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Further biological activities of vincaleukoblastine—an alkaloid isolated from Vinca rosea (L.)
Biochemical Pharmacology, 1958. Vol. I, pp. 347-357. Pergamon Press Ltd., Printed in Great Britain
SHORT COMMUNICATIONS Further biological activities of vincaleukoblastine-an
alkaloid isolated from Vinccl rosea (L.)
OVER a number of years interest in this laboratory has centred on possible biological activity of extracts of the plant Vincu YOSPU.In 1955 the preparation and partial purification of an extract containing a principle causing a fall in circulating leukocytes in the rat was reported.’ Further studies showed that, while the myelocytes were particularly affected, extensive changes in the bone marrow were induced.“* 3 Recently it was reported that a highly active alkaloid, vincaleukoblastine, had been isolated as the crystalline sulphate which in a dose of 0.35 mg/kg administered intraperitoneally caused typical haematological changes in mice, rats or guinea pigs and had little effect on the gut. The blood changes could not be altered by treatment with folic or folinic acid.” Other impure fractions, if given in larger doses, had a similar effect on the haemopoetic system. One fraction was found at a dose level of from 2.5 to 3 mgikg also to antagonize the effect of oestrogen on the rat uterus, and this effect could be prevented by folinic acid.3 In a few studies crude fractions of the extract were found to exhibit some carcinostatic action against a transplanted mammary tumor in DBA mice, and against a transplantable rat sarcoma.4 In view of the apparent high degree of activity of extracts of Vinra msea against the blood forming organs, experiments have been extended to include leukemia and other tumours. Only preliminary observations have been made to date in view of the limited quantity of vincaleukoblastine available. It is apparent, however, that this substance can readily inhibit the growth of a number of animal turnours. ]t was noted that intraperitoneal administration of an aqueous solution of the alkaloid sulphate gave somewhat variable effects on the blood count. However, reproducible results were obtained if the alkaloid was administered as a solution of the free base in aqueous propylene glycol. The sulphate was therefore neutralized (pH 7.0) with dilute sodium bicarbonate and the resulting suspension of the free base diluted with 2 ~01s. of propylene glycol. Appropriate amounts of the resulting clear solution were diluted with propylene glycol before administration. Anti-tumour activity of vincaleukoblastine has been demonstrated against Ll210 and PI534 leukaemia transplanted in BDF hybrid mice, AKr leukaemia transplanted in AK mice, and sarcoma 180in Swiss mice. Limited studies have been made with an acute leukemia in Fischer line 344 rats (leukaemia IRC 741/1398).* Generally, treatment of groups of five mice WdS instituted 24 hr after inoculation of the animals, and consisted of daily intraperitoneal injections (0.2 cmY) for 10 days. Control animals received daily injections of the same amounts of propylene glycol. Previous studies had indicated that single doses above 1.5 mg/kg of body weight were toxic for rats and mice. However, from 3 to 4 times this amount was tolerated with little indication of toxicity. if administered over a period of IO days. The results obtained are summarized below and show that vincaleukoblastine has an inhibitory effect on the development of these tumours. In the case of ~1210 leukaemia a comparison has been made with the activity of methotrexate. Tests with myeloid leukaemia IRC741 This tumour has been maintained by serial transplant in duplicate, and the duplicate animal only has been available for testing. Consequently treatment with Vlb. has not been instituted until the leukaemia was well advanced (7-10 days after inoculation). Many of the animals were acutely ill. Although survival time has not been significantly increased (15 days for treated vs. 13 days for untreated (three) animals), there has been pronounced alteration of the leukaemic process in peripheral blood. At the time therapy was instituted, the animals showed elevated total white counts (from 30,000 cells/mm” to 200,000 cells/mm”) with the presence of leukaemic cells (5-24 per cent). Within 3 days of the administration of a single dose of Vlb. (0.2-0.75 mgig) the count dropped sharply to less than 50 per cent, accompanied by disappearance of the leukaemic cells. In one rat the peripheral * Kindly supplied by Dr. W. F. DUNNING, University of Miami 347
Short
34x
communlc~~lion~
count was maintained at less than 8000 cells mm” with no showed a slight reduction in size of the peripheral tumour Further work on vincaleukoblastine is in progress to affect tumours. A number of other fractions have been blood picture and these are being investigated for their
I. ANTI-TUMOUR
TABLE
EFFECT OF VINC‘,U.I i,l
Survival Treated
DOSC
Tumour
Drug
(mg;kg)
Vlb.
L I210
Methotrexate
P 1534
Vlb.
Vlb.
AKr
leukaemic cells. for 5 days. This mimal ;\I\o (from 4.5 cm diameter to 3.0 cm diameter). determine the optimum dosage regimen to obtained from V;~I~NYOYO(Iwhich affect the possible action on cxp:rimcntal tumours.
0.3 0.4
9 IO
12.0
0.4 0.6
IO IO
13.1) 13.5
0.4 0.6
IO I5
*
0.3
IO
time (days) Control
~_____
I I.8
’ I
*
1 Increase (7”)
74 7.7
59 56
IO.2 9.8
33 38
16.5 I
* One of 5 animals at the lower dose died on day 28. The remaining animals are living and well at 42 days. t One inadvertently killed on day 4, one died at day 12 showing no evidence of tumour.
TABLE 2. EFFECTS ON TUMOUK S180
Material
Vlb
( -0.4
Dose (mg:kg)
I IO
Take time (days)* Treated Control mean mean
Survival time (days) Treated Control mean mean
19.5 (2)l
26 (2,t
8 (5)
* Time for tumour t3 reach I cm diameter. 7 Three failed to develop tumours. There was no evidence when autopsied at 80 days.
Aciinow/~d~c,m~nr-We Dynes for valuable Institute of Canada during the tenure of Cutts on a Research
23.4 (5)
of turnout
would like to thank Mr. J. Purko, M.Sc.. Mr. G. Carpenter and assistance and the National Research Council, Ottawa and National for financial support of this project. Dr. Beer has worked on this a Medical Research Associateship of the National Research Council Fellowship of the National Cancer Institute of Canada. R. L. NOBLE C. T. BEER J. H. CUTTS
REFERENCES I. 2. 3. 4.
C. J. J. R.
T. H. H. L.
BEER, Brirish Empire, Caner C’ampai,rn 33~d Ann. Rep. p. 487 (I 955). CUTTS, C. T. BEER and R. L. NoaLE, Rw. C‘ctnnd. Biol. 16, 476 (1957). Cus-rs. Plot. Amer. A.ss. Camw Res. 2, 289 ( 1958). NOBLE, C. T. BEER and J. H. CUTTS, Ann. N. Y. Acud. SC,;. 76, 882 (195X).
Miss M. Cancer problem and Dr.
SHORT COMMUNICATIONS Further biological activities of vincaleukoblastine-an
alkaloid isolated from Vinccl rosea (L.)
OVER a number of years interest in this laboratory has centred on possible biological activity of extracts of the plant Vincu YOSPU.In 1955 the preparation and partial purification of an extract containing a principle causing a fall in circulating leukocytes in the rat was reported.’ Further studies showed that, while the myelocytes were particularly affected, extensive changes in the bone marrow were induced.“* 3 Recently it was reported that a highly active alkaloid, vincaleukoblastine, had been isolated as the crystalline sulphate which in a dose of 0.35 mg/kg administered intraperitoneally caused typical haematological changes in mice, rats or guinea pigs and had little effect on the gut. The blood changes could not be altered by treatment with folic or folinic acid.” Other impure fractions, if given in larger doses, had a similar effect on the haemopoetic system. One fraction was found at a dose level of from 2.5 to 3 mgikg also to antagonize the effect of oestrogen on the rat uterus, and this effect could be prevented by folinic acid.3 In a few studies crude fractions of the extract were found to exhibit some carcinostatic action against a transplanted mammary tumor in DBA mice, and against a transplantable rat sarcoma.4 In view of the apparent high degree of activity of extracts of Vinra msea against the blood forming organs, experiments have been extended to include leukemia and other tumours. Only preliminary observations have been made to date in view of the limited quantity of vincaleukoblastine available. It is apparent, however, that this substance can readily inhibit the growth of a number of animal turnours. ]t was noted that intraperitoneal administration of an aqueous solution of the alkaloid sulphate gave somewhat variable effects on the blood count. However, reproducible results were obtained if the alkaloid was administered as a solution of the free base in aqueous propylene glycol. The sulphate was therefore neutralized (pH 7.0) with dilute sodium bicarbonate and the resulting suspension of the free base diluted with 2 ~01s. of propylene glycol. Appropriate amounts of the resulting clear solution were diluted with propylene glycol before administration. Anti-tumour activity of vincaleukoblastine has been demonstrated against Ll210 and PI534 leukaemia transplanted in BDF hybrid mice, AKr leukaemia transplanted in AK mice, and sarcoma 180in Swiss mice. Limited studies have been made with an acute leukemia in Fischer line 344 rats (leukaemia IRC 741/1398).* Generally, treatment of groups of five mice WdS instituted 24 hr after inoculation of the animals, and consisted of daily intraperitoneal injections (0.2 cmY) for 10 days. Control animals received daily injections of the same amounts of propylene glycol. Previous studies had indicated that single doses above 1.5 mg/kg of body weight were toxic for rats and mice. However, from 3 to 4 times this amount was tolerated with little indication of toxicity. if administered over a period of IO days. The results obtained are summarized below and show that vincaleukoblastine has an inhibitory effect on the development of these tumours. In the case of ~1210 leukaemia a comparison has been made with the activity of methotrexate. Tests with myeloid leukaemia IRC741 This tumour has been maintained by serial transplant in duplicate, and the duplicate animal only has been available for testing. Consequently treatment with Vlb. has not been instituted until the leukaemia was well advanced (7-10 days after inoculation). Many of the animals were acutely ill. Although survival time has not been significantly increased (15 days for treated vs. 13 days for untreated (three) animals), there has been pronounced alteration of the leukaemic process in peripheral blood. At the time therapy was instituted, the animals showed elevated total white counts (from 30,000 cells/mm” to 200,000 cells/mm”) with the presence of leukaemic cells (5-24 per cent). Within 3 days of the administration of a single dose of Vlb. (0.2-0.75 mgig) the count dropped sharply to less than 50 per cent, accompanied by disappearance of the leukaemic cells. In one rat the peripheral * Kindly supplied by Dr. W. F. DUNNING, University of Miami 347
Short
34x
communlc~~lion~
count was maintained at less than 8000 cells mm” with no showed a slight reduction in size of the peripheral tumour Further work on vincaleukoblastine is in progress to affect tumours. A number of other fractions have been blood picture and these are being investigated for their
I. ANTI-TUMOUR
TABLE
EFFECT OF VINC‘,U.I i,l
Survival Treated
DOSC
Tumour
Drug
(mg;kg)
Vlb.
L I210
Methotrexate
P 1534
Vlb.
Vlb.
AKr
leukaemic cells. for 5 days. This mimal ;\I\o (from 4.5 cm diameter to 3.0 cm diameter). determine the optimum dosage regimen to obtained from V;~I~NYOYO(Iwhich affect the possible action on cxp:rimcntal tumours.
0.3 0.4
9 IO
12.0
0.4 0.6
IO IO
13.1) 13.5
0.4 0.6
IO I5
*
0.3
IO
time (days) Control
~_____
I I.8
’ I
*
1 Increase (7”)
74 7.7
59 56
IO.2 9.8
33 38
16.5 I
* One of 5 animals at the lower dose died on day 28. The remaining animals are living and well at 42 days. t One inadvertently killed on day 4, one died at day 12 showing no evidence of tumour.
TABLE 2. EFFECTS ON TUMOUK S180
Material
Vlb
( -0.4
Dose (mg:kg)
I IO
Take time (days)* Treated Control mean mean
Survival time (days) Treated Control mean mean
19.5 (2)l
26 (2,t
8 (5)
* Time for tumour t3 reach I cm diameter. 7 Three failed to develop tumours. There was no evidence when autopsied at 80 days.
Aciinow/~d~c,m~nr-We Dynes for valuable Institute of Canada during the tenure of Cutts on a Research
23.4 (5)
of turnout
would like to thank Mr. J. Purko, M.Sc.. Mr. G. Carpenter and assistance and the National Research Council, Ottawa and National for financial support of this project. Dr. Beer has worked on this a Medical Research Associateship of the National Research Council Fellowship of the National Cancer Institute of Canada. R. L. NOBLE C. T. BEER J. H. CUTTS
REFERENCES I. 2. 3. 4.
C. J. J. R.
T. H. H. L.
BEER, Brirish Empire, Caner C’ampai,rn 33~d Ann. Rep. p. 487 (I 955). CUTTS, C. T. BEER and R. L. NoaLE, Rw. C‘ctnnd. Biol. 16, 476 (1957). Cus-rs. Plot. Amer. A.ss. Camw Res. 2, 289 ( 1958). NOBLE, C. T. BEER and J. H. CUTTS, Ann. N. Y. Acud. SC,;. 76, 882 (195X).
Miss M. Cancer problem and Dr.