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Studies on the intracerebral injection of vincristine free and entrapped within liposomes in the rat
Journal of the Neurological Sciences, 1985, 68:25-30 Elsevier
25
Studies on the Intracerebral Injection of Vincristine Free and Entrapped Within Liposomes in the Rat A.S. Oliver 1, G. Firth 2 and R.O. McKeran 1'3 IDepartment of Biochemistry, St. George's Hospital Medical School, London, 2Hurstwood Park Neurological Centre, Sussex and the University of Surrey and 3Department of Neurology, Atkinson Morley's Hospital London (U.K.) (Received 10 July, 1984) (Revised, received 2 November, 1984) (Accepted 5 November, 1984)
SUMMARY
The cerebral tissue response and behaviour of rats injected with vincristine of increasing concentration, free and entrapped within fiposomes was studied. In separate experiments, blood, urine and brain levels of vineristine were measured after intracerebral injection of free and fiposome-entrapped vincristine. When entrapped within liposomes, vineristine clearance from the brain was significantly reduced and the amount of tissue damage was directly related to the amount of drug given, being slightly greater at the same dose when entrapped. These results indicate a potential application for drugs entrapped within liposomes acting as a depot preparation in the treatment of cerebral gliomas.
Key words: Antimitotic drugs- Human cerebral gliomas- Liposomes- Vincristine
INTRODUCTION
Since human malignant gliomas are uniformly fatal and treatment has only a marginal effect on longevity, it is important to consider new ways of treating this
This study was supported financiallyby the St. George's Hospital Medical Research Committee and the South West Thames Locally Organised Research Scheme. Correspondence to: Dr. R.O. McKeran, Department of Neurology, Atkinson Morley's Hospital, 31 Copse Hill, Wimbledon, SW 20, U.K. 0022-410X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
26 condition. Many drugs when given systemically do not reach the brain in high enough and sustained concentration to produce a significant effect, and when attempts are made to correct the deficiency by increasing the dose of drug, systemic toxicity invariably curtails treatment. We have recently determined the optimal conditions for producing maximum incorporation within liposomes and sustained release of two cell cycle specific antimitotic drugs (bleomycin and vincristine) used in the treatment of human cerebral gliomas (Firth et al. 1984a), and have demonstrated dose-response curves are similar for the two drugs whether free or entrapped within liposomes when applied to a human glioma cell line (U251-MG). When bleomycin is entrapped within liposomes and injected intracerebrally, it is of low toxicity to normal rat cerebral tissue and is cleared more slowly from the injection site than when the free drug is injected (Firth et al. 1984b). These results have suggested a potential application for drugs entrapped within liposomes acting as a depot preparation in the treatment of human cerebral gliomas which we have confirmed by our preliminary observations in human subjects (McKeran et al. 1984). Since antimitotic drugs are used in combination in the treatment of human tumours, it was important to establish whether similar considerations applied to other drugs and we have, therefore, extended our studies to include vincristine. MATERIALS AND METHODS
Materials Cholesterol (C), dipalmitoyl phosphatidyl choline (D) and dipalmitoyl phosphatidic acid (P) of highest obtainable purity were purchased from Sigma Chemical Company. Vincristine was donated by Ely Lily. Sepharose B6 was obtained from Pharmacia Ltd. [3H]Vincristine was obtained from Amersham International and vincristine antiserum was supplied by Guildhay, University of Surrey. All other reagents were obtained through BDH Ltd.
Preparation of liposomes Liposomes were prepared by the reverse phase method described by Szoka and Papahadjopaulos (1978).
Assay of vincristine The vincristine radioimmunoassay was performed following the method of Teale et al. (1977) with minor modifications.
Cerebral wxicity studies Reverse phase prepared liposomes were made containing either buffer or vincristine and dilutions made using 0.02 M pH 7.4 phosphate buffer (Firth et al. 1984a). Male Wistar rats weighing approximately 250 g were anaesthetised using an air/nitrous oxide/halothane mixture. A l-ram diameter hole was drilled through the skull 2.7 mm anterior from the bregma point and 1.5 mm laterally over the left hemisphere using a stereotactic manipulator. Suitable dilutions in a total volume of 5 and 10/~1
27 o f free l i p o s o m e a n d o f free a n d l i p o s o m e - e n t r a p p e d v i n c r i s t i n e w e r e i n j e c t e d at a d e p t h o f 2 m m in the c e r e b r a l h e m i s p h e r e c o r t e x u s i n g a h a m i l t o n syringe a n d the s t e r e o t a c t i c m a n i p u l a t o r . T w o a n i m a l s w e r e studied at e a c h c o n c e n t r a t i o n o f lipid a n d o f v i n c r i s t i n e w h e n e n t r a p p e d within l i p o s o m e s w i t h t w o buffer o n l y c o n t r o l s . T h e rats w e r e a l l o w e d to r e c o v e r in m e t a b o l i c c a g e s w h e r e f o o d c o n s u m p t i o n , u r i n e a n d faeces p r o d u c t i o n a n d g e n e r a l b e h a v i o u r w e r e o b s e r v e d for 7 days. T h e a n i m a l s w e r e t h e n killed a n d the b r a i n r e m o v e d after it h a d b e e n p e r f u s e d w i t h h e p a r i n i s e d saline f o l l o w e d by buffered f o r m o l saline. T h e b r a i n s w e r e s t o r e d in b u f f e r e d f o r m o l saline at 4 ° C until p r o c e s s e d for histology. Serial a c t i o n s w e r e cut a n d stained w i t h h a e m a t o x y l i n a n d eosin. T h e serial sections w e r e c o d e d a n d the degree o f c e r e b r a l d a m a g e w a s a s s e s s e d w i t h light m i c r o s c o p y by t w o o b s e r v e r s w h o h a d n o k n o w l e d g e o f t h e c o d e . T h e d e g r e e o f histological d a m a g e w a s g r a d e d o n a 6 - p o i n t scale ( T a b l e 1).
Tissue distribution studies of vincristine after intracerebral injectionfree and entrapped within liposomes M a l e W i s t a r rats a n a e s t h e t i s e d w i t h p e n t a b a r b i t o n e w e r e i n j e c t e d i n t r a c e r e b r a l l y w i t h either free vincristine o r vincristine e n t r a p p e d within l i p o s o m e s in d u p l i c a t e by the TABLE 1 CEREBRAL HISTOLOGICAL APPEARANCE AFTER INTRACEREBRAL INJECTION OF VINCRISTINE IN LIPOSOMES Lipid content (/~moles)
Total vincristine injected (/~g)
Histology
Behavioural response
3.3 1.65 6.6 3.3 3.3
_ -m m --
Needle tract Needle tract Needle tract Needle tract Needle tract
No No No No No
125.0 9.4 1.25
+ + + + + + + + + + + + +
No change (1 animal killed at 5 days)
x x x x ×
10- i 10- ~ 10 -2 10 -2 10 -3
w
3.3 4.4 4.4 4.4
x l 0 -~ x 10 -2 x 10 -3 x 10 -4
9.4 1.25 1.25 x 10 -1 1.25 x 10 -2 Buffer only
+ + + + + + + + + +
+ + + + +
+ + + + + + +
= = = = = = =
change change change change change
++++++ + + + + + + + +
No change
Minimal local reaction around needle tract. Local reaction around needle tract. Marked local reaction around needle tract, no necrosis. Marked local reaction with necrosis. 25% of the cerebral hemisphere necrotic, marked inflammatory response. 50% of the cerebral hemisphere necrotic, marked inflammatory response. 50% of the cerebral hemisphere necrotic, haemorrhage, marked inflammatory response.
28 technique described above (0.1 mg of vincristine per animal). Repeated blood and urine samples were obtained over a period of 4 h and assayed for vincristine. The animals were sacrificed at 4 h, and tissue samples were taken and stored at - 20 ° C until assayed for vincristine. Additional similar longer term experiments were performed in duplicate after intracerebral injection of free vincristine and vincristine entrapped within liposomes in rats anaesthetised with an air/nitrous oxide/halothane mixture. These animals were sacrificed at 24 and 72 h and 7 days with blood, urine and tissue samples obtained at these stages.
Assay of vincristine Blood, urine and brain levels of vincristine were assayed. The brain samples were homogenized in 3 ml of phosphate buffer as used in the radioimmunoassay using a glass hand homogenizer. An aliquot of the supernatant was removed after centrifuging at 8500 x g for 2 min. RESULTS
Cerebral toxicity studies No alteration of behaviour, food consumption, urine or faecal production was detected in any of the animals after intracerebral injection of reverse phase prepared liposomes containing either buffer or vincristine over a period of 1 week's observation except at the higher concentration of vincristine when given either as the free drug or entrapped within liposomes. One of the animals injected with the highest dose of free vincristine was sacrificed at 5 days owing to marked behavioural changes. The cerebral histological changes observed after intracerebral injection of free vincristine or reverse phase prepared liposomes containing either buffer or vincristine are shown in Table 1. As the concentration of vincristine was increased, the evidence of histological damage also increased correspondingly. Only at low doses of vincristine was the amount of damage to normal brain tissues sufficiently restricted therefore to consider a future possible therapeutic use in human studies. Tissue distribution studies No vincristine could be detected in the serum at the dose levels used which is compatible with the known sensitivity of the radioimmunoassay method. Low levels of vincristine could be detected in the urine at 4 h but not at 24 h after intracerebral injection, there being no significant difference between the results obtained after injection of the free or liposome-entrapped drug. At 4 h after intracerebral injection, there was twice as much vincristine still in the brain, mainly localised to the hemisphere of injection, in those animals which had been injected with vincristine entrapped within liposomes as compared to those receiving the free drug. At 24 h, no vincristine could be detected in the brains of those rats injected with free drug, whereas there was still residual activity detectable in animals receiving the drug entrapped within liposomes. After 24 h, no vincristine could be detected in the brains of any animals (Table 2).
29 TABLE 2 VINCRISTINE LEVELSMEASURED IN BRAIN, URINE AND SERUM AFTER INTRACEREBRALINJECTION OF FREE AND LIPOSOME ENTRAPPED VINCRISTINE Free drug (~o of total amount injected, mean, n = 2)
Liposome-entrappeddrug (~ of total amount injected, mean, n = 2)
4h 4h 4h 4h
20.2 2.9 None detected 23.1
35.6 6.9 0.7 43.2
4h 4h 24 h
1.6 None detected None detected
1.3 None detected 2.5
Site
Lt. brain Rt. brain Rear brain Total Urine Serum Brain
DISCUSSION Vincristine is an alkaloid agent derived from the plant Cantharanthus roseus which has been widely used in cancer chemotherapy particularly in the treatment ofleukaemia and lymphoma. The major antitumour effect of this drug appears to be related to its high-affinity binding to tubulin, the basic protein subunit of microtubules, resulting in the disruption of the mitotic spindle assembly and arrest of cells in metaphase (Owellen et al. 1976). In vitro studies have suggested a reversibility of mitotic effect in tissue culture (Tucker et al. 1977) and that its effectiveness as an antitumour agent could be enhanced if methods were developed to prolong exposure of neoplastic tissues for longer periods of time than currently produced by conventional methods of administration (Jackson and Bender 1979). The antitumour effect ofvincristine is critically dependent on both concentration and duration of exposure. However, neurotoxicity is frequently encountered and is the limiting factor in its clinical use (Jackson and Bender 1978). This is characterised by a peripheral mixed sensory and motor neuropathy, confusion, depression, agitation, insomnia, hallucinations, psychosis and hyponatraemia resulting from an inappropriate antidiuretic hormone secretion. Biphasic curves for the disappearance of tritiated vincristine from the blood in the rat and dog have been described with an initial half-life of approximately 15 rain and a secondary half-life of approximately 75 min (Castle et al. 1976). Tissue levels were high in the rat at 1 h, declining rapidly except in the brain where low levels were found throughout the length of the experiment (24 h). The bile was found to be the major site of excretion. Vincristine penetrates the CSF of rhesus monkeys rapidly following intravenous injection and low, rather constant, levels of vincristine and its metabolites are maintained in the CSF over 72 h and may explain the neurotoxicity of the drug (Jackson et al. 1980). Measurable blood levels of vincristine and its products have been demon-
30
strated in man for up to 3 days following intravenous injection, possibly due to extensive protein and tissue binding and enterohepatic recirculation (Jackson and Bender 1978). We have recently confn'med the antitumour effect of vincristine in vitro against a human glioma cell line (U251-MG) and demonstrated no loss of effectiveness when incorporated within negatively charged liposomes applied to this cell line (Firth et al. 1984a). The sustained release of vincristine from these liposomes suggested that it might be used therapeutically incorporated within liposomes and injected into a glioma tumour bed with sustained subtoxic release of drug at a therapeutic concentration to the tumour tissue. This paper, however, demonstrates the extreme dose-dependent neurotoxicity of vincristine when injected intracerebrally in the rat both as the free and liposomeentrapped drug which appears to be enhanced by incorporating the drug within liposomes. The liposome-encapsulated drug was cleared more slowly from the brain than the free drug. These studies have demonstrated a potential application for the use of vincristine entrapped within liposomes in the treatment of human cerebral gliomas although the dose will have to be carefully assessed in human studies to avoid neurotoxicity. REFERENCES Castle, M, C., D. A. Margileth and V.T. Oliverio (1976) Distribution and excretion of [aH]vincristine in the rat and the dog, Cancer Res., 36: 3684-3689. Firth, G., A. S. Oliver and R. O. McKeran (1984a) Studies on the use of antimitotic drugs entrapped within liposomes and of their action on a human glioma cell line, J. Neurol. Sci., 63: 153-165. Firth, G., A. S. Oliver and R. O. McKeran (1984b) Studies on the intracerebral injection of bleomycin free and entrapped within liposomes in the rat, J. Neurol. Neurosurg. Psychiat., 47: 585-589. Jackson, D.V. and R. A. Bender (1978)The clinical pharmacology of the vinca alkaloids epipodophyllotoxins and maytansine. In: H. M. Pine,do (Ed.), Clinical Pharmacology of Ana-Neoplastic Drugs, Elsevier/NorthHolland Biomedical Press, Amersterdam, pp. 277-294. Jackson, D.V. and R.A. Bender (1979) Cytotoxic thresholds of vincristine in a routine and a human leukaemia cell line in vitro, Cancer Res., 39: 4346-4349. Jackson, D.V., M. C. Castle, D.G. Poplack and R. A. Bender (1980) Pharmacokinctics of vincristine in the cerebrospinal fluid of subhuman primates, Cancer Res., 40: 722-724. McKeran, R.O., G. Firth, A.S. Oliver, D. Uttley and S. O'Laoire (1984) A potential application for the intracerebralinjection of drugs entrapped within liposomes in the treatment of human cerebral gliomas, J. Neurol. Neurosurg. Psychiat., in press. Owellen, R.J., C.A. Hartke, R.M. Dickerson and F.O. Hains (1976) Inhibition of tubulin-microtubule polymerization by drugs of the vinca alkaloid class, Cancer Res., 36: 1499-1592. Szoka, F. and D. Papahadjapoulos (1978) Procedure for the preparation of liposomes with large internal aqueous space and high capture by reverse phase evaporation, Prec. Nat. Acad. Sci. (USA), 75: 4194-4198. Teale, J.D., J.M. Clough and V. Marks (1977) Radioimmunoassay of vinblastine and vincristine, Brit. J. Clin. Pharmae., 4: 169-172. Tucker, R.W., R.J. Owellen and S.B. Harris (1977) Correlation of cytotoxicity and muscle spindle dissolution by vinblastine in mammalian cells, Cancer Res., 37: 4346-4351.
25
Studies on the Intracerebral Injection of Vincristine Free and Entrapped Within Liposomes in the Rat A.S. Oliver 1, G. Firth 2 and R.O. McKeran 1'3 IDepartment of Biochemistry, St. George's Hospital Medical School, London, 2Hurstwood Park Neurological Centre, Sussex and the University of Surrey and 3Department of Neurology, Atkinson Morley's Hospital London (U.K.) (Received 10 July, 1984) (Revised, received 2 November, 1984) (Accepted 5 November, 1984)
SUMMARY
The cerebral tissue response and behaviour of rats injected with vincristine of increasing concentration, free and entrapped within fiposomes was studied. In separate experiments, blood, urine and brain levels of vineristine were measured after intracerebral injection of free and fiposome-entrapped vincristine. When entrapped within liposomes, vineristine clearance from the brain was significantly reduced and the amount of tissue damage was directly related to the amount of drug given, being slightly greater at the same dose when entrapped. These results indicate a potential application for drugs entrapped within liposomes acting as a depot preparation in the treatment of cerebral gliomas.
Key words: Antimitotic drugs- Human cerebral gliomas- Liposomes- Vincristine
INTRODUCTION
Since human malignant gliomas are uniformly fatal and treatment has only a marginal effect on longevity, it is important to consider new ways of treating this
This study was supported financiallyby the St. George's Hospital Medical Research Committee and the South West Thames Locally Organised Research Scheme. Correspondence to: Dr. R.O. McKeran, Department of Neurology, Atkinson Morley's Hospital, 31 Copse Hill, Wimbledon, SW 20, U.K. 0022-410X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
26 condition. Many drugs when given systemically do not reach the brain in high enough and sustained concentration to produce a significant effect, and when attempts are made to correct the deficiency by increasing the dose of drug, systemic toxicity invariably curtails treatment. We have recently determined the optimal conditions for producing maximum incorporation within liposomes and sustained release of two cell cycle specific antimitotic drugs (bleomycin and vincristine) used in the treatment of human cerebral gliomas (Firth et al. 1984a), and have demonstrated dose-response curves are similar for the two drugs whether free or entrapped within liposomes when applied to a human glioma cell line (U251-MG). When bleomycin is entrapped within liposomes and injected intracerebrally, it is of low toxicity to normal rat cerebral tissue and is cleared more slowly from the injection site than when the free drug is injected (Firth et al. 1984b). These results have suggested a potential application for drugs entrapped within liposomes acting as a depot preparation in the treatment of human cerebral gliomas which we have confirmed by our preliminary observations in human subjects (McKeran et al. 1984). Since antimitotic drugs are used in combination in the treatment of human tumours, it was important to establish whether similar considerations applied to other drugs and we have, therefore, extended our studies to include vincristine. MATERIALS AND METHODS
Materials Cholesterol (C), dipalmitoyl phosphatidyl choline (D) and dipalmitoyl phosphatidic acid (P) of highest obtainable purity were purchased from Sigma Chemical Company. Vincristine was donated by Ely Lily. Sepharose B6 was obtained from Pharmacia Ltd. [3H]Vincristine was obtained from Amersham International and vincristine antiserum was supplied by Guildhay, University of Surrey. All other reagents were obtained through BDH Ltd.
Preparation of liposomes Liposomes were prepared by the reverse phase method described by Szoka and Papahadjopaulos (1978).
Assay of vincristine The vincristine radioimmunoassay was performed following the method of Teale et al. (1977) with minor modifications.
Cerebral wxicity studies Reverse phase prepared liposomes were made containing either buffer or vincristine and dilutions made using 0.02 M pH 7.4 phosphate buffer (Firth et al. 1984a). Male Wistar rats weighing approximately 250 g were anaesthetised using an air/nitrous oxide/halothane mixture. A l-ram diameter hole was drilled through the skull 2.7 mm anterior from the bregma point and 1.5 mm laterally over the left hemisphere using a stereotactic manipulator. Suitable dilutions in a total volume of 5 and 10/~1
27 o f free l i p o s o m e a n d o f free a n d l i p o s o m e - e n t r a p p e d v i n c r i s t i n e w e r e i n j e c t e d at a d e p t h o f 2 m m in the c e r e b r a l h e m i s p h e r e c o r t e x u s i n g a h a m i l t o n syringe a n d the s t e r e o t a c t i c m a n i p u l a t o r . T w o a n i m a l s w e r e studied at e a c h c o n c e n t r a t i o n o f lipid a n d o f v i n c r i s t i n e w h e n e n t r a p p e d within l i p o s o m e s w i t h t w o buffer o n l y c o n t r o l s . T h e rats w e r e a l l o w e d to r e c o v e r in m e t a b o l i c c a g e s w h e r e f o o d c o n s u m p t i o n , u r i n e a n d faeces p r o d u c t i o n a n d g e n e r a l b e h a v i o u r w e r e o b s e r v e d for 7 days. T h e a n i m a l s w e r e t h e n killed a n d the b r a i n r e m o v e d after it h a d b e e n p e r f u s e d w i t h h e p a r i n i s e d saline f o l l o w e d by buffered f o r m o l saline. T h e b r a i n s w e r e s t o r e d in b u f f e r e d f o r m o l saline at 4 ° C until p r o c e s s e d for histology. Serial a c t i o n s w e r e cut a n d stained w i t h h a e m a t o x y l i n a n d eosin. T h e serial sections w e r e c o d e d a n d the degree o f c e r e b r a l d a m a g e w a s a s s e s s e d w i t h light m i c r o s c o p y by t w o o b s e r v e r s w h o h a d n o k n o w l e d g e o f t h e c o d e . T h e d e g r e e o f histological d a m a g e w a s g r a d e d o n a 6 - p o i n t scale ( T a b l e 1).
Tissue distribution studies of vincristine after intracerebral injectionfree and entrapped within liposomes M a l e W i s t a r rats a n a e s t h e t i s e d w i t h p e n t a b a r b i t o n e w e r e i n j e c t e d i n t r a c e r e b r a l l y w i t h either free vincristine o r vincristine e n t r a p p e d within l i p o s o m e s in d u p l i c a t e by the TABLE 1 CEREBRAL HISTOLOGICAL APPEARANCE AFTER INTRACEREBRAL INJECTION OF VINCRISTINE IN LIPOSOMES Lipid content (/~moles)
Total vincristine injected (/~g)
Histology
Behavioural response
3.3 1.65 6.6 3.3 3.3
_ -m m --
Needle tract Needle tract Needle tract Needle tract Needle tract
No No No No No
125.0 9.4 1.25
+ + + + + + + + + + + + +
No change (1 animal killed at 5 days)
x x x x ×
10- i 10- ~ 10 -2 10 -2 10 -3
w
3.3 4.4 4.4 4.4
x l 0 -~ x 10 -2 x 10 -3 x 10 -4
9.4 1.25 1.25 x 10 -1 1.25 x 10 -2 Buffer only
+ + + + + + + + + +
+ + + + +
+ + + + + + +
= = = = = = =
change change change change change
++++++ + + + + + + + +
No change
Minimal local reaction around needle tract. Local reaction around needle tract. Marked local reaction around needle tract, no necrosis. Marked local reaction with necrosis. 25% of the cerebral hemisphere necrotic, marked inflammatory response. 50% of the cerebral hemisphere necrotic, marked inflammatory response. 50% of the cerebral hemisphere necrotic, haemorrhage, marked inflammatory response.
28 technique described above (0.1 mg of vincristine per animal). Repeated blood and urine samples were obtained over a period of 4 h and assayed for vincristine. The animals were sacrificed at 4 h, and tissue samples were taken and stored at - 20 ° C until assayed for vincristine. Additional similar longer term experiments were performed in duplicate after intracerebral injection of free vincristine and vincristine entrapped within liposomes in rats anaesthetised with an air/nitrous oxide/halothane mixture. These animals were sacrificed at 24 and 72 h and 7 days with blood, urine and tissue samples obtained at these stages.
Assay of vincristine Blood, urine and brain levels of vincristine were assayed. The brain samples were homogenized in 3 ml of phosphate buffer as used in the radioimmunoassay using a glass hand homogenizer. An aliquot of the supernatant was removed after centrifuging at 8500 x g for 2 min. RESULTS
Cerebral toxicity studies No alteration of behaviour, food consumption, urine or faecal production was detected in any of the animals after intracerebral injection of reverse phase prepared liposomes containing either buffer or vincristine over a period of 1 week's observation except at the higher concentration of vincristine when given either as the free drug or entrapped within liposomes. One of the animals injected with the highest dose of free vincristine was sacrificed at 5 days owing to marked behavioural changes. The cerebral histological changes observed after intracerebral injection of free vincristine or reverse phase prepared liposomes containing either buffer or vincristine are shown in Table 1. As the concentration of vincristine was increased, the evidence of histological damage also increased correspondingly. Only at low doses of vincristine was the amount of damage to normal brain tissues sufficiently restricted therefore to consider a future possible therapeutic use in human studies. Tissue distribution studies No vincristine could be detected in the serum at the dose levels used which is compatible with the known sensitivity of the radioimmunoassay method. Low levels of vincristine could be detected in the urine at 4 h but not at 24 h after intracerebral injection, there being no significant difference between the results obtained after injection of the free or liposome-entrapped drug. At 4 h after intracerebral injection, there was twice as much vincristine still in the brain, mainly localised to the hemisphere of injection, in those animals which had been injected with vincristine entrapped within liposomes as compared to those receiving the free drug. At 24 h, no vincristine could be detected in the brains of those rats injected with free drug, whereas there was still residual activity detectable in animals receiving the drug entrapped within liposomes. After 24 h, no vincristine could be detected in the brains of any animals (Table 2).
29 TABLE 2 VINCRISTINE LEVELSMEASURED IN BRAIN, URINE AND SERUM AFTER INTRACEREBRALINJECTION OF FREE AND LIPOSOME ENTRAPPED VINCRISTINE Free drug (~o of total amount injected, mean, n = 2)
Liposome-entrappeddrug (~ of total amount injected, mean, n = 2)
4h 4h 4h 4h
20.2 2.9 None detected 23.1
35.6 6.9 0.7 43.2
4h 4h 24 h
1.6 None detected None detected
1.3 None detected 2.5
Site
Lt. brain Rt. brain Rear brain Total Urine Serum Brain
DISCUSSION Vincristine is an alkaloid agent derived from the plant Cantharanthus roseus which has been widely used in cancer chemotherapy particularly in the treatment ofleukaemia and lymphoma. The major antitumour effect of this drug appears to be related to its high-affinity binding to tubulin, the basic protein subunit of microtubules, resulting in the disruption of the mitotic spindle assembly and arrest of cells in metaphase (Owellen et al. 1976). In vitro studies have suggested a reversibility of mitotic effect in tissue culture (Tucker et al. 1977) and that its effectiveness as an antitumour agent could be enhanced if methods were developed to prolong exposure of neoplastic tissues for longer periods of time than currently produced by conventional methods of administration (Jackson and Bender 1979). The antitumour effect ofvincristine is critically dependent on both concentration and duration of exposure. However, neurotoxicity is frequently encountered and is the limiting factor in its clinical use (Jackson and Bender 1978). This is characterised by a peripheral mixed sensory and motor neuropathy, confusion, depression, agitation, insomnia, hallucinations, psychosis and hyponatraemia resulting from an inappropriate antidiuretic hormone secretion. Biphasic curves for the disappearance of tritiated vincristine from the blood in the rat and dog have been described with an initial half-life of approximately 15 rain and a secondary half-life of approximately 75 min (Castle et al. 1976). Tissue levels were high in the rat at 1 h, declining rapidly except in the brain where low levels were found throughout the length of the experiment (24 h). The bile was found to be the major site of excretion. Vincristine penetrates the CSF of rhesus monkeys rapidly following intravenous injection and low, rather constant, levels of vincristine and its metabolites are maintained in the CSF over 72 h and may explain the neurotoxicity of the drug (Jackson et al. 1980). Measurable blood levels of vincristine and its products have been demon-
30
strated in man for up to 3 days following intravenous injection, possibly due to extensive protein and tissue binding and enterohepatic recirculation (Jackson and Bender 1978). We have recently confn'med the antitumour effect of vincristine in vitro against a human glioma cell line (U251-MG) and demonstrated no loss of effectiveness when incorporated within negatively charged liposomes applied to this cell line (Firth et al. 1984a). The sustained release of vincristine from these liposomes suggested that it might be used therapeutically incorporated within liposomes and injected into a glioma tumour bed with sustained subtoxic release of drug at a therapeutic concentration to the tumour tissue. This paper, however, demonstrates the extreme dose-dependent neurotoxicity of vincristine when injected intracerebrally in the rat both as the free and liposomeentrapped drug which appears to be enhanced by incorporating the drug within liposomes. The liposome-encapsulated drug was cleared more slowly from the brain than the free drug. These studies have demonstrated a potential application for the use of vincristine entrapped within liposomes in the treatment of human cerebral gliomas although the dose will have to be carefully assessed in human studies to avoid neurotoxicity. REFERENCES Castle, M, C., D. A. Margileth and V.T. Oliverio (1976) Distribution and excretion of [aH]vincristine in the rat and the dog, Cancer Res., 36: 3684-3689. Firth, G., A. S. Oliver and R. O. McKeran (1984a) Studies on the use of antimitotic drugs entrapped within liposomes and of their action on a human glioma cell line, J. Neurol. Sci., 63: 153-165. Firth, G., A. S. Oliver and R. O. McKeran (1984b) Studies on the intracerebral injection of bleomycin free and entrapped within liposomes in the rat, J. Neurol. Neurosurg. Psychiat., 47: 585-589. Jackson, D.V. and R. A. Bender (1978)The clinical pharmacology of the vinca alkaloids epipodophyllotoxins and maytansine. In: H. M. Pine,do (Ed.), Clinical Pharmacology of Ana-Neoplastic Drugs, Elsevier/NorthHolland Biomedical Press, Amersterdam, pp. 277-294. Jackson, D.V. and R.A. Bender (1979) Cytotoxic thresholds of vincristine in a routine and a human leukaemia cell line in vitro, Cancer Res., 39: 4346-4349. Jackson, D.V., M. C. Castle, D.G. Poplack and R. A. Bender (1980) Pharmacokinctics of vincristine in the cerebrospinal fluid of subhuman primates, Cancer Res., 40: 722-724. McKeran, R.O., G. Firth, A.S. Oliver, D. Uttley and S. O'Laoire (1984) A potential application for the intracerebralinjection of drugs entrapped within liposomes in the treatment of human cerebral gliomas, J. Neurol. Neurosurg. Psychiat., in press. Owellen, R.J., C.A. Hartke, R.M. Dickerson and F.O. Hains (1976) Inhibition of tubulin-microtubule polymerization by drugs of the vinca alkaloid class, Cancer Res., 36: 1499-1592. Szoka, F. and D. Papahadjapoulos (1978) Procedure for the preparation of liposomes with large internal aqueous space and high capture by reverse phase evaporation, Prec. Nat. Acad. Sci. (USA), 75: 4194-4198. Teale, J.D., J.M. Clough and V. Marks (1977) Radioimmunoassay of vinblastine and vincristine, Brit. J. Clin. Pharmae., 4: 169-172. Tucker, R.W., R.J. Owellen and S.B. Harris (1977) Correlation of cytotoxicity and muscle spindle dissolution by vinblastine in mammalian cells, Cancer Res., 37: 4346-4351.