 0.05 mg. per kilogram of body weight given1 to 3 times weekly to an average total dose of0.5 mg. per kilogram of body weight. The drugwas discontinued when toxicity or an adequatetherapeutic response occurred. Toxic effects con -sisted of leukopenia, thnombocytopenia, alopecia,and cellulitis following extravasation of the drug.Transient urinary symptoms such as dysunia, frequency, urgency and polyunia without microscopicabnormalities occurred in 5 patients. Ptosis of thelids with no other meningeal signs occurred in 5patients. . - . In 1 of 4 children with Wilm's Tumonan abdominal mass disappeared without subjective benefit; 1 child with Hodgkin's disease,@ of3 children with embryonal Rhabdomyosarcoma, 1

of@ with neuroblastoma and 1 child with embryonal adenocarcinoma had objective regression withclinical benefits continuing for 1 to@ months.â€•

Only in these two reports (9, 79) from the SloanKettering Institute have leukopenia and thrombocytopenia been classed as prominent side-effects.It should be understood that only in these twoseries were doses of vincnistine often administeredmore frequently than once every 7 days. Otherinvestigators have commented rather on the relative freedom from marrow toxicity which vincnistine affords. Significantly, these others have notregularly spaced their doses so closely as did theSloan-Kettering investigators. From this it appears that there is merit in not giving vincnistinedoses more frequently than once every 7 days.

Selawny et al. (69) of Roswell Park MemorialInstitute gave vincnistine in weekly doses of 1.5â€”5mg/sq meter, averaging 3 mg/sq meter of bodysurface, to fourteen children. â€œObjective tumorresponse was noted in 4/5 patients with acute lym

phoblastic leukemia . . . @/4patients with neuroblastoma . . . and 1/1 patient with ovarian dysgenminoma. . . . Noteworthy was the absence ofthrombocytopenia. Weight loss of 13â€”@5per centduring 5â€”10weeks of drug administration constituted a serious problem in 5 patients and appearedto be unrelated to G.I. toxicity. This drug seemsto have a wide spectrum of activity in childhoodtumors. . . .â€œ

Weight loss of this magnitude has also been reported by the National Cancer Institute group butnot by others. It may be significant that both theRoswell Park Memorial Institute and the NationalCancer Institute groups employed weekly dosesfor some patients in excess of 3 mg/sq meter ofbody surface. Further, the National Cancer Institute workers have examined their data for anyinfluence which dosage magnitude might have onweight change. They found that patients who received weekly doses in excess of 0.10 mg/kg lostweight, whereas those who received weekly dosessmaller than 0.09 mg/kg gained weight. From thisthere would seem to be merit in giving a weeklyvincnistine dose no larger than 0.10 mg/kg to leukemic children. Also, to avoid excessive neurotoxicity, it seems advisable not to increase the weeklydose, if possible, above 0.0@5 mg/kg for patientswith solid tumors. It remains to be establishedwhether some lymphoma and solid-tumor patientsmight benefit from higher weekly dosage than0.0@5 mg/kg if no response is obtained with thisdose.

Costa et al. of Roswell Park Memorial Institutehave reported that patchy liven necrosis was seenat autopsy in some of their vincristine-treatedpatients (14). However, they also noted that:â€œTheliver function tests performed in these pa@tients had not been altered by drug administnation.â€•

Costa et a!. (14) reported in addition that:â€œPartial remissions were observed in single instances in carcinoma of the cervix, choriocarcinoma, anaplastic carcinoma, testicular teratocarcinoma, and carcinoma of the prostate.â€• Theycontinued : â€œTheactivity in carcinoma of the cervix is worthy of particular attention, since it nepnesents one of the human neoplasms singularly resistant to chemotherapy. Confirmation of suchactivity of vincnistine in carcinoma of the cervixhas been obtained in a larger series of patients.â€•



Cancer Research Vol. @3,September 196314@20

The duration of dosage before complete remission occurred in 37 children with acute leukemiaaveraged 88 days (range, 11â€”106days).'9 Patientswith Hodgkin's disease or lymphosarcoma usually begin responding to vincnistme within a dayafter the first or second dose. For this reason itseems advisable to increase the fluid intake ofpatients at the time of the first few doses to avoidexcessive elevation of the blood uric acid. It remains to be established how late one can expect aresponse in the various other solid tumors, buttumor shrinkage has been variously reported tooccur as soon as a few days after the first dose and

as late as after the sixth weekly dose of vincnistinein some patients with various types of carcinoma.Since significant benefit and gross tumor shrinkagehave occurred in some patients only after 1@weeksof dosage with the similar alkaloid vinblastine (4,43), it would seem that a similar period of trialmight be appropriate with vincnistine.

As with vinblastine, withdrawal of vincnistinedosage has in several cases been followed by thereappearance of tumor growth within @â€”3weeks

of the last dose.19 For this reason it would seemadvisable to continue with weekly vincristine maintenance dosage for as long as a patient derivesbenefit. It is not known whether smaller weeklydoses than are necessary to produce tumor shrinkage can be relied upon to maintain a beneficialresponse once it has occurred. A cooperative groupstudy of this problem would be important, becauseit has been observed that downward adjustmentof dosage magnitude in some of the patients ofArmstrong et al. has been followed by reversal orreduction in neurotoxicity.

In that vincristine causes little if any depressionof bone-marrow function (3, 11, 13, 14, 44, 45, 66,69), this drug should be relatively safe for use inconjunction with radiation therapy.

Like vinbiastine (5Q, 91), vincnistine has beenadministered intra-artenially with beneficial resultsto some patients with brain tumors.2' Vincnistinehas also been infused directly into the hepatic antery of a patient with primary carcinoma of theliver, a procedure which was followed by tumorshrinkage and clinical improvement.2' A few otherpatients with primary liver carcinoma have beentreated with vincnistine administered intravenously rather than into the hepatic artery. None of thelatter patients have shown any response.

The doses of vinbiastine or vincnistine infusedintra-arterially into tumor-bearing pants of thebody have been several times larger than can begiven intravenously@'@ (37, 5@, 91) . In all cases

either no toxicity or much less toxicity occurredthan would be expected from such doses given intravenously. This suggests that both vinblastineand vincristine are cleared from the blood andfixed rapidly on malignant tissue when administered intra-artenially.

Beer has shown that, when material derived bytitration of vinblastine was injected intravenously into rats, it was cleared very rapidly from theblood.@ Within minutes of injection much of theradioactivity was detected in the liver; and in lessthan an hour the blood was clear. The radioactivity was subsequently traced from the liver throughthe biliany system into the gut. Only 5 per cent ofthe radioactivity appeared in the urine.

With vincnistine it has been shown that dosageis more toxic to rats which have undergone ligationof the common bile ducts than to rats with patentbiliary tnacts.@ It has also been reported that patients with obstructive jaundice experienced moresevere side-effects than patients without bilianyobstruction Here again then, as in the case ofvinblastine, we have some evidence that vincnistine may be largely and rapidly excreted by theliver. A summary of the differences and similar actions of vinblastine and vincnistine in clinical useas understood in December, 196@, is shown inTable @6.

DISCUSSION

Included in the invitation to present this reviewwas a paragraph indicating that the purpose ofthis presentation, as well as of the entire symposium, was to document progress; and in addition topoint up problems which remain to be solved, todepict the nature of the obstacles which appear tobe impeding further progress, and to re-evaluateexisting concepts. Participants were urged not

only to be candid and objective, but also to bespeculative in approach. Recognizing these objectives and the limitations of time and space, wehave attempted to document the present state ofour knowledge concerning the production, chemistry, toxicology, experimental activity, mechanismof action, and clinical efficacy of these new agents,emphasizing what we view as areas requiring furthen exploration.

SOME ASPECTS OF STRUCTURE AND MODIFICATION

The structures of vinblastine, vincnistine, andto a lesser extent, vinleunosine, are reasonably wellestablished. Modification of these large alkaloidalmolecules is difficult. One such modification whichexperimentally appeared to be useful was not con

32 Personal communication, Dr. C. T. Beer, University of

British Columbia.31 Unpublished data, Dr. James G. Armstrong, Lilly Lab

oratories for Clinical Research.



CharacteristicVLBVCR

TABLE 26

COMPARISON OF THE CLINICAL CHARACTERISTICS OF VINBLASTINE SULFATE (VLB)

AND VINCRISTINE SULFATE (YC R) AS UNDERSTOOD IN DECEMBER, 1962

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

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

Weekly oral dosage Possibly 2â€”3Xthe intravenous dose; Not studiedremains 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 reversible

Constipation Only rarely occurs Almost invariable if preventive medication 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 perafter 8â€”12weeks' dosage in about centage of patients30 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 knownage; more evaluation needed

Carcinoma of cervix As above Benefit reported; more evaluationneeded

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

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

Chorionepithelioma and other tro- Frequently beneficial Frequently beneficialphoblastic tumors

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

Primary brain tumors (intra-arteri- Reported as frequently clinically Reported as frequently clinicallyal dosage) beneficial beneficial

Mycosis fungoides Some complete remissions reported Not known

Hand SchUllerChristian Disease Usefulremissionsreported Not knownand Letterer-Siwe Disease

Neuroblastoma Benefit reported Benefit reported

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



Cancer Research14@2 Vol. @23,September 1963

firmed in the clinic, but does illustrate some interesting features of even slight modification of thesecompounds.

Preliminary clinical evaluation of vinleurosinehad not yet been initiated when it was observedexperimentally that the methiodide of vinleurosinegave a striking increase in activity over the parentcompound when tested in the P-1534 system. Prolongations of 70â€”160per cent were obtained ovena dose range of 30â€”60mg/kg, and in additionmarked prolongation and survival were obtainedin near-terminal animals. In contrast to the increase in P-1534 activity, there was a loss of nesponse in all other tumors carried in our expenimental spectrum. This was interpreted to indicatean increase in specificity, and for this reason yinleurosine methiodide was selected for preliminaryclinical evaluation. An additional pharmacologicaldifference of some interest was the effect on peripheral white blood cell counts. As has long beenrecognized, vinblastine and, to a lesser extent, yin

leurosine cause leukopenia as a result of bone-manrow depression. The methiodide of vinleurosine, incontrast, was not observed to produce leukopeniaunder the conditions of our investigation. On thecontrary, there was noted an induction of leukocytosis in nonleukemic cancer patients. Althoughsome evidence of minimal tumor shrinkage wasnoted clinically, vinleunosine methiodide has notbeen pursued further in humans because of theseverity of side-effects not encountered with the

other clinically active Vinca alkaloids.These marked differences in activity and side

effects after only minor changes in the molecule

emphasize the importance of even minute changes

in configuration of these compounds. Continuedstructure analysis, both of the parent compoundsand products produced due to their being metabolized, is essential for the understanding of themechanism of their activity. The striking difference in clinical activity of vinblastine and vincnistine in the acute leukemias of childhood must berelated to the presence of an N-formyl group invincnistine instead of the N-methyl group foundin vinblastine.

Observations on molecular modifications sometimes give rise to additional problems. In the citedcase of vinleurosine and its methiodide, vinleunosine was temporarily withheld from a clinical evaluation, and the methiodide given priority, becausethe quatennary compound appeared superior interms of P-1534 activity. However, our subsequentexperiences, just described, serve to emphasizethat an enormity of effort can come to noughtwhen the clinical trial fails to substantiate leadssuggested by experimental evaluation in animals.

To add to our problems, many of these compoundsare obtained from the Vinca plant in low yield.Problems of yield are recurrent and chronic. Before rejection of a compound, we must study itfor any evidence of activity to determine if attempts to increase the yield are worthwhile. Itwould be gratifying if some of the more abundantly available, but less active or inactive, compounds could be converted to active materials. Onpaper a number of alterations seem feasible-forexample, the oxidation of an N-methyl group of

vinleurosine. However, with any method proposedto accomplish a useful change, many reactive centens on these complex molecules are readily affected, as for example, in the oxidation of the basicnitrogen in the catharanthine moiety to an amide,all biological activity is lost.

PHARMACOLOGY

A number of problems which basically have abearing on toxicology of these compounds deservefull discussion and some ne-evaluation. All the active antitumon alkaloids appear to have some capability for arresting mitosis of the mammalian cellin vitro and in vivo (1@,16, 39, 4@,6@,81). A numben of amino acids including glutamic, ornithine,citrulline, arginine, aspartic, and tryptophan have

been reported from this laboratory to reverse orblock the TCD50 dose of vinblastine in 1-weekgrowth curves in tissue culture (4@).

These observations were extended in vivo in theP-1534 system and were found to block either theantitumon effect or toxicity in nonleukemic animals given an ED,0 of vinblastine. Cutts (16)found a reduced percentage of cells in mitosis,under certain conditions of administration, in anin i@ivosystem following treatment with vinblastine, if glutamic acid and tryptophan were alsoadministered. The effects observed by Cutts weresimilar to those observed clinically by Vaitkevicius(81) with glutamic acid.

Two points need to be reconsidered in the relationship of these amino acids to the biologicalaction of vinbiastine. First, in this laboratory,effects of these amino acids in nitro were observedonly at 50 per cent end-point levels of vinblastine,and this was also true of in vivo work. The factthat optimal doses of vinblastine were unaffectedsuggests that the amino acids play only an ancillary role on vinblastine action. While the suggestion was made by one of the authors (39, 4@) thatthe antituinon mechanism of vinblastine might beby interference with metabolic pathways involvingthese amino acids, these observations obviouslydo not present a complete picture.

Secondly, however, as far as we are aware, there



Dose(mg Xkg

x I)RouteAv.

life(days)Per

cent ofprolonga

tion*Indefinitesurvivors(73days)1

.31.31.3SalineIntraperitoneal

SubcutaneousIntravenousAll S routes16

2326160

46534

250

JOHNSON et al.â€”Vinca Alkaloids 14@3

has been little effort by clinical investigators, otherthan the limited work of Vaitkevicius and Armstrong to study or take advantage of the possibleuse of these amino acids in host-protection studieswith vinbiastine. In parallel studies with P-1534-beaning and tumor-free mice, it was observed thatglutamic acid offered host protection and blockedantitumor effect, whereas tryptophan blocked antitumor effect but offered no host protection.Aspartic acid, however, did not reverse antitumoreffects and did offer host protection (39). It wouldseem that a greater clinical effort to explore these

relationships in terms of host protection is warranted.

Clinically the use of vincnistine is associatedwith neuromuscular manifestations which havenot as yet been fully studied experimentally. Parenthetically, it is interesting that similar neunomuscular manifestations have been reported insome malignant conditions without treatment ofany kind. Whether or not the neuromuscular manifestations following vincristine therapy are indeedrelated to the parent compound or to its metabolic products which subsequently may pass theso-called blood-brain barrier; or whether the compound exerts some more fundamental effect oftriggering a common, unrecognized mechanismsometimes found in certain malignant conditionswithout therapy, remains to be clarified. A difficulty has been the lack of a convenient expenimental system in which these same features can

be studied. The muscular weakness and head-dropobserved in rabbits following administration ofvincristine might be such a system and deservefurther exploration. Recent observations in thislaboratory of a host-protection effect of folinic acidwithout reversal of the anti-P-1534 activity ofvincristine would also appear to deserve clinicalinvestigation.

Leukemia of the central nervous system hasbeen observed in patients in hematological remission following vincristine therapy. Thus, it hasbeen suggested that vincnistine might not pass theblood-brain barrier. The results of recent investigation of vincnistine therapy in intracranially implanted P-1534 mice demonstrate that vincnistinecan be â€œcurativeâ€•in this experimental CNS leukemia (Table @7).From the work of Costa it mightbe implied that the neuromuscular effects of thiscompound are due to direct action of vincnistineon the CNS. This implication could perhaps besuggested by his comparison of toxicity of vincnistine administered by intravenous, intrapenitoneal,and intracisternal routes in dogs. In this study it

â€w̃as found that there was a marked increase in

toxicity by the intracisternal route. It is interest

ing to note that it has previously been shown thatboth vinblastineand vincnistine potentiate barbiturate anesthesia in animals, and Noble@ has reported increased toxicity and mortality in ratstreated with vinblastine when they were underbarbiturate anesthesia. From these considerations,it would seem appropriate to determine whetherthe increased toxicity observed by Costa in dogsmight be associated with the fact that the animalsreceiving vincnistine intracistennally were also under barbiturate anesthesia. In addition, there havebeen interesting differences reported in LD50's ofvinblastine given by different routes. This compound is approximately 6 times more toxic in ratsby the intrapenitoneal route than when administered intravenously. Thus, the question of toxicityof these compounds by different routes in the pres

TABLE 27

EFFECT OF VINCRISTINE THERAPY ONINTRACRANIAL P-1534 LEUKEMIA

Ten mice per group

* Based on animals which died only.

ence and absence of anesthesia and in differentspecies, would seem to require more extensiye investigation, and in addition it would appear appropriate to observe whether on not intrathecaluse of folinic acid with vincnistine might not decrease toxicity observed by the use of this route.

POSSIBLE ADDITIONAL CLINICAL APPLICATIONS

There are four other areas in which a more extensive clinical investigation of these compoundsseems warranted, although in some of these situations it has been difficult to find useful experimentILl systems to provide a basis for direct extensionof these clinical studies. This would not seem tobe the case, however, in Noble's finding that ratsof different sex and different age groups, in thecase of females, differ in tolerance to yinblastine.This work and his additional finding of the effectsof estrogens on vinblastine tolerance has alreadybeen alluded to, and is an example of a problemwhich deserves a more intensive clinical investigation. This finding is of obvious importance inevaluating the effect of vinblastine, and perhapsvincristine, in carcinoma of the breast and perhaps



Cancer Research Vol. @3,September_196314@4

other tumors as well. Evaluation of vinblastine inthe treatment of carcinoma of the breast serves toillustrate an additional obstacle to clinical progness in determining the extent of usefulness ofthese new agents. It would seem desirable to studya much larger number of breast carcinoma patientstreated for atleast 1@ weeks with vinblastine. How

ever, the Breast Carcinoma Cooperative Chemotherapy Groups tend to focus their attention onsteroidal compounds leaving few uncommitted patients or investigators to investigate nonsteroidalbut obviously active compounds such as vinblastine.

A second inadequately studied area is the possible use of the Vinca alkaloids in combinationwith or as an adjunct to radiation therapy. Sincevincristine is not a severe depressor of the bonemarrow, it would seem to be a particularly goodcandidate, but it would seem equally appropriateto investigate low dosage levels of vinblastine aswell. Some drugs have been demonstrated clinically to render radiation-resistant tumors sensitiveto radiation therapy. The usefulness of the Vincaalkaloids in this area has not been adequately investigated. Various modalities in terms of time of

radiation in reference to states of metaphase annest should be borne in mind during any such investigation.

There has been no effort to investigate thesecompounds as surgical adjuncts. Noble (17) hasreported the clearing of leukemic cells from the

peniphenal circulation of rats by vinblastine. Thecomplete obliteration of P-1534 leukemias in animals in which vincnistine therapy was delayeduntil the animals were in a near-terminal state ofgeneralized disease has already been described in

this repont. Recently, Schabel33 has investigatedthe effect of vinblastine in a series of hamster tumors which are characterized by wide-spread metastases. Vinblastine decreased the incidence ofmetastases of some of these tumors at times in theabsence of any effect on the growth of the primarytumor. Tumors in which such effects have beenreported include : adenocarcinoma of the pancreas(Pan. 1B), cystadenocancinoma of the liven (H. Ep.1B), melanotic melanoma (M. Mel. 1), adenocan

cinoma of the kidney (R. Ca. 1), pnostatic cancinoma (Prost. 1) and fibrosancoma (Fi. Sa. @).

This study is in need of further exploration aswell as confirmation, but these experimental nesuits as well as available clinical reports would

argue for expansion of investigations of the Vinca

alkaloids as surgical adjuncts. Again, because ofits relative lack of bone-marrow depression vim

cristine should be of obvious interest, but vimblastine should probably also be considered.

Lastly are the effects of prolonged administration of vinbiastine at low dosage levels. We havecommented on the importance of maintenancetherapy with these compounds and, in addition,the duration of therapy required for a useful effectof vinblastine in carcinoma of the breast and perhaps of the bronchus. In the latter case, it wasstated that it appeared possible to obtain a significant increase in survival in the absence of tumorshrinkage. Recently, Walker and Wright (8@)havemade an observation in tissue culture of primaryexplants from a variety of human neoplasms which

may also lend support to the concept of prolongednontoxic therapy with vinblastine At subtoxiclevels of vinblastine, these workers observed morphological alteration of these cultures from pleomorphic multipolan elongated cells to an epithelialtype. These workers took note of the fact that, ingeneral, normal cell lines have polygonal or epithelial-like cells which might change to a fusiform,fibroblast-like morphology, and that the effect ofvinblastine in these cultures appeared to be thereversal of this type of transformation which isalso frequently observed in virus-induced tnansformation. One could interpret their observationsas a â€œre-differentiationâ€•from a state of â€œde-differentiation.â€• In any event, the clinical evaluation ofprolongedlow-level therapy with vinblastine whereclinically feasible seems warranted.

ACKNOWLEDGMENTS

The authors would like to thank the following individualsfor technical assistance: Miss Louise Wood, Mr. H. F. Wright,Mr. G. A. Poore, Mr. C. P. Dorsey, and Mr. L. A. Baker. Wewould like to thank Mrs. P. Steuernagel and Mr. L. T. Willisfor assistance with the manuscript, and C. J. Jansen, M.D.,for critically reviewing certain portions of it. In addition, theisolation and chemical work described herein were carried outin cooperation with Drs. Gordon Svoboda, Norbert Neuss,H. E. Boaz, and Mr. A. J. Barnesof the Lilly Laboratories,and a special acknowledgment is given to Prof. K. Biemannof M.I.T. for recording and interpretation of mass spectraldata. We would like to thank P. J. Fouts, M.D., and R. W.Dyke, M.D., for clinical assistance.

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1963;23:1390-1427. Cancer Res Irving S. Johnson, James G. Armstrong, Marvin Gorman, et al. The Vinca Alkaloids: A New Class of Oncolytic Agents

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