LETTERS TO THE EDITORS
was prepared by the Department of Pharmacy, St
Bartholomew's Hospital.
337
m-tyramine and metasaminol. Life Sci, 5, 501-507. GHOSE, K., TURNER, P. & COPPEN, A. (1975).
KARABI GHOSE, L.A. GIFFORD & P. TURNER Department of Clinical Pharmacology, St Bartholomew's Hospital, London EC1A 7BE
Intravenous tyramine pressor response in depression. Lancet, i, 1317-1318. GIFFORD, L.A., TURNER, P. & PARE, C.M.B. (1975).
Sensitive method for the routine determination of tricyclic antidepressants in plasma using a specific nitrogen detector J. Chromatography, 105, 107-113. MITCHELL, J.R., CAVANAUGH, J.H., ARIAS, L. &
MONICA LEIGHTON Computing Unit for Medical Sciences, St Bartholomew's Hospital, London ECIA 7BE Received October 16, 1975 References ALEXANDERSON, B. & SJOQVIST, F. (1971). Individual differences in the pharmacokinetics of monomethylated tricyclic antidepressants: role of genetic and environmental factors and clinical importance. Ann. N.Y. Acad. Sci., 179, 1739-1751. FREYSCHUSS, A., SJOQVIST, F. & TUCK, D. (1970). Tyramine pressor effects in man before and during treatment with nortriptyline or E.C.T: Correlation between plasma level and effect of nortriptyline. Pharmacologia Clinica, 2, 72-78. GESSA, G.L., VARGILL, L. & CRABAI, F. (1966). Interaction of desmethylimipramine with the adrenergic and NE releasing actions of tyramine, a-methyl-
OATES, J.A. (1970). Guanethidine and related agents: Antagonism by drugs which inhibit the norepinephrine pump in man. Clin. Invest., 49, 1596-1904. NACHEV, C., COLLIER, J. & ROBINSON, B. (1971). Simplified method for measuring compliance of superficial veins. Cardiovascular Res., 5, 147-159. SCHILDKRAUT, J.J. (1970). Biochemistry, schizophrenias and affective illnesses, ed. Himwick, H.E. Baltimore: Williams & Wilcins. SILVERSTONE, T. & TURNER, P. (1974). Drug treatment in psychiatry. London and Boston: Routledge & Kegan Paul Ltd. SIWERS, B., TUCK, D., FREYSCHUSS, U., AZAR-
NOFF, D.L. & SJOQVIST, F. (1969). A novel approach to the phase I clinical trial of tricyclic antidepressant drugs. Abstract for Fourth International Congress on Pharmacology, Basle, Switzerland. WHITE, C. de B. & UDWADIA, B.P. (1975). cs-adrenoceptors in the human dorsal vein and the effects of propranolol and practolol on venous sensitivity to noradrenaline. Br. J. clin. Pharmac., 2, 99-105.
SENSITIVE METHOD FOR THE DETERMINATION OF TANDAMINE HYDROCHLORIDE IN PLASMA USING A SPECIFIC NITROGEN DETECTOR Recently a new class of compounds, having a substituted thiopyrano-indole ring structure has been synthesized of which tandamine hydrochloride (Figure 1) is an example that is now being investigated. Animal studies (Ayerst Research Laboratories) suggest that tandamine hydrochloride might have potential use as an antidepressant drug and studies of in vivo pharmacokinetics required a sensitive and specific method of assay. The sensitivity of the alkali flame ionization detector to the tricyclic antidepressant drugs has recently been shown to provide a suitable method for the determination of these drugs in routine plasma samples (Gifford, Turner & Pare, 1975). The method presented here, is used to estimate plasma levels of this new tricyclic antidepressant after a single oral dose. Analyses were performed using an isothermal chromatograph with heated nitrogen detector (Perkin-Elmer, Beaconsfield, Great Britain) and
S N
CIH2
CH3 CH2CH2N..CH3 - HCI
CH3 Figure 1
Structural formula of tandamine.
linear recorder. The back flush system previously reported in the study on tricyclic antidepressants was used in conjunction with a 9ft x J in o.d. glass column (Gifford et al., 1975). The column was silanized with a 5% solution of dimethyldichlorosilane in chloroform prior to packing with 3% OV-1 7 on Chromasorb W, 100-120 mesh. The packed column was conditioned at 300 C for 24 h with nitrogen
338
LETTERS TO THE EDITORS
carrier gas flowing and silylated with N, o-bis(trimethylsilyl)-triflu,oroacetamide prior to use. The operating conditions were fixed with the back flush valve set so as to allow passage of the total sample through the detector. The carrier gas flow-rate through the column was adjusted to 30 ml/min, hydrogen flow-rate to 3 ml/min and air flow rate to 60 ml/min. The column oven temperature was maintained at 270 C and the injector temperature at 2800 C. The temperature of the rubidium silicate detector bead was adjusted to provide maximum response from an injection of 2 Ml of a 25 ,ug/ml solution of tandamine hydrochloride in methanol. Samples were injected with the back flush valve in the purge mode. After the solvent front and interfering compounds had eluted from the column the position of the valve was changed to the conventional mode of operation and the drugs recorded as normal. To plasma (4 ml) containing tandamine hydrochloride was added 1 ml of aqueous internal standard (desmethylimipramine, 250 ng/ml). This addition of a known concentration of standard to the plasma before the extraction procedure is used to make possible the quantitative determination of the drug after recovery. The plasma was then diluted with distilled water (4 ml) and made alkaline by the addition of 1 OM NaOH (1 ml). The drugs were extracted into n-heptane (10 ml) by gently shaking on a horizontal shaker for 20 min. The organic phase was then separated by centrifuging for 10 min and 7 ml of the upper organic phase aspirated. To the organic phase iso-amyl alcohol (100 ,l) and glacial acetic acid (100 ,l) were added prior to evaporation in order to reduce volatilization of the tandamine during the concentration procedure. The organic extract was evaporated to a final volume of 50 Ml in a constant temperature water bath at 650 C with the aid of a gentle stream of nitrogen. Aliquots (2 l) of the final extract were injected into the chromatograph for separation and quantitation. The expression of the heights of the twin peaks produced on the chromatogram as a ratio circumvents the otherwise impractical situation
c
s)100
a) 80
E 60
co 40 c CZ
cu 20
E co
I
.
.
12345678
18
12
24
Time (h) Figure 2 Plasma concentration v time in 62 kg male volunteer after 50 mg oral dose of tandamine hydrochloride.
of maintaining an identical extraction procedure for each sample. The reproducibility of the procedure was tested at three concentrations of tandamine hydrochloride in plasma over the expected therapeutic range. Standard curves were prepared each day using blood bank plasma containing tandamine hydrochloride (0-300 ng/ml). The test solutions and calibration standards were extracted simultaneously thus eliminating variation due to extraction conditions. The ratio of tandamine peak height to desmethylimipramine peak height was calculated and compared with the values obtained from the standard solutions. As expected no peak was obtained in the absence of any drug. The results from six determinations of each of the three concentrations studied are shown in Table 1. Coefficients of variation for concentrations in the range 50-150 ng/ml were found to be 5-8% and recoveries based on the results were in the range 96-98%. The plasma concentration v time curve obtained in a male volunteer after a single 50 mg oral dose is shown in Figure 2. From the curve it can be seen that peak plasma levels were achieved within 2 h and thereafter the plasma level decreased rapidly up to 8 h, with an apparent half life of 3 hours. However, there was a significant but low plasma level at 24 h which
Table 1 Reproducibility of tandamine estimation from plasma. Each of the results is the mean of six determinations of each tandamine concentration. Tandamine HCI concentration
Amount found
(ng/ml)
(ng/mlJ
Recovery (%)
%sad.
50 100 150
49.0 95.7 144.5
98 96 96
5.6 7.7 5.5
LETTERS TO THE EDITORS
would indicate the presence of a second and longer elimination half life. Any accurate assessment of this longer half life could not be made due to the lack of information between the 8 and 24 h samples. It is clear from this study that the proposed method is capable of simply estimating plasma levels over an extended period after a single oral dose of this drug.
339
D.J. WITTS Department of Clinical Pharmacology, St Bartholomews. Hospital, London ECIA 7BE Received October 29, 1975
The authors wish to thank Professor P. Turner for his encouragement and the use of facilities to carry out the work and to Ayerst Laboratories for samples of tandamine hydrochloride.
References
L.A. GIFFORD
GIFFORD, L.A., TURNER, P. & PARE, C.M.B. (1975).
Department of Chemistry, Loughborough University of Technology, Loughborough, Leicester LEII 3TU
AYERST RESEARCH LABORATORIES. Reports, Animal Health Division.
Internal
Sensitive method for the routine determination of tricyclic antidepressants in plasma using a specific nitrogen detectorJ. Chromatogr., 105, 107-113.
RECTAL ADMINISTRATION OF FEPRAZONE IN HEALTHY VOLUNTEERS Drugs given rectally in the form of suppositories are used for local therapy, for systemic effects or for situations in which the oral route is unsatisfactory. Dissolution of drug from a suppository may be the rate-limiting step for the overall absorption process and therefore particle size and suppositories formulations become significant factors affecting the absorption rate. The purpose of the study was to compare the bioavailability of the feprazone (4 - prenyl - 1,2 - diphenyl - 3,5 pyrazolidinedione), effective drug in rheumatoid arthritis (Cherie Ligniere, Colombo, Carabba, Ferrari & Robotti, 1972; Billings, Burry, Grahame & Berry, 1975; Fletcher, Loebl & Scott, 1975; Sturrock, Isaacs & Hart, 1975), after rectal administration of three lots of suppositories with different formulations of the base and different particle size. Six healthy volunteers (three males and three females ranging in age from 21-25 years and in body weight from 50-82 kg) entered the trial after their informed consent. The volunteers had not consumed any drug substances for at least one week prior to suppositories administration and during the study itself. All subjects were fasted ovemight and 4 h post dosing. No enema was given before drug administration. The subjects received suppositories containing feprazone (300 mg). The code of treatment, the suppositories base and particle size are respectively: A = Witepsol
(1390 mg), soya lecithin (5.7 mg) and particle size 18.4 ,u; B = Suppocire BML (1400 mg) and particle size 18.4,u; C= Suppocire BML (1520 mg) and particle size 8.8 ,u. The study has been planned as balanced incomplete block design (Li, 1964). Each subject received two of the three treatments on two different occasions with a wash-out period of 7 days between administrations. Blood samples (10 ml) were drawn from each subject at 3, 6, 9, 24, 27, 32 h after insertion of the suppository. The samples were centrifuged as soon as possible after collection and the serum samples were assayed using a specific gas-chromatographic method (Perego, Gallazzi, Vanoni & Lucarella, 1972). The mean serum levels of feprazone for each of three formulations are in Figure 1. The first step of data analysis is carried out on the area under serum levels, on the peak height of the serum levels and on the time to reach the peak, which are generally considered as parameters describing bioavailability (Ritschel, 1972). The differences in the bioavailability parameters among the three formulations are not statistically significant. On the other hand the figure shows the possibility that the formulation A might have a serum levels pattem different from those of the formulations B and C. The second step of data analysis is to compare the shape of the three serum level patterns. The outcome of the analysis of variance for testing these differences (Westlake,
was prepared by the Department of Pharmacy, St
Bartholomew's Hospital.
337
m-tyramine and metasaminol. Life Sci, 5, 501-507. GHOSE, K., TURNER, P. & COPPEN, A. (1975).
KARABI GHOSE, L.A. GIFFORD & P. TURNER Department of Clinical Pharmacology, St Bartholomew's Hospital, London EC1A 7BE
Intravenous tyramine pressor response in depression. Lancet, i, 1317-1318. GIFFORD, L.A., TURNER, P. & PARE, C.M.B. (1975).
Sensitive method for the routine determination of tricyclic antidepressants in plasma using a specific nitrogen detector J. Chromatography, 105, 107-113. MITCHELL, J.R., CAVANAUGH, J.H., ARIAS, L. &
MONICA LEIGHTON Computing Unit for Medical Sciences, St Bartholomew's Hospital, London ECIA 7BE Received October 16, 1975 References ALEXANDERSON, B. & SJOQVIST, F. (1971). Individual differences in the pharmacokinetics of monomethylated tricyclic antidepressants: role of genetic and environmental factors and clinical importance. Ann. N.Y. Acad. Sci., 179, 1739-1751. FREYSCHUSS, A., SJOQVIST, F. & TUCK, D. (1970). Tyramine pressor effects in man before and during treatment with nortriptyline or E.C.T: Correlation between plasma level and effect of nortriptyline. Pharmacologia Clinica, 2, 72-78. GESSA, G.L., VARGILL, L. & CRABAI, F. (1966). Interaction of desmethylimipramine with the adrenergic and NE releasing actions of tyramine, a-methyl-
OATES, J.A. (1970). Guanethidine and related agents: Antagonism by drugs which inhibit the norepinephrine pump in man. Clin. Invest., 49, 1596-1904. NACHEV, C., COLLIER, J. & ROBINSON, B. (1971). Simplified method for measuring compliance of superficial veins. Cardiovascular Res., 5, 147-159. SCHILDKRAUT, J.J. (1970). Biochemistry, schizophrenias and affective illnesses, ed. Himwick, H.E. Baltimore: Williams & Wilcins. SILVERSTONE, T. & TURNER, P. (1974). Drug treatment in psychiatry. London and Boston: Routledge & Kegan Paul Ltd. SIWERS, B., TUCK, D., FREYSCHUSS, U., AZAR-
NOFF, D.L. & SJOQVIST, F. (1969). A novel approach to the phase I clinical trial of tricyclic antidepressant drugs. Abstract for Fourth International Congress on Pharmacology, Basle, Switzerland. WHITE, C. de B. & UDWADIA, B.P. (1975). cs-adrenoceptors in the human dorsal vein and the effects of propranolol and practolol on venous sensitivity to noradrenaline. Br. J. clin. Pharmac., 2, 99-105.
SENSITIVE METHOD FOR THE DETERMINATION OF TANDAMINE HYDROCHLORIDE IN PLASMA USING A SPECIFIC NITROGEN DETECTOR Recently a new class of compounds, having a substituted thiopyrano-indole ring structure has been synthesized of which tandamine hydrochloride (Figure 1) is an example that is now being investigated. Animal studies (Ayerst Research Laboratories) suggest that tandamine hydrochloride might have potential use as an antidepressant drug and studies of in vivo pharmacokinetics required a sensitive and specific method of assay. The sensitivity of the alkali flame ionization detector to the tricyclic antidepressant drugs has recently been shown to provide a suitable method for the determination of these drugs in routine plasma samples (Gifford, Turner & Pare, 1975). The method presented here, is used to estimate plasma levels of this new tricyclic antidepressant after a single oral dose. Analyses were performed using an isothermal chromatograph with heated nitrogen detector (Perkin-Elmer, Beaconsfield, Great Britain) and
S N
CIH2
CH3 CH2CH2N..CH3 - HCI
CH3 Figure 1
Structural formula of tandamine.
linear recorder. The back flush system previously reported in the study on tricyclic antidepressants was used in conjunction with a 9ft x J in o.d. glass column (Gifford et al., 1975). The column was silanized with a 5% solution of dimethyldichlorosilane in chloroform prior to packing with 3% OV-1 7 on Chromasorb W, 100-120 mesh. The packed column was conditioned at 300 C for 24 h with nitrogen
338
LETTERS TO THE EDITORS
carrier gas flowing and silylated with N, o-bis(trimethylsilyl)-triflu,oroacetamide prior to use. The operating conditions were fixed with the back flush valve set so as to allow passage of the total sample through the detector. The carrier gas flow-rate through the column was adjusted to 30 ml/min, hydrogen flow-rate to 3 ml/min and air flow rate to 60 ml/min. The column oven temperature was maintained at 270 C and the injector temperature at 2800 C. The temperature of the rubidium silicate detector bead was adjusted to provide maximum response from an injection of 2 Ml of a 25 ,ug/ml solution of tandamine hydrochloride in methanol. Samples were injected with the back flush valve in the purge mode. After the solvent front and interfering compounds had eluted from the column the position of the valve was changed to the conventional mode of operation and the drugs recorded as normal. To plasma (4 ml) containing tandamine hydrochloride was added 1 ml of aqueous internal standard (desmethylimipramine, 250 ng/ml). This addition of a known concentration of standard to the plasma before the extraction procedure is used to make possible the quantitative determination of the drug after recovery. The plasma was then diluted with distilled water (4 ml) and made alkaline by the addition of 1 OM NaOH (1 ml). The drugs were extracted into n-heptane (10 ml) by gently shaking on a horizontal shaker for 20 min. The organic phase was then separated by centrifuging for 10 min and 7 ml of the upper organic phase aspirated. To the organic phase iso-amyl alcohol (100 ,l) and glacial acetic acid (100 ,l) were added prior to evaporation in order to reduce volatilization of the tandamine during the concentration procedure. The organic extract was evaporated to a final volume of 50 Ml in a constant temperature water bath at 650 C with the aid of a gentle stream of nitrogen. Aliquots (2 l) of the final extract were injected into the chromatograph for separation and quantitation. The expression of the heights of the twin peaks produced on the chromatogram as a ratio circumvents the otherwise impractical situation
c
s)100
a) 80
E 60
co 40 c CZ
cu 20
E co
I
.
.
12345678
18
12
24
Time (h) Figure 2 Plasma concentration v time in 62 kg male volunteer after 50 mg oral dose of tandamine hydrochloride.
of maintaining an identical extraction procedure for each sample. The reproducibility of the procedure was tested at three concentrations of tandamine hydrochloride in plasma over the expected therapeutic range. Standard curves were prepared each day using blood bank plasma containing tandamine hydrochloride (0-300 ng/ml). The test solutions and calibration standards were extracted simultaneously thus eliminating variation due to extraction conditions. The ratio of tandamine peak height to desmethylimipramine peak height was calculated and compared with the values obtained from the standard solutions. As expected no peak was obtained in the absence of any drug. The results from six determinations of each of the three concentrations studied are shown in Table 1. Coefficients of variation for concentrations in the range 50-150 ng/ml were found to be 5-8% and recoveries based on the results were in the range 96-98%. The plasma concentration v time curve obtained in a male volunteer after a single 50 mg oral dose is shown in Figure 2. From the curve it can be seen that peak plasma levels were achieved within 2 h and thereafter the plasma level decreased rapidly up to 8 h, with an apparent half life of 3 hours. However, there was a significant but low plasma level at 24 h which
Table 1 Reproducibility of tandamine estimation from plasma. Each of the results is the mean of six determinations of each tandamine concentration. Tandamine HCI concentration
Amount found
(ng/ml)
(ng/mlJ
Recovery (%)
%sad.
50 100 150
49.0 95.7 144.5
98 96 96
5.6 7.7 5.5
LETTERS TO THE EDITORS
would indicate the presence of a second and longer elimination half life. Any accurate assessment of this longer half life could not be made due to the lack of information between the 8 and 24 h samples. It is clear from this study that the proposed method is capable of simply estimating plasma levels over an extended period after a single oral dose of this drug.
339
D.J. WITTS Department of Clinical Pharmacology, St Bartholomews. Hospital, London ECIA 7BE Received October 29, 1975
The authors wish to thank Professor P. Turner for his encouragement and the use of facilities to carry out the work and to Ayerst Laboratories for samples of tandamine hydrochloride.
References
L.A. GIFFORD
GIFFORD, L.A., TURNER, P. & PARE, C.M.B. (1975).
Department of Chemistry, Loughborough University of Technology, Loughborough, Leicester LEII 3TU
AYERST RESEARCH LABORATORIES. Reports, Animal Health Division.
Internal
Sensitive method for the routine determination of tricyclic antidepressants in plasma using a specific nitrogen detectorJ. Chromatogr., 105, 107-113.
RECTAL ADMINISTRATION OF FEPRAZONE IN HEALTHY VOLUNTEERS Drugs given rectally in the form of suppositories are used for local therapy, for systemic effects or for situations in which the oral route is unsatisfactory. Dissolution of drug from a suppository may be the rate-limiting step for the overall absorption process and therefore particle size and suppositories formulations become significant factors affecting the absorption rate. The purpose of the study was to compare the bioavailability of the feprazone (4 - prenyl - 1,2 - diphenyl - 3,5 pyrazolidinedione), effective drug in rheumatoid arthritis (Cherie Ligniere, Colombo, Carabba, Ferrari & Robotti, 1972; Billings, Burry, Grahame & Berry, 1975; Fletcher, Loebl & Scott, 1975; Sturrock, Isaacs & Hart, 1975), after rectal administration of three lots of suppositories with different formulations of the base and different particle size. Six healthy volunteers (three males and three females ranging in age from 21-25 years and in body weight from 50-82 kg) entered the trial after their informed consent. The volunteers had not consumed any drug substances for at least one week prior to suppositories administration and during the study itself. All subjects were fasted ovemight and 4 h post dosing. No enema was given before drug administration. The subjects received suppositories containing feprazone (300 mg). The code of treatment, the suppositories base and particle size are respectively: A = Witepsol
(1390 mg), soya lecithin (5.7 mg) and particle size 18.4 ,u; B = Suppocire BML (1400 mg) and particle size 18.4,u; C= Suppocire BML (1520 mg) and particle size 8.8 ,u. The study has been planned as balanced incomplete block design (Li, 1964). Each subject received two of the three treatments on two different occasions with a wash-out period of 7 days between administrations. Blood samples (10 ml) were drawn from each subject at 3, 6, 9, 24, 27, 32 h after insertion of the suppository. The samples were centrifuged as soon as possible after collection and the serum samples were assayed using a specific gas-chromatographic method (Perego, Gallazzi, Vanoni & Lucarella, 1972). The mean serum levels of feprazone for each of three formulations are in Figure 1. The first step of data analysis is carried out on the area under serum levels, on the peak height of the serum levels and on the time to reach the peak, which are generally considered as parameters describing bioavailability (Ritschel, 1972). The differences in the bioavailability parameters among the three formulations are not statistically significant. On the other hand the figure shows the possibility that the formulation A might have a serum levels pattem different from those of the formulations B and C. The second step of data analysis is to compare the shape of the three serum level patterns. The outcome of the analysis of variance for testing these differences (Westlake,
