Investigations on the metabolic pathways of cyclosporine: I. Excretion of cyclosporine and its metabolites in human bile—isolation of 12 new cyclosporine metabolites

1. Cyclosporine metabolites of known and unknown structures were isolated, by semi-preparative?h.p.l.c., from human bile from the T-tube of liver-grafted patients, who received cyclosporine treatment. Their structures were elucidated by FAB mass spectrometry and ¹H-n.m.r. spectroscopy.

2. Twelve of the cyclosporine metabolites, with known chemical structures, were isolated and identified using authentic standard material.

3. Four isolated fractions contained tri-hydroxylated metabolites; two fractions contained di-hydroxylated, demethylated metabolites; one fraction contained a trihydroxylated, demethylated metabolite; and one fraction a mono-hydroxylated, demethylated metabolite. The exact metabolism sites were partially defined.

4. Two carboxylated cyclosporine metabolites, of which one was hydroxylated in an unknown position, were isolated.

5. One new metabolite proved to be a glucuronylated phase II metabolite. Deglucuronylation of this metabolite by β-glururonidase yielded metabolite AM1c. The proposed structure was AM1c-Gic; is a proposed extension of the Hawk's Cay nomenclature of the cyclosporine metabolites for glucuronylated metabolites.

6. One of the unknown metabolites was hydroxylated in two positions of amino acid 1. The proposed nomenclature was ‘AM11d’, where ‘1d’ indicates hydroxylation at the δC of amino acid 1.

7. A metabolite with an aldehyde functional group at amino acid 1, which had two isomeric forms, was isolated. I.r.-spectroscopy indicated that isomerism may be caused by conjugation of the aldehyde group with the double bond between C6 and C7 of amino acid 1.     

Investigations on the metabolic pathways of cyclosporine: II. Elucidation of the metabolic pathways in vitro by human liver microsomes.

1. Cyclosporine and its metabolites, isolated from human bile and identified by FAB mass spectrometry and 1H-n.m.r. spectroscopy, were metabolized by human liver microsomes for the identification of new cyclosporine metabolites. From these data a metabolic pathway for cyclosporine, which includes these new cyclosporine metabolites, has been proposed. The new metabolites were isolated by semi-preparative h.p.l.c. and their chemical structures were elucidated by FAB mass spectrometry. These isolated metabolites were further metabolized and the products identified by FAB mass spectrometry. 2. Fourteen metabolites, whose structure has not yet been elucidated, were isolated after metabolism of structurally identified cyclosporine metabolites, and chemical structures for five of these metabolites were proposed. 3. The structures of the new cyclosporine metabolites were: (i) a N-demethylated, carboxylated derivative (AM1A4N), (ii) a di-hydroxylated, N-demethylated derivative (AM14N9), (iii) a hydroxylated and carboxylated derivative (AM1A9), (iv) a di-hydroxylated, cyclized and N-demethylated derivative (AM1c4N9) and (v) a cyclized and carboxylated (AM1cA) derivative. 4. A proposed cyclosporine metabolic pathway comprises a total of 29 metabolites. It consists of four main branches originating from metabolites AM1, AM1c, AM9 and AM4N.     

Investigations on the metabolic pathways of cyclosporine: II. Elucidation of the metabolic pathways in vitro by human liver microsomes

1. Cyclosporine and its metabolites, isolated from human bile and identified by FAB mass spectrometry and ¹H-n.m.r. spectroscopy, were metabolized by human liver microsomes for the identification of new cyclosporine metabolites. From these data a metabolic pathway for cyclosporine, which includes these new cyclosporine metabolites, has been proposed. The new metabolites were isolated by semi-preparative?h.p.l.c. and their chemical structures were elucidated by FAB mass spectrometry. These isolated metabolites were further metabolized and the products identified by FAB mass spectrometry.

2. Fourteen metabolites, whose structure has not yet been elucidated, were isolated after metabolism of structurally identified cyclosporine metabolites, and chemical structures for five of these metabolites were proposed.

3. The structures of the new cyclosporine metabolites were: (i) a N-demethylated, carboxylated derivative (AM1A4N), (ii) a di-hydroxylated, N-demethylated derivative (AM14N9), (iii) a hydroxylated and carboxylated derivative (AM1A9), (iv) a di-hydroxylated, cyclized and N-demethylated derivative (AM1c4N9) and (v) a cyclized and carboxylated (AM1cA) derivative.

4. A proposed cyclosporine metabolic pathway comprises a total of 29 metabolites. It consists of four main branches originating from metabolites AM1, AM1c, AM9 and AM4N.     

Toxicity of cyclosporine metabolites

Eight cyclosporine (CsA) metabolites were isolated from the urine of renal transplant recipients. The structure and purity of the metabolites were characterized by fast atom bombardment/mass spectroscopy as well as by proton and 13C nuclear magnetic resonance. The in vitro toxicity of the metabolites were tested using a porcine renal epithelial cell line (LLC-PK1). None of the metabolites was as effective as CsA in inhibiting cell growth and DNA, RNA, or protein synthesis, with the majority of them exhibiting activity less than 10% of that of CsA when the IC50 (the concentration required for 50% inhibition of that particular metabolic function) values were compared. The exception to this was the demethylated metabolite M-21, which exhibited a potency of 17-50% of CsA for the various metabolic parameters examined. The results suggest that the immunosuppressive activity of metabolites may be dissociated from their toxicity. Morphologically, CsA and the metabolite M-21 resulted in changes consistent with the vacuolization seen in tubular cells exposed to CsA in vivo. In contrast, M-17 up to the maximum concentration tested (25,000 micrograms/L) was found not to cause such changes.     

Diltiazem increases blood concentrations of cyclized cyclosporine metabolites resulting in different cyclosporine metabolite patterns in stable male and female renal allograft recipients

1 Six male and six female stable renal allograft recipients under cyclosporine immunosuppression and without concomitant therapy with drugs known either to induce or inhibit CYP3A enzymes were included in the study and received 180  mg day⁻¹ diltiazem for 1 week in a two-period cross-over fashion. Cyclosporine (352±56  mg day⁻¹) was given in two daily oral doses. The daily doses were not changed during the study. Blood samples were collected for 12  h after receiving cyclosporine alone and after receiving diltiazem in addition for 1 week. Cyclosporine and nine of its metabolites were quantified using h.p.l.c.
2 Co-administration of diltiazem caused a 1.6 fold increase of the AUC(0,12  h) of cyclosporine and a 1.7 fold increase of the AUC(0, 12  h) of its metabolites. Analysis of the metabolite patterns showed an over-proportional increase of the AUC(0, 12  h) of the cyclized metabolites AM1c (2.6 fold) and AM1c9 (2.2 fold). The AUC(0, 12  h) values of cyclosporine and the hydroxylated metabolites increased less than two fold.
3 Differences of the AUC(0, 12  h) values of cyclosporine with and without diltiazem were significantly higher in female than in male patients ( P <0.02). The differences in the AUC(0, 12  h) values of the metabolites, especially AM1c, tended to be higher in female patients as well.
4 It is concluded that coadministration of diltiazem not only increases the blood concentration of cyclosporine but also those of its metabolites, leads to a shift of the metabolite pattern towards cyclized metabolites, and that the pharmacokinetic changes under diltiazem administration are more prominent in female than in male patients.     

Disposition of cyclosporine in several animal species and man. I. Structural elucidation of its metabolites

Nine ether-extractable metabolites of cyclosporine were isolated from urine of dog and man and from rat bile and feces and purified by preparative HPLC and TLC. Structural assignments were mainly based on spectroscopic data (1H NMR, 13C NMR, MS) and the results of the amino acid analysis after hydrolysis with hydrochloric acid. All the identified metabolites retained the intact cyclic oligopeptide structure of the parent drug. Structural modifications originated from enzymatic oxidation at specific sites of the peptide subunits. Transformation processes principally involved hydroxylation at the terminal carbon atom (eta-position) of the C9-amino acid 1 and the gamma-position of the N-methylleucines 4, 6, and 9, as well as N-demethylation of the N-methylleucine 4. Regioisomeric monohydroxylated cyclosporines (metabolites 1, and 17) and N-demethylcyclosporine (metabolite 21) were the primary metabolites resulting from hydroxylation of the C9-amino-acid 1 and the N-methylleucine 9, and from N-demethylation of the N-methylleucine 4. Dihydroxylated derivatives of cyclosporine (metabolites 8, 10, and 16) were generated by further oxidation of metabolite 1 on one of the other N-methylleucines (4 or 6) or on the C9-amino acid 1, or of metabolite 17 on the N-methylleucine 9. More extensive modifications were observed for metabolite 9, a dihydroxy-N-demethylcyclosporine, which could have been formed from the dihydroxy derivative 16 by N-demethylation or from the N-demethylcyclosporine 21 by dihydroxylation. Metabolite 18 differed from 17 (monohydroxycyclosporine) by the presence of a cyclic ether moiety, formally derived by intramolecular addition of the beta-hydroxyl group to the double bond of the C9-amino acid 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of two immunosuppressive metabolites after in vitro metabolism of rapamycin.

Rapamycin was incubated with human liver microsomes and an NADPH regenerating system, the metabolites were purified by semipreparative HPLC, and their structures were elucidated by direct chemical ionization and FAB-MS. At least six fractions were isolated containing rapamycin metabolites, indicating that rapamycin is metabolized by the human liver cytochrome P-450 system. One of these metabolites was identified as 41-O-demethyl-rapamycin. A second metabolite was hydroxylated in a yet unknown position. These two metabolites retained immunosuppressive activity in a phytohemagglutinin-stimulated human lymphocyte assay with IC50S of 1 and 1.5 nmol/liter, respectively. Rapamycin was metabolized by rat small intestinal microsomes to at least two metabolites, indicating extra-hepatic metabolism of rapamycin.     

Isolation of 10 cyclosporine metabolites from human bile

Ten metabolites of cyclosporine were isolate from the ethyl ether extract of bile from four liver transplant patients receiving cyclosporine. Two of the metabolites were unique and previously unidentified. Liquid-liquid partitioning into diethyl ether with subsequent defatting with n-hexane was used for the initial extraction from bile. Separation of the individual metabolites (A-J) was performed using a Sephadex LH-20 column and a gradient high performance liquid chromatographic method. The molecular weights of the isolated metabolites were determined by fast atom bombardment/mass spectrometry. Gas chromatography with mass spectrometric amino acid analysis was also used to identify the amino acid composition and the hydroxylation position of metabolites A, B, C, D, and G. Proton nuclear magnetic resonance spectra were utilized to distinguish the chemical shifts of N-CH3 singlets and NH doublets of metabolites A, B, C, and D. Metabolites A, E, F, H, I, and J were reported previously in human urine and animal bile. Metabolites C and D are dihydroxylated compounds which cannot be clearly described as previously isolated compounds. Metabolites B and G are novel metabolites with a mass fragment which corresponded to a loss of 131 Da from the protonated molecular ion (MH+) in the fast atom bombardment/mass spectrometry, suggesting that the double bond in amino acid 1 has been modified. Metabolites B and G were primarily isolated from the bile of one of the liver transplant patients which contained abnormally high concentrations of these two metabolites. The method described is an efficient procedure for isolating milligram quantities of the major metabolites with greater than 95% purity.     

Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: implications for immunoassays

Abstract: Cyclosporine exhibits a wide spectrum of metabolites that vary considerably in the extent to which they interfere with the various parent drug monitoring immunoassays. There is no consensus regarding the clinical significance of metabolites. Cyclosporine exerts its immunosuppressive action by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites in this perspective? The aim of the present study was to determine the concentration of cyclosporine (by means of three different assay methods) and the four most significant metabolites (AM1, AM4N, AM9, and AM1C) in relation to calcineurin phosphatase inhibition. Twelve randomly selected cyclosporine‐treated renal transplant patients were included in the study. Blood samples were drawn before, 1, 2, 3, 4, 6, 8, and 12 hr after oral intake of cyclosporine. Parent drug and metabolites were determined by liquid chromatography/tandem mass spectrometry (LC/MSMS). Additionally, cyclosporine concentration was determined by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19‐amino acid peptide. We found that calcineurin phosphatase inhibition correlates strongly with parent cyclosporine metabolites concentrations determined by all three assay methods. Determination methods that took metabolites into consideration exhibit stronger correlations with calcineurin phosphatase inhibition (sum of cyclosporin plus metabolites r=?0.93, LC/MSMS; pFPIA r=?0.94, P≤0.001), compared with methods that measure exclusively the parent drug (EMIT: ?0.84; LC/MS‐MS: ?0.81, P≤0.05). Our results indicate that the immunosuppressive role of cyclosporines metabolites should not be considered valueless per se. Further research is required in order to verify the potential clinical importance of our observations.     

The in vitro activity, radioimmunoassay cross-reactivity, and molecular weight of thirteen rabbit cyclosporine metabolites

Thirteen metabolites of cyclosporine were isolated from the bile of rabbits receiving intravenous cyclosporine. The molecular weights of these metabolites were determined by fast atom bombardment mass spectrometry. These molecular weights were consistent with hydroxylated, N-demethylated, and carboxylated metabolites of cyclosporine as described previously. The in vitro activities of the metabolites were established using mitogen-stimulated lymphocyte proliferation assays. Only the two monohydroxylated metabolites were found to have significant activity, this being between 5 and 10% of that of the parent drug. The metabolites were also compared with cyclosporine in two commercial radioimmunoassay kits. The metabolites were found to cross-react with the parent drug in amounts ranging from 20 to 100%, with the least polar metabolites cross-reacting the most strongly. It is concluded that the cross-reacting metabolites measured by the presently available radioimmunoassays for cyclosporine probably do not represent significant additional immunosuppressive activity in vivo.     

Influence of cyclosporine on the serum concentration and biliary excretion of mycophenolic acid and 7-O-mycophenolic acid glucuronide

The authors have investigated whether cyclosporine decreases the serum concentration of mycophenolic acid, the active principle of the immunosuppressant mycophenolate mofetil, and increases that of the inactive metabolite 7-O-mycophenolic acid glucuronide by reducing their enterohepatic recirculation. Rats were treated daily with methylcellulose (1.66 mL/kg PO) plus 0.9% NaCl (6 mL/kg IP), mycophenolate mofetil (20 mg/kg PO) plus 0.9% NaCl (6 mL/kg IP), methylcellulose (1.66 mL/kg PO) plus cyclosporine (5 mg/kg IP), and mycophenolate mofetil (20 mg/kg PO) plus cyclosporine (5 mg/kg IP). After 14 days a bile fistula was installed to measure the biliary excretion of the immunosuppressants and their metabolites. After 90 minutes blood was taken to determine their concentrations in blood or serum by HPLC. Cyclosporine significantly decreased the serum concentration of mycophenolic acid by 39% and increased, not significantly, that of 7-O-mycophenolic acid glucuronide by 53%. The biliary excretion of 7-O-mycophenolic acid glucuronide was significantly reduced by cyclosporine by 57%, whereas that of mycophenolic acid was not affected. Mycophenolate mofetil did not show a significant effect on either the blood concentration or the biliary excretion of cyclosporine and its metabolites AM1, AM9, AM1c, and AM4N. Cyclosporine significantly decreased the serum concentration of active mycophenolate acid and increased, not significantly, the serum concentration of inactive 7-O-mycophenolic acid glucuronide, presumably by reducing the biliary excretion of this inactive metabolite.     

More acidic metabolites of delta1-tetrahydrocannabinol isolated from rabbit urine.

The in vivo metabolism of delta1-tetrahydrocannabinol (delta1-THC) was further investigated in the rabbit after i.v. administration. Nine acidic metabolites were isolated from a previously not investigated fraction of the urine and identified by gas chromatography-mass spectrometry and by proton magnetic resonance spectroscopy. The major metabolites were side-chain hydroxylated monocarboxylic acids. Three side-chains monocarboxylic acids hydroxylated in allylic positions in the isoprene moiety were also characterized. The metabolites 4'-hydroxy-delta1-THC-7-oic acid and 7-hydroxy-4',5'-bisnor-delta1-THC-3'-oic acid were hitherto not identified. An earlier described dicarboxylic metabolite was present in high concentration. Further, the identity of an O-glucuronide as an in vivo urinary metabolite of delta1-THC was here for the first time unambiguously established by m.s. and p.m.r.     

Time course of cyclosporine and its metabolites in blood,liver and spleen of naive lewis rats: Comparison with preliminary data obtained in transplanted animals

The time course of intravenously administered cyclosporine (1mg kg^?1) and its metabolites AM1, AM9, and AM1c were examined in the blood, liver, and spleen of naive Lewis rats. Cyclosporine concentration versus time data for all three tissues were qualitatively similar, following a biexponential model C = Ae + Be with maximum cylosporine concentrations reached at 1h. Whole-blood cyclosporine clearance, terminal half-life, mean residence time, steady state volume of distribution, and hepatic extraction ratio (calculated from blood data) were similar to previously reported results. Cyclosporine in the liver showed the largest area under the concentration—time curve, mean residence time, and disposition and terminal half-lives. Spleen cyclosporine mean residence time and terminal half-life were not significantly different from blood parameters. Metabolites AM1, AM9, and AM1c showed almost parallel time courses in all three tissues. The hydroxylated derivative AM9 was the major metabolite found in all tissues, with twofold greater levels in the liver compared to the blood and the spleen. Slightly less AM1 was found in the liver relative to blood and spleen, where it was present in equal amounts. AM1c levels in the liver were not different from those in the spleen and were greater than observed for blood. The results obtained above were reflected in preliminary studies using liver transplanted rats treated with multiple doses of cyclosporine. Both blood and liver biopsy levels of CyA, AM1, and AM9 post-transplant showed twofold to fourfold decreases from day 3 (samples taken 4h post-CyA-dose) to day 7 (samples taken 24h post-CyA-dose) and concentrations were not significantly different from similarly sampled naive controls. More importantly, the metabolite/CyA ratios did not vary significantly between liver and blood in the two groups. For naive rats, and liver transplanted animals not undergoing rejection, changes in blood cyclosporine levels seem to predict variations in tissue concentrations.     

Metabolism of a new synthetic opioid tetrahydrofuranylfentanyl in fresh isolated human hepatocytes: Detection and confirmation of ring‐opened metabolites

The metabolism of a new synthetic opioid tetrahydrofuranylfentanyl (THF‐fentanyl) was investigated using fresh human hepatocytes. Fourteen metabolites of THF‐fentanyl, such as tetrahydrofuran ring‐opened metabolites, desphenethylated metabolites, hydroxylated metabolites, and hydroxylated and methoxylated metabolites and their glucuronides, were detected in the culture medium of hepatocytes incubated with THF‐fentanyl. Six metabolites, i.e. desphenethylated metabolite, 4′‐hydroxy‐THF‐fentanyl, β‐hydroxy‐THF‐fentanyl, 4′‐hydroxy‐3′‐methoxy‐THF‐fentanyl, ring‐opened alcohol metabolite, and ring‐opened carboxylic acid metabolite, were identified via chemically synthesized authentic standards. A ring‐opened alcohol metabolite and a ring‐opened carboxylic acid metabolite are thought to be formed by reduction or oxidation of the intermediate aldehyde, which was formed by ring‐opening of the metabolite hydroxylated at the carbon atom adjacent to the oxygen atom of the tetrahydrofuran ring. A ring‐opened carboxylic acid metabolite was the main metabolite of THF‐fentanyl based on the peak intensity.     

The kinetics of cyclosporine and its metabolites in bone marrow transplant patients.

1. The pharmacokinetics of cyclosporine (CsA) and the time course of CsA metabolites were studied in five bone marrow transplant patients after intravenous (i.v.) administration on two separate occasions and once after oral CsA administration. 2. Cyclosporine and cyclosporine metabolites were measured in whole blood by h.p.l.c. 3. Cyclosporine clearance after i.v. administration decreased from 3.9 +/- 1.7 ml min-1 kg-1 to 2.0 +/- 0.6 ml min-1 kg-1 after 14 days of treatment. The mean +/- s.d. absolute oral bioavailability of cyclosporine was 17 +/- 11%. 4. Hydroxylated CsA (M-17) was the major metabolite in blood. There were no significant differences in the mean metabolite/CsA AUC ratios between the first and second i.v. studies. 5. After oral administration, the metabolite to CsA AUC ratios were higher for most metabolites compared to those observed in the second i.v. study, suggesting a contribution of intestinal metabolism to the clearance of CsA.     

Monitoring cyclosporine of pre-dose and post-dose samples using nonextraction homogeneous immunoassay

A nonextraction homogeneous immunoassay (CEDIA Cyclosporine Plus Assay) has been developed for the measurement of cyclosporine in predose (trough) and post-dose (C2 to C8) whole-blood samples. The method includes a low-range assay that measures cyclosporine from 25 to 450 ng/mL in pre-dose samples and a high-range assay that detects cyclosporine from 450 to 2000 ng/mL in post-dose samples. The high-range assay allows a direct measurement of post-dose samples without a dilution step. Alternatively, post-dose samples can be correctly measured by the low-range assay following a twofold dilution. Using an NCCLS precision protocol, the assay exhibited less than 10% CV or error less than the functional sensitivity. Functional sensitivity of the low-range assay was demonstrated at 20 ng/mL cyclosporine. Cross-reactivity was measured in the presence of cyclosporine and was found to be 4.4%, 19.8%, 16.4%, 0.9%, 1.0%, and 1.6% for metabolites AM1, AM9, AM4n, AM19, AM4n9, and AM1c, respectively. When 53 samples were evaluated using an HPLC method, the three most significant cross-reactive metabolites, AM1, AM4n, and AM9, exhibited an average concentration profile of 123%, 19%, and 0.06% of the parent cyclosporine, respectively. The average total contribution to cyclosporine quantification from these metabolites was estimated at 7.2% based on the percentage cross-reactivity of each metabolite in the CEDIA assay and the concentration of each metabolite as determined by HPLC. The method comparison study revealed a linear regression correlation of CEDIA = 1.095 x HPLC + 6.6, r = 0.972, for the low-range assay, and CEDIA = 1.018 x HPLC - 36.4, r = 0.968, for the high-range assay. In conclusion, the CEDIA Cyclosporine Plus Assay is a precise and accurate method for quantification of cyclosporine in pre-dose and post-dose samples.     

The subcellular distribution of 14C-lidamidine and its metabolites

Examination of the subcellular distribution of 14C-lidamidine and its metabolites in rat liver showed that the majority of radioactivity appeared in the postmicrosomal supernatant fraction, with lysosomes and microsomes having the highest relative specific activity (RSA) of the particulate fractions. When the subcellular distribution pattern was corrected for cross-contamination, based on the distribution of subcellular fraction marker enzymes, there was a significant decrease in the lysosomal RSA with an attendant increase in the microsomal RSA. Thin-layer chromatography of subcellular fraction extracts revealed different distribution patterns for lidamidine and metabolites in each fraction. The whole homogenate and cytosol fraction contained mostly polar metabolites (76-91%), whereas the particulate fractions contained 37-50% of their radioactivity as polar metabolites. The highest percentages of unchanged lidamidine and its more pharmacologically active, demethylated metabolite were associated with the mitochondrial and microsomal fractions.     

Biotransformation of diclofenac sodium (Voltaren) in animals and in man. I. Isolation and identification of principal metabolites.

1. The anti-inflammatory agent diclofenac sodium (o-[(2,6-dichlorophenyl)amino]phenylacetic acid sodium salt) is extensively metabolized by rat, dog, baboon and man. The main metabolites were isolated from the urine of all species and from the bile of rat and dog and identified by spectroscopy. 2. Metabolism involves direct conjugation of the unchanged drug, or oxidation of the aromatic rings usually followed by conjugation. Sites of oxidation are either position 3' or 4' of the dichlorophenyl ring or, alternatively, position 5 of the phenyl ring attached to the acetic acid moiety. 3. In the urine of rat, baboon and man conjugates of the hydroxylated metabolites predominate, but the major metabolite in dog urine is the taurine conjugate of unchanged diclofenac. 4. In the bile of rat and dog, the main metabolite is the ester glucuroniade of unchanged diclofenac.     

Pharmacokinetics of amitriptyline and its demethylated and hydroxylated metabolites in streptozocin-induced diabetic rats

Plasma and brain levels of amitriptyline (AMI), its demethylated and hydroxylated metabolites were determined after acute IP administration of AMI (20 mg/kg) in streptozocin-induced diabetic Sprague-Dawley rats. Results showed

• 1.1. in plasma: rapid AMI absorption, but slow elimination; the proportion of AMI similar to those of the rest of compounds; the proportion of its demethylated metabolite, nortriptyline, 1.8-fold higher than that of 10-hydroxy-nortriptyline.
• 2.2. in brain: the proportions of AMI and nortriptyline were 9.5- and 2.6-fold higher respectively, than those of whole hydroxylated metabolites, which represented 7.4% of the total amount.

     

Urinary metabolites of doxapram in premature neonates.

1. Urine samples from 20 premature neonates who received doxapram by i.v. infusion were analysed for drug metabolites by g.l.c-mass spectrometry. 2. In addition to doxapram, all urines contained at least one metabolite, but the known metabolite, 3-ketodoxapram, was detected in only 50% of the samples, and in some instances only in trace amounts. 3. Significant inter-individual differences in the metabolic pathways of doxapram were observed. 4. A total of six metabolites of doxapram were isolated three of which have not been observed previously in human or in dog. 5. Appropriate structures for the new metabolites have been deduced from their mass spectral fragmentation pathways, and are 1-ethyl-4-[2-(N-formyl-N-(2-hydroxy-ethyl)amino)ethyl]-3,3-diphenyl-2- pyrrolidinone (VII), 1-ethyl-4-[2-(4-morpholin-2-onyl)ethyl]-3,3-diphenyl-2-pyrro lidinone (IX) and 4-ethenyl-1-ethyl-3,3-diphenyl-2-pyrrolidinone (X).