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Formulation for slow release of oral radiation-protection drugs
Workin Pro~rt!ss 1nr. J. Nucl. Med. Eiol. Vol. 1I. No. 1, pp. 53-54, 1984 Pergamon Press Ltd 1984. Printed in Great Britain. 0047~0740/84 $3.00 t 0.00
Formulation for Slow Release of Oral Radiation-Protection Drugs
M. ABDULRAZIK, J. SHANI, S. BENITA, A. SAMUNI. M. DONBROW and A. YERUSHALMI Departments of Pharmacology, Pharmacy and Molecular Biology, The Hebrew University Faculty of Medicine, Jerusalem, and Radiation Unit, The Weizmann Institute of Science, Rehovot, Israel (Received 6 June 1983)
Cysteine and cysteamine, two effective radioprotectants in mammals, were compressed into tablets with a matrix composed of ethylcellulose and stearic acid at various proportions. The concentration of the unoxidized drug was monitored continuously for 8-16 h, as a parameter for the residual drug in the tablets with time, in vitro. Also, in oivo studies with tablets implanted into stomachs of rats, at various time intervals before their whole-body irradiation, were carried out. A correlation was obtained in the in vitro studies between the stearic acid/ethylcellulose ratio and the release rate of both radioprotectants, and the Higuchi’s diffusional mechanism was demonstrated. In vivo, the best survival ratios were obtained when 8 h lapsed between implantation of the tablets in the stomachs and the irradiation. It is concluded that there is a distinct correlation between the in vitro release of the drugs and their in viva efficacy as radioprotectants.
and mixed in a mortar with stearic acid (Merck, conforms to DAB 7) and ethylcellulose (Hercules N-100, with ethoxy content of 47.5-49.0x), in various proportions. The structural formulae of these four compounds are given in Fig. I. 20% cysteine or 20% cysteamine were prepared in defined ratios of the matrix mixture and compressed into cylindrical tablets of 13.1 mm in diameter and mean surface area of 4.0cm’, in a laboratory press at various pressure values between 2 and 8 tons.‘*’ Release of the drug was measured spectrophotometrically at 240 nm, using a rotating basket dissolution apparatus. The dissolution medium (phosphate buffer pH 7.4, containing 8 x lo-’ M K,HPO, and 2 x lo-?M KH,PO,) was kept under nitrogen. Exactly 0.5 L of the buffer, previously heated and maintained at 37°C was used for each experiment. The basket was immersed in the buffer, and rotated at 100 rpm. The concentration of the unoxidized drug was monitored continuously for 8-16 h, using a 10 mm flow-cell fed by a peristaltic pump at a flow rate of 25mL/min. Experimental results obtained spectrophotometrically were plotted against the square root of time, using a program to fit Higuchi’s equation!‘) The parabolic U.V.absorption curve, which demonstrates the accumulated released drug when plotted on a linear scale of time, is converted into a linear correlation when plotted against the square root of time, following the Higuchi law. For this calculation, the molar extinction coefficients for cysteine and cysteamine were determined under nitrogen. In vivo studies included implanting the sustained-release tablets in stomachs of albino Lewis rats under ether anaesthesia. At certain time intervals after their recovery from the anaesthesia, the rats were whole-body irradiated with 1000 rad, with a @Co Atomic Energy of Canada y-beam of 150 A. The daily survival rates of the rats were monitored for 14 days post irradiation. Survival rates of 50% of the rats (SR,, values) were obtained from the curves describing the survival rate at any given time, up to 14 days post irradiation. The composition of the matrix in the in viva experiment was steaxic acid:ethylcellulose = 3: I.
NH2
CYSTEINE
Introduction Many thousands of compounds have been synthesized during the past two decades aimed at obtaining a superior radioprotective agent. These compounds mainly act as scavengers of primary free radicals, and are intended to be used in cases of nuclear emergencies and atomic threats. A major survey in this field was published in 1979”’and lists over 4400 of the compounds synthesized and tested in Walter Reed Research Institute, for radiation-protection capacity, by oral feeding and by i.p. administration to laboratory animals. It occurred to us that due to the high toxicity, excretion and degradation of the radioprotective thiols and the high doses required for their effectivity, it would be desirable to formulate a sustained-release radioprotective tablet, enabling achievement of a serum level of the drug sufficient for protection against accidental irradiation, but well below the level causing systemic toxicity. Cysteine and cysteamine, two of the most effective radioprotectants explored in mammals, were used as model drugs in this study, and their in vitro release and in vivo efficacy evaluated in rats. The ultimate goal of this study is to better control the bioavailability of the drug in the serum, without markedly diminishing its protective index.
FREE
-
( B -mercaptoalonina
bH - COOH
HCl hydrote, L-cys)
bH,
RADICAL SCAVENGERS
I
CYSTEAMINE
STEARIC MATRIX
&H. HCI
or
ACID
( Octadecanoic m. p. 69 - 70pC
acid,
CH3 (kH2),6
I
)
COOH
INGREDIENS
and ETHYLCELLULOSE Cellulose _ethocel
ethyl
ether
- OC2 ; - OCz -I-OC2Hs 2 - OCn -I - OC2 %-OCeHs - OC2
H5 Hg Hg H5 Hs
Fig. 1. The two radioprotective agents tested in this study (cysteine and cysteamine) and the two components of the matrix mixture.
Materials and Methods Cysteine HCl and Cysteamine HCl (Sigma) were dried 53
54
Work in progress CQ-
$
75
Release -
rate
Correlation
=610% x mencoeff = 0999
‘k?
**
w \rl
.4*
I
.9-.*
ih
Schemotlc
Tablet
Fig. 2. A sustained-release of cysteine from a tablet composed of 20% cysteine in the matrix mixture, analyzed according to the Higuchi diffusional mechanism.
before
, drug
dissolutton
orocess
during
after
and
1s dissolved
Fig. 3. A graphic presentation of a sustained-release tablet before, during and after dissolution of the active ingredient.
RSUltS
Discussion
A very definite correlation was obtained in the in vitro study between the steak acid/ethylcellulose ratios and the release rate of the 20”/, cysteine, pressed at 4 tons, fr;om the tablets. The mean release rate constant (percent/mini x 102) at five increasing ratios of steak acid were 6.1, 5.1, 3.7, 2.9 and 2.4 for 0, 25, 50, 75 and 85% steak acid in the matrix. Tablets of 100% steak acid in the matrix liquified during preparation, and could not be manufactured. A Higuchi diffusional mechanism of 20% cysteine release from matrix is shown in Fig. 2. In general, the release rates of 20% cysteamine from similar matrix mixtures were much higher than those of cysteine: 10.4 x 10e2 and 6.1 x 10m2for 0 and 50% steak acid in the matrix. Tablet formation pressure had controversial influence on release rate. The in viuo studies were aimed at establishing the duration of the effect of the drug, i.e. the length of time that may lapse between implantation of the tablet and irradiation of the rat, giving optimal survival rates. The results are summarized in Table 1. In summary, increase in the ethylcellulose ratio increased the release rate of the two aminothiols linearily, while the concentrations of the two radioprotectants did not affect their own release rate from the matrix tablet. When a tablet containing 50% cysteine was implanted into the stomach of a rat 8 h before its whole-body irradiation, survival rate increased by 70°%as compared to non-protected rats.
In our in vitro studies, 75% stearic acid in the matrix composition seems to be the best formulation of all ratios investigated. It was shown that the dissolution of a soluble drug at high concentration from an insoluble matrix follows the Higuchi square root equation, except during an initial lag phase and a terminal diffusion phase.14’The main assumptions made in this study are that the twodimensional cross-sectional porosity has the same mean as the volumetric porosity, and that the dissolving substance is sufficiently dilute not to affect the porosity. A graphic demonstration of a tablet before, during and after the dissolution of the drug is given in Fig. 3.14’ Although both components of the matrix are hydrophobic, steak acid is much more hydrophobic than ethylcellulose, due to its waxy character, and this quality may be the reason for the decrease in porosity of its matrix, thus slowing down the release of the radioprotectant. The in uiuo results suggest that the formulation encompassing 50% cysteine not only protects effectively against ionizing irradiation, but also prolongs the release of the drug sufficiently to supply it at a protective level for at least 8 h. The release of cysteine in this study is so slow, that the levels of the drug after 2 and 6 h are still not high enough and those obtained after 16 h are already too low to protect the animals. These results suggests that there is a correlation between the in oitro profile of release and the in uiuo efficacy of cysteine, due to its prolonged release, via this new pharmaceutical formulation.
Table 1. Survival rates (SR,) of rats irradiated 1OOOrad at various time intervals after implantation of matrix of sustained-release tablet containing 50% cysteine
Time interval (h) Matrix only 2 6 8 16
SRm (days) 8.9 9.5 12.0 14.8 11.0
References Sweeney T. R. A Survey of Compoundsfrom
the Aniiradiation Drug Development Program of the US Army Medical R & D Command (Walter Reed Research
Institute, Washington, D.C.) (i979). Parrott E. L. and Sharma V. L. J. Pharm. Sci. 56, 1341 (1967). Higuchi T. J. Pharm. Sci. 52, 1145 (1963). Fessi H., Marty J. P. et a/. Int. J. Pharm. 1, 265 (1978).
Formulation for Slow Release of Oral Radiation-Protection Drugs
M. ABDULRAZIK, J. SHANI, S. BENITA, A. SAMUNI. M. DONBROW and A. YERUSHALMI Departments of Pharmacology, Pharmacy and Molecular Biology, The Hebrew University Faculty of Medicine, Jerusalem, and Radiation Unit, The Weizmann Institute of Science, Rehovot, Israel (Received 6 June 1983)
Cysteine and cysteamine, two effective radioprotectants in mammals, were compressed into tablets with a matrix composed of ethylcellulose and stearic acid at various proportions. The concentration of the unoxidized drug was monitored continuously for 8-16 h, as a parameter for the residual drug in the tablets with time, in vitro. Also, in oivo studies with tablets implanted into stomachs of rats, at various time intervals before their whole-body irradiation, were carried out. A correlation was obtained in the in vitro studies between the stearic acid/ethylcellulose ratio and the release rate of both radioprotectants, and the Higuchi’s diffusional mechanism was demonstrated. In vivo, the best survival ratios were obtained when 8 h lapsed between implantation of the tablets in the stomachs and the irradiation. It is concluded that there is a distinct correlation between the in vitro release of the drugs and their in viva efficacy as radioprotectants.
and mixed in a mortar with stearic acid (Merck, conforms to DAB 7) and ethylcellulose (Hercules N-100, with ethoxy content of 47.5-49.0x), in various proportions. The structural formulae of these four compounds are given in Fig. I. 20% cysteine or 20% cysteamine were prepared in defined ratios of the matrix mixture and compressed into cylindrical tablets of 13.1 mm in diameter and mean surface area of 4.0cm’, in a laboratory press at various pressure values between 2 and 8 tons.‘*’ Release of the drug was measured spectrophotometrically at 240 nm, using a rotating basket dissolution apparatus. The dissolution medium (phosphate buffer pH 7.4, containing 8 x lo-’ M K,HPO, and 2 x lo-?M KH,PO,) was kept under nitrogen. Exactly 0.5 L of the buffer, previously heated and maintained at 37°C was used for each experiment. The basket was immersed in the buffer, and rotated at 100 rpm. The concentration of the unoxidized drug was monitored continuously for 8-16 h, using a 10 mm flow-cell fed by a peristaltic pump at a flow rate of 25mL/min. Experimental results obtained spectrophotometrically were plotted against the square root of time, using a program to fit Higuchi’s equation!‘) The parabolic U.V.absorption curve, which demonstrates the accumulated released drug when plotted on a linear scale of time, is converted into a linear correlation when plotted against the square root of time, following the Higuchi law. For this calculation, the molar extinction coefficients for cysteine and cysteamine were determined under nitrogen. In vivo studies included implanting the sustained-release tablets in stomachs of albino Lewis rats under ether anaesthesia. At certain time intervals after their recovery from the anaesthesia, the rats were whole-body irradiated with 1000 rad, with a @Co Atomic Energy of Canada y-beam of 150 A. The daily survival rates of the rats were monitored for 14 days post irradiation. Survival rates of 50% of the rats (SR,, values) were obtained from the curves describing the survival rate at any given time, up to 14 days post irradiation. The composition of the matrix in the in viva experiment was steaxic acid:ethylcellulose = 3: I.
NH2
CYSTEINE
Introduction Many thousands of compounds have been synthesized during the past two decades aimed at obtaining a superior radioprotective agent. These compounds mainly act as scavengers of primary free radicals, and are intended to be used in cases of nuclear emergencies and atomic threats. A major survey in this field was published in 1979”’and lists over 4400 of the compounds synthesized and tested in Walter Reed Research Institute, for radiation-protection capacity, by oral feeding and by i.p. administration to laboratory animals. It occurred to us that due to the high toxicity, excretion and degradation of the radioprotective thiols and the high doses required for their effectivity, it would be desirable to formulate a sustained-release radioprotective tablet, enabling achievement of a serum level of the drug sufficient for protection against accidental irradiation, but well below the level causing systemic toxicity. Cysteine and cysteamine, two of the most effective radioprotectants explored in mammals, were used as model drugs in this study, and their in vitro release and in vivo efficacy evaluated in rats. The ultimate goal of this study is to better control the bioavailability of the drug in the serum, without markedly diminishing its protective index.
FREE
-
( B -mercaptoalonina
bH - COOH
HCl hydrote, L-cys)
bH,
RADICAL SCAVENGERS
I
CYSTEAMINE
STEARIC MATRIX
&H. HCI
or
ACID
( Octadecanoic m. p. 69 - 70pC
acid,
CH3 (kH2),6
I
)
COOH
INGREDIENS
and ETHYLCELLULOSE Cellulose _ethocel
ethyl
ether
- OC2 ; - OCz -I-OC2Hs 2 - OCn -I - OC2 %-OCeHs - OC2
H5 Hg Hg H5 Hs
Fig. 1. The two radioprotective agents tested in this study (cysteine and cysteamine) and the two components of the matrix mixture.
Materials and Methods Cysteine HCl and Cysteamine HCl (Sigma) were dried 53
54
Work in progress CQ-
$
75
Release -
rate
Correlation
=610% x mencoeff = 0999
‘k?
**
w \rl
.4*
I
.9-.*
ih
Schemotlc
Tablet
Fig. 2. A sustained-release of cysteine from a tablet composed of 20% cysteine in the matrix mixture, analyzed according to the Higuchi diffusional mechanism.
before
, drug
dissolutton
orocess
during
after
and
1s dissolved
Fig. 3. A graphic presentation of a sustained-release tablet before, during and after dissolution of the active ingredient.
RSUltS
Discussion
A very definite correlation was obtained in the in vitro study between the steak acid/ethylcellulose ratios and the release rate of the 20”/, cysteine, pressed at 4 tons, fr;om the tablets. The mean release rate constant (percent/mini x 102) at five increasing ratios of steak acid were 6.1, 5.1, 3.7, 2.9 and 2.4 for 0, 25, 50, 75 and 85% steak acid in the matrix. Tablets of 100% steak acid in the matrix liquified during preparation, and could not be manufactured. A Higuchi diffusional mechanism of 20% cysteine release from matrix is shown in Fig. 2. In general, the release rates of 20% cysteamine from similar matrix mixtures were much higher than those of cysteine: 10.4 x 10e2 and 6.1 x 10m2for 0 and 50% steak acid in the matrix. Tablet formation pressure had controversial influence on release rate. The in viuo studies were aimed at establishing the duration of the effect of the drug, i.e. the length of time that may lapse between implantation of the tablet and irradiation of the rat, giving optimal survival rates. The results are summarized in Table 1. In summary, increase in the ethylcellulose ratio increased the release rate of the two aminothiols linearily, while the concentrations of the two radioprotectants did not affect their own release rate from the matrix tablet. When a tablet containing 50% cysteine was implanted into the stomach of a rat 8 h before its whole-body irradiation, survival rate increased by 70°%as compared to non-protected rats.
In our in vitro studies, 75% stearic acid in the matrix composition seems to be the best formulation of all ratios investigated. It was shown that the dissolution of a soluble drug at high concentration from an insoluble matrix follows the Higuchi square root equation, except during an initial lag phase and a terminal diffusion phase.14’The main assumptions made in this study are that the twodimensional cross-sectional porosity has the same mean as the volumetric porosity, and that the dissolving substance is sufficiently dilute not to affect the porosity. A graphic demonstration of a tablet before, during and after the dissolution of the drug is given in Fig. 3.14’ Although both components of the matrix are hydrophobic, steak acid is much more hydrophobic than ethylcellulose, due to its waxy character, and this quality may be the reason for the decrease in porosity of its matrix, thus slowing down the release of the radioprotectant. The in uiuo results suggest that the formulation encompassing 50% cysteine not only protects effectively against ionizing irradiation, but also prolongs the release of the drug sufficiently to supply it at a protective level for at least 8 h. The release of cysteine in this study is so slow, that the levels of the drug after 2 and 6 h are still not high enough and those obtained after 16 h are already too low to protect the animals. These results suggests that there is a correlation between the in oitro profile of release and the in uiuo efficacy of cysteine, due to its prolonged release, via this new pharmaceutical formulation.
Table 1. Survival rates (SR,) of rats irradiated 1OOOrad at various time intervals after implantation of matrix of sustained-release tablet containing 50% cysteine
Time interval (h) Matrix only 2 6 8 16
SRm (days) 8.9 9.5 12.0 14.8 11.0
References Sweeney T. R. A Survey of Compoundsfrom
the Aniiradiation Drug Development Program of the US Army Medical R & D Command (Walter Reed Research
Institute, Washington, D.C.) (i979). Parrott E. L. and Sharma V. L. J. Pharm. Sci. 56, 1341 (1967). Higuchi T. J. Pharm. Sci. 52, 1145 (1963). Fessi H., Marty J. P. et a/. Int. J. Pharm. 1, 265 (1978).