XENOBIOTICA,
1991, VOL. 21,
NO.
7, 827-837
Pharmacokinetics and fate of 3H-trospectomycin sulphate, a novel aminocyclitol antibiotic, in male and female rats D. J. NICHOLS?, M. BURROWS, A. BYE, D. A. CONSTABLE, L. G. DRING and P. JEFFREY
Xenobiotica Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
Drug Investigation and Clinical Research, Upjohn Ltd, Fleming Way, Crawley, West Sussex, U K
Received 5 December 1989, accepted 24 January 1991
I . The pharmacokinetics and fate of 3H-trospectomycin sulphate, a novel aminocyclitol antibiotic, were examined in male and female rats after intramuscular (i.m.), intravenous (i.v.) and subcutaneous (s.c.) dosing. 2. Total radioactivity levels in plasma were associated with unchanged trospectomycin. Two radioactive components were found in urine, one was indistinguishable from trospectomycin and the other was probably a degradation product formed after excretion or during storage rather than a metabolite. 3. T h e disappearance of drug from plasma followed a biphasic pattern with half lives of 0,344h and 45-80 h and a large distribution volume, which indicated some retention of drug by tissues. Clearance rates were within the normal range for glomerular filtration rate, which indicated that the primary process of elimination is filtration of unchanged drug. 4. Excretion was initially rapid (> 40% by 4 h) and mainly into urine (faecal excretion 95% as determined by t.1.c. and h.p.1.c. All references to the mass or concentration of trospectomycin refer to the hydrated drug sulphate, not the free base. To convert to free base all doses and concentrations are multiplied by 0.625.
t T o whom correspondence should be addressed. 0049-8254/91 $340
0 1991 Taylor & Francis Ltd.
D . J . Nichols et al.
828
CH3
-
5 H,O
Xenobiotica Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
Figure 1. Chemical structure of trospectomycin sulphate.
Animals Male and female Sprague-Dawley rats (Charles River UK Ltd, Manston Rd, Margate, Kent, UK) of body weight 200-300g, were held for at least 5 days before use. Room temperature was maintained between 19°C and 20"C, relative humidity was 40-65% and 12 h-12 h light-dark photoperiod was used. All animals had free access to food (SDS diet No. 1, Wm. Lillico and Sons, Betchworth, UK) and fresh water was available ad libitum. Experimental design Excretion. T o examine the effects of route of administration on the disposition of trospectomycin, male and female rats (four per treatment) were housed individually in metabolism cages suitable for the collection of urine and faeces, and given trospectomycin sulphate (5 mglkg, 4 4 3 MBqIkg body weight) in 7&13Opl isotonic saline s.c., i.m. or i.v. Urine was collected for the periods@2,24,4-6,6-24 h and daily thereafter until 168 h post-dose. Faeces were collected every 24 h. All samples were stored at - 20°C until analysis. Pharmacokinetics. T o examine the pharmacokinetics by different routes of administration, male and female rats (four per treatment) were housed in cages with raised grid floors and given trospectomycin sulphate (5 mg/kg body weight, 15.70 MBq/kg) in 7&130pl isotonic saline s.c., i.m. or i.v. Blood samples ( 2 5 0 ~ 1were ) taken into heparinized microcentrifuge tubes from the tail vein of each rat at the following times post-dose: 15,30 and 45 min and 1 , l . S . 2 and 3 h. A 400 pl sample was taken at 4,6,8,24,48 and 72 h post-dose. At 96 h each rat was killed and a large terminal blood sample was taken from the inferior vena cava. Whole blood was aliquoted in duplicate (lop1up to 3 h, 25 p1 thereafter) to measure 'H content. The remaining blood was centrifuged and the plasma stored frozen at - 20°C until analysis. Dose proportionalily. T o examine the linearity of the kinetics with dose, male and female rats (four per dose), housed in cages with raised grid floors, were given trospectomycin sulphate ( S O , 100 or 200mgikg body weight, 14.4, 13.4 and 16.9MRq respectively) in 37G700pI sterile water S.C.(over the shoulder region). Blood samples ( 2 5 0 ~ 1were ) collected into heparinized microcentrifuge tubes from the tail vein of each rat at the following times: 15,30and 45 min and 1, 1 . 5 , 2 , 3 , 4 , 6 , 8 , 2 4 , 4 8 , 7 2 , 9 6and 120 h. T h e blood was centrifuged and the plasma was stored at - 20°C until analysis. Radiochemical techniques Urine and Plasma. Urine and plasma samples (l&lOOOpI) were mixed with water to give a total volume of 300-15OOp1 and agitated with 3 ml Optiphase X scintillation fluid (Fisons plc, Loughborough, Leics., UK) in plastic minivials. Radioactive counts were measured with an LKB liquid scintillation counter using the external standard ratio method for quench correction. Plasma concentration of trospectomycin sulphate was calculated from the counts in the radioactive areas of the t.1.c. plate (see below) for the study to compare different routes of administration. Because there was little difference between the counts after t.1.c. and the total counts, total radioactivity was used to calculate the plasma concentrations in the dose proportionality study. A preliminary experiment was conducted to ascertain whether tritium exchange/ metabolic instability would be a problem (Constable et al. 1990). No tritiated water was found in the expired air and there was no significant loss of radioactivity from urine subjected to freeze drying. Faeces. The faeces were homogenized, combusted in a Packard 306 Sample Oxidizer and mixed with scintillant (Monophase-40 Plus, Packard Instruments, Caversham, Reading, Berks., U K ; 15 ml for 300mg faecal homogenate). The combusted samples were counted in 20ml glass vials using a Packard 3385 scintillation counter with the external standard ratio method for quench correction. Unchanged trospectomycin and metabolitesldegradation products Urine. Urine (10-20p1) was applied directly to t.1.c. plates and run in solvent system A or B (see below). After drying, the plates were sprayed with EN3HANCE (NEN, Dreieich, Germany), dried for 1 h and
Pharmacokinetics of trospectomycin in rat
829
placed in apposition with an X-Omat AR film (Kodak Ltd, Hemel Hempstead, Herts., UK), for about 2 weeks. The film was developed, the radioactive areas located, the silica gel corresponding to each radioactive area was scraped off into minivials containing 1.5 ml water and mixed with Optiphase X to form a stable gel which was counted as described above.
Xenobiotica Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
Plasma. Five microlitres of H,SO, and 0.5ml methanol were vortex-mixed with 1 5 0 ~ plasma, 1 then centrifuged for 10 min. T h e supernatant was removed and the pellet was resuspended in methanol-water (1 : 4 v/v, containing 2 0 ~ 1of S M H,SO,/lOml), vortex-mixed and the precipitate removed. The supernatant was combined with the first, evaporated to dryness at 30°C by vacuum vortex and resuspended in the acidified aqueous methanol. T h e fine precipitate was removed by centrifugation and lop1 of the supernatant was subjected to t.1.c. in solvent system A. The radioactive areas were located on the plate by removing bands of silica gel 2 cm wide by 5 mm deep, transferring to minivials, sonicating for 5 min with 0 5 ml of 5 M H,SO,, mixed with 1 ml water and Optiphase X to form a stable gel, and counted. Chromatography Thin-layer chromatography. T.1.c. was performed on glass plates pre-coated with Keiselgel 60 FZ5,, thickness 0.25 mm (E. Merck, Darmstadt, Germany) using either solvent system A: butal-1-01-acetic acid-water (5 : 1 : 2 by vol.) or B: methanol-chloroform-ammonium hydroxide (sp. gr. 0.88) (4 : 3 : 2 by vol.). The solvent front was allowed to run for 16 cm from the origin. Trospectomycin could be visualized as an orange spot by spraying with 2.4-dinitrophenylhydrazine (05% w/v in 2 M HCI). High-performance liquid chromatography. Either plasma or urine samples were injected into an h.p.1.c. system which consisted of an M730 Data Module (Waters Associates Ltd, Northwich, Cheshire, UK), a Waters Wisp 710B autoinjection unit, a 720 system controller, a Waters 6000A solvent delivery system, a Waters 441 absorbance detector, a Shimadzu column oven CTO-2A (Shimadzu Corp., Kyoto, Japan) in association with a programmable Superrac 221 1 fraction collector (LKB Instruments Ltd). T h e column was an APS-Hypersil (3 pm particle size; Shandon Southern Products Ltd, Runcorn, Cheshire, U K ) 200 mm by 4.6 mm analytical column. T h e mobile phase used was acetonitrile (700 ml) and 0.1 M KH,PO, (350ml) adjusted to p H 2 with phosphoric acid. This was degassed with helium and used at a flow rate of 1 ml/min. The column oven was maintained at a temperature of 50°C. T h e radioactivity eluted between 7 and 13 min. Ten-second samples were collected over this time period and the radioactive elution profile was determined by scintillation counting. T h e only occasion that the h.p.1.c. method was used quantitatively was in the determination of radiochemical purity. In this case the trospectomycin peak was collected, counted and compared with the counts actually put on the column. Enzyme hydrolyszs. Urine (1 ml) was mixed with an equal volume of acetate buffer ( 0 . 2 ~ pH , 5.0) containing 8-glucuronidase type H-2 (ex. Helix pomatia, containing some arylsulphatase activity; Sigma Chemical Co. Ltd, Poole, Dorset, UK). The mixture was incubated overnight at 3 7 T , with suitable controls; namely, either the mixture lacking enzyme or with phenolphthalein glucuronic acid (50 pl; 0-03M). Before t.1.c. of the hydrolysed urine the excess protein in the incubation mixture was removed by adding an equal volume of methanol and vortex-mixing. After 1 h at 4°C the mixture was centrifuged and the supernatant stored at - 20°C overnight. The sample was centrifuged, and supernatant removed, dried under nitrogen, reconstituted in 1 ml methanol and subjected to t.1.c. Pharmacokinetic analysis. The concentration of trospectomycin sulphate were fitted by a twocompartment open model with first-order rate constants with NONLIN84 (Metzler and Weiner 1984). Pharmacokinetic parameters were calculated in NONLIN84; k01, k12, K21 and k10 are the rates of absorption, intercompartment transport and elimination, V1 is the volume of the first compartment, Vss is the total volume of distribution, A UC is the area under the timeplasma concentration curve, CL is the plasma clearance rate (CL= Dose x f / A U C ) where f is the fraction of drug absorbed? CLr is the renal clearance rate ( C L r = A e , , , f / A U C , , , where Ae,6Mis the total amount of drug lost in urine to 168 h and AUC,,, is the area under the plasma concentration-time curve from 0 to 168h), MRT is the mean residence time ( M R T = Vss/CL)and t l i Z aand t l l Z Bare the initial and terminal half-lives respectively. The i.v. input was as a bolus but the S.C. and i.m. data were best described by first-order input. Statistical differences in pharmacokinetic parameters between doses, sexes and routes of administration were analysed by ANOVA using SAS (SAS Institute Inc, Box 8000, Cary, NC, USA).
Results Metabolism T h e total radioactivity values for trospectomycin in plasma were not different from the values found after t.l.c., indicating the apparent absence of metabolism in the rat. Therefore, the total radioactive counts in plasma were used as a measure of unchanged drug. T.1.c. was performed on pooled plasma samples up to 24 h from animals dosed by the three routes. It was not technically feasible to go beyond this
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830
D. J. Nichols et al.
timepoint because there were insufficient counts in the samples for t.1.c. T.1.c. of the urine and autoradiography and/or counts of the t.1.c. regions revealed only two major bands of radioactivity; one which corresponded to unchanged trospectomycin (R,0.3 1 and 0.64 in solvents A and B respectively) and one which had an R,of 0.24 in solvent system A and an R,of 0.40 in solvent system B. Only two radioactive peaks were seen after h.p.1.c.: one with a retention time of 6.25mi1-1,corresponding to unchanged trospectomycin, and one at 7.0 min. These R,values and retention times indicate that the second component is probably 6'-propyl-actinospectinoic acid. Additionally, enzyme hydrolysis with the P-glucuronidaselsulphatase preparation did not affect the R,value of the compound with an R,value of 0 2 4 in system A; it is therefore unlikely to be either a glucuronide or sulphate conjugate. Quantitatively, approximately equal amounts of unchanged drug and metabolite/degradation product were seen in each urine sample.
Pharmacokinetics and disposition Trospectomycin concentration in plasma disappeared in a bi-exponential manner with a rapid initial phase ( t I l 2 . 03-0-4 h) and a slow terminal decline ( t l l z B 45-80 h; figures 2 and 3). This is unlikely to be due to tritiated water; although there was insufficient plasma for a freeze-drying experiment no tritiated water was detected in either expired air or in urine and it is unlikely that there was any in plasma. The distribution volume was large ( x 15 times body mass) which indicates that there is retention of drug in the tissues. T h e pharmacokinetic parameters are summarized in table 1. T h e major route of excretion of radioactivity was the urine with > 60% of the dose excreted in 48 h (figure 4). Faeces were only a minor route of excretion (15-20% dose by 168 h). T h e total recovery was >SO% of the dose. In a separate experiment the distribution of total radioactivity in various tissues up to 120h was examined. Even at the last time period appreciable quantities of radioactivity were seen in the tissues with the liver containing the largest quantity (almost 4% of the dose).
Effect of route of administration The bioavailability of trospectomycin, based on the A U C value, was 100% following an i.m. injection but only 66% (females) and 85% (males) after an S.C. injection (table 1). There was a significantly lower (P
1991, VOL. 21,
NO.
7, 827-837
Pharmacokinetics and fate of 3H-trospectomycin sulphate, a novel aminocyclitol antibiotic, in male and female rats D. J. NICHOLS?, M. BURROWS, A. BYE, D. A. CONSTABLE, L. G. DRING and P. JEFFREY
Xenobiotica Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
Drug Investigation and Clinical Research, Upjohn Ltd, Fleming Way, Crawley, West Sussex, U K
Received 5 December 1989, accepted 24 January 1991
I . The pharmacokinetics and fate of 3H-trospectomycin sulphate, a novel aminocyclitol antibiotic, were examined in male and female rats after intramuscular (i.m.), intravenous (i.v.) and subcutaneous (s.c.) dosing. 2. Total radioactivity levels in plasma were associated with unchanged trospectomycin. Two radioactive components were found in urine, one was indistinguishable from trospectomycin and the other was probably a degradation product formed after excretion or during storage rather than a metabolite. 3. T h e disappearance of drug from plasma followed a biphasic pattern with half lives of 0,344h and 45-80 h and a large distribution volume, which indicated some retention of drug by tissues. Clearance rates were within the normal range for glomerular filtration rate, which indicated that the primary process of elimination is filtration of unchanged drug. 4. Excretion was initially rapid (> 40% by 4 h) and mainly into urine (faecal excretion 95% as determined by t.1.c. and h.p.1.c. All references to the mass or concentration of trospectomycin refer to the hydrated drug sulphate, not the free base. To convert to free base all doses and concentrations are multiplied by 0.625.
t T o whom correspondence should be addressed. 0049-8254/91 $340
0 1991 Taylor & Francis Ltd.
D . J . Nichols et al.
828
CH3
-
5 H,O
Xenobiotica Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
Figure 1. Chemical structure of trospectomycin sulphate.
Animals Male and female Sprague-Dawley rats (Charles River UK Ltd, Manston Rd, Margate, Kent, UK) of body weight 200-300g, were held for at least 5 days before use. Room temperature was maintained between 19°C and 20"C, relative humidity was 40-65% and 12 h-12 h light-dark photoperiod was used. All animals had free access to food (SDS diet No. 1, Wm. Lillico and Sons, Betchworth, UK) and fresh water was available ad libitum. Experimental design Excretion. T o examine the effects of route of administration on the disposition of trospectomycin, male and female rats (four per treatment) were housed individually in metabolism cages suitable for the collection of urine and faeces, and given trospectomycin sulphate (5 mglkg, 4 4 3 MBqIkg body weight) in 7&13Opl isotonic saline s.c., i.m. or i.v. Urine was collected for the periods@2,24,4-6,6-24 h and daily thereafter until 168 h post-dose. Faeces were collected every 24 h. All samples were stored at - 20°C until analysis. Pharmacokinetics. T o examine the pharmacokinetics by different routes of administration, male and female rats (four per treatment) were housed in cages with raised grid floors and given trospectomycin sulphate (5 mg/kg body weight, 15.70 MBq/kg) in 7&130pl isotonic saline s.c., i.m. or i.v. Blood samples ( 2 5 0 ~ 1were ) taken into heparinized microcentrifuge tubes from the tail vein of each rat at the following times post-dose: 15,30 and 45 min and 1 , l . S . 2 and 3 h. A 400 pl sample was taken at 4,6,8,24,48 and 72 h post-dose. At 96 h each rat was killed and a large terminal blood sample was taken from the inferior vena cava. Whole blood was aliquoted in duplicate (lop1up to 3 h, 25 p1 thereafter) to measure 'H content. The remaining blood was centrifuged and the plasma stored frozen at - 20°C until analysis. Dose proportionalily. T o examine the linearity of the kinetics with dose, male and female rats (four per dose), housed in cages with raised grid floors, were given trospectomycin sulphate ( S O , 100 or 200mgikg body weight, 14.4, 13.4 and 16.9MRq respectively) in 37G700pI sterile water S.C.(over the shoulder region). Blood samples ( 2 5 0 ~ 1were ) collected into heparinized microcentrifuge tubes from the tail vein of each rat at the following times: 15,30and 45 min and 1, 1 . 5 , 2 , 3 , 4 , 6 , 8 , 2 4 , 4 8 , 7 2 , 9 6and 120 h. T h e blood was centrifuged and the plasma was stored at - 20°C until analysis. Radiochemical techniques Urine and Plasma. Urine and plasma samples (l&lOOOpI) were mixed with water to give a total volume of 300-15OOp1 and agitated with 3 ml Optiphase X scintillation fluid (Fisons plc, Loughborough, Leics., UK) in plastic minivials. Radioactive counts were measured with an LKB liquid scintillation counter using the external standard ratio method for quench correction. Plasma concentration of trospectomycin sulphate was calculated from the counts in the radioactive areas of the t.1.c. plate (see below) for the study to compare different routes of administration. Because there was little difference between the counts after t.1.c. and the total counts, total radioactivity was used to calculate the plasma concentrations in the dose proportionality study. A preliminary experiment was conducted to ascertain whether tritium exchange/ metabolic instability would be a problem (Constable et al. 1990). No tritiated water was found in the expired air and there was no significant loss of radioactivity from urine subjected to freeze drying. Faeces. The faeces were homogenized, combusted in a Packard 306 Sample Oxidizer and mixed with scintillant (Monophase-40 Plus, Packard Instruments, Caversham, Reading, Berks., U K ; 15 ml for 300mg faecal homogenate). The combusted samples were counted in 20ml glass vials using a Packard 3385 scintillation counter with the external standard ratio method for quench correction. Unchanged trospectomycin and metabolitesldegradation products Urine. Urine (10-20p1) was applied directly to t.1.c. plates and run in solvent system A or B (see below). After drying, the plates were sprayed with EN3HANCE (NEN, Dreieich, Germany), dried for 1 h and
Pharmacokinetics of trospectomycin in rat
829
placed in apposition with an X-Omat AR film (Kodak Ltd, Hemel Hempstead, Herts., UK), for about 2 weeks. The film was developed, the radioactive areas located, the silica gel corresponding to each radioactive area was scraped off into minivials containing 1.5 ml water and mixed with Optiphase X to form a stable gel which was counted as described above.
Xenobiotica Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
Plasma. Five microlitres of H,SO, and 0.5ml methanol were vortex-mixed with 1 5 0 ~ plasma, 1 then centrifuged for 10 min. T h e supernatant was removed and the pellet was resuspended in methanol-water (1 : 4 v/v, containing 2 0 ~ 1of S M H,SO,/lOml), vortex-mixed and the precipitate removed. The supernatant was combined with the first, evaporated to dryness at 30°C by vacuum vortex and resuspended in the acidified aqueous methanol. T h e fine precipitate was removed by centrifugation and lop1 of the supernatant was subjected to t.1.c. in solvent system A. The radioactive areas were located on the plate by removing bands of silica gel 2 cm wide by 5 mm deep, transferring to minivials, sonicating for 5 min with 0 5 ml of 5 M H,SO,, mixed with 1 ml water and Optiphase X to form a stable gel, and counted. Chromatography Thin-layer chromatography. T.1.c. was performed on glass plates pre-coated with Keiselgel 60 FZ5,, thickness 0.25 mm (E. Merck, Darmstadt, Germany) using either solvent system A: butal-1-01-acetic acid-water (5 : 1 : 2 by vol.) or B: methanol-chloroform-ammonium hydroxide (sp. gr. 0.88) (4 : 3 : 2 by vol.). The solvent front was allowed to run for 16 cm from the origin. Trospectomycin could be visualized as an orange spot by spraying with 2.4-dinitrophenylhydrazine (05% w/v in 2 M HCI). High-performance liquid chromatography. Either plasma or urine samples were injected into an h.p.1.c. system which consisted of an M730 Data Module (Waters Associates Ltd, Northwich, Cheshire, UK), a Waters Wisp 710B autoinjection unit, a 720 system controller, a Waters 6000A solvent delivery system, a Waters 441 absorbance detector, a Shimadzu column oven CTO-2A (Shimadzu Corp., Kyoto, Japan) in association with a programmable Superrac 221 1 fraction collector (LKB Instruments Ltd). T h e column was an APS-Hypersil (3 pm particle size; Shandon Southern Products Ltd, Runcorn, Cheshire, U K ) 200 mm by 4.6 mm analytical column. T h e mobile phase used was acetonitrile (700 ml) and 0.1 M KH,PO, (350ml) adjusted to p H 2 with phosphoric acid. This was degassed with helium and used at a flow rate of 1 ml/min. The column oven was maintained at a temperature of 50°C. T h e radioactivity eluted between 7 and 13 min. Ten-second samples were collected over this time period and the radioactive elution profile was determined by scintillation counting. T h e only occasion that the h.p.1.c. method was used quantitatively was in the determination of radiochemical purity. In this case the trospectomycin peak was collected, counted and compared with the counts actually put on the column. Enzyme hydrolyszs. Urine (1 ml) was mixed with an equal volume of acetate buffer ( 0 . 2 ~ pH , 5.0) containing 8-glucuronidase type H-2 (ex. Helix pomatia, containing some arylsulphatase activity; Sigma Chemical Co. Ltd, Poole, Dorset, UK). The mixture was incubated overnight at 3 7 T , with suitable controls; namely, either the mixture lacking enzyme or with phenolphthalein glucuronic acid (50 pl; 0-03M). Before t.1.c. of the hydrolysed urine the excess protein in the incubation mixture was removed by adding an equal volume of methanol and vortex-mixing. After 1 h at 4°C the mixture was centrifuged and the supernatant stored at - 20°C overnight. The sample was centrifuged, and supernatant removed, dried under nitrogen, reconstituted in 1 ml methanol and subjected to t.1.c. Pharmacokinetic analysis. The concentration of trospectomycin sulphate were fitted by a twocompartment open model with first-order rate constants with NONLIN84 (Metzler and Weiner 1984). Pharmacokinetic parameters were calculated in NONLIN84; k01, k12, K21 and k10 are the rates of absorption, intercompartment transport and elimination, V1 is the volume of the first compartment, Vss is the total volume of distribution, A UC is the area under the timeplasma concentration curve, CL is the plasma clearance rate (CL= Dose x f / A U C ) where f is the fraction of drug absorbed? CLr is the renal clearance rate ( C L r = A e , , , f / A U C , , , where Ae,6Mis the total amount of drug lost in urine to 168 h and AUC,,, is the area under the plasma concentration-time curve from 0 to 168h), MRT is the mean residence time ( M R T = Vss/CL)and t l i Z aand t l l Z Bare the initial and terminal half-lives respectively. The i.v. input was as a bolus but the S.C. and i.m. data were best described by first-order input. Statistical differences in pharmacokinetic parameters between doses, sexes and routes of administration were analysed by ANOVA using SAS (SAS Institute Inc, Box 8000, Cary, NC, USA).
Results Metabolism T h e total radioactivity values for trospectomycin in plasma were not different from the values found after t.l.c., indicating the apparent absence of metabolism in the rat. Therefore, the total radioactive counts in plasma were used as a measure of unchanged drug. T.1.c. was performed on pooled plasma samples up to 24 h from animals dosed by the three routes. It was not technically feasible to go beyond this
Xenobiotica Downloaded from informahealthcare.com by University of Sydney on 01/01/15 For personal use only.
830
D. J. Nichols et al.
timepoint because there were insufficient counts in the samples for t.1.c. T.1.c. of the urine and autoradiography and/or counts of the t.1.c. regions revealed only two major bands of radioactivity; one which corresponded to unchanged trospectomycin (R,0.3 1 and 0.64 in solvents A and B respectively) and one which had an R,of 0.24 in solvent system A and an R,of 0.40 in solvent system B. Only two radioactive peaks were seen after h.p.1.c.: one with a retention time of 6.25mi1-1,corresponding to unchanged trospectomycin, and one at 7.0 min. These R,values and retention times indicate that the second component is probably 6'-propyl-actinospectinoic acid. Additionally, enzyme hydrolysis with the P-glucuronidaselsulphatase preparation did not affect the R,value of the compound with an R,value of 0 2 4 in system A; it is therefore unlikely to be either a glucuronide or sulphate conjugate. Quantitatively, approximately equal amounts of unchanged drug and metabolite/degradation product were seen in each urine sample.
Pharmacokinetics and disposition Trospectomycin concentration in plasma disappeared in a bi-exponential manner with a rapid initial phase ( t I l 2 . 03-0-4 h) and a slow terminal decline ( t l l z B 45-80 h; figures 2 and 3). This is unlikely to be due to tritiated water; although there was insufficient plasma for a freeze-drying experiment no tritiated water was detected in either expired air or in urine and it is unlikely that there was any in plasma. The distribution volume was large ( x 15 times body mass) which indicates that there is retention of drug in the tissues. T h e pharmacokinetic parameters are summarized in table 1. T h e major route of excretion of radioactivity was the urine with > 60% of the dose excreted in 48 h (figure 4). Faeces were only a minor route of excretion (15-20% dose by 168 h). T h e total recovery was >SO% of the dose. In a separate experiment the distribution of total radioactivity in various tissues up to 120h was examined. Even at the last time period appreciable quantities of radioactivity were seen in the tissues with the liver containing the largest quantity (almost 4% of the dose).
Effect of route of administration The bioavailability of trospectomycin, based on the A U C value, was 100% following an i.m. injection but only 66% (females) and 85% (males) after an S.C. injection (table 1). There was a significantly lower (P
