Increase of CD8+ T-effector memory cells in peripheral blood of patients with relapsing-remitting multiple sclerosis compared to healthy controls.

CCR7 and CD45RA expression on CD4+ and CD8+ T-cells in blood (PB) of 16 patients with multiple sclerosis (MS) and 16 healthy controls and cerebrospinal fluid (CSF) of 10 patients suffering from MS were analysed by flow cytometric measurements. T-cells were divided by their distinct homing potentials and effector-functions in three groups: naïve T-cells (CCR7+, CD45RA+), central memory T-cells (TCM) (CCR7+, CD45RA-) and effector memory T-cells (TEM) (CCR7-, CD45RA-). There was a significant increase of CD8+ TEM-cells in PB of MS patients compared to healthy controls, indicating systemic immune activation. Further we found a relative depletion of CD8+ TEM-cells in CSF of MS patients compared to matching blood samples, suggesting that these cells represent the effector arm of the immune response and infiltrate the brain tissue at the sites of inflammation.

[Improvements in detection of rectal cancer recurrence by multiplanar reconstruction]. / Verbesserte Rezidiverkennbarkeit mittels multiplanarer Rekonstruktion beim Rektumkarzinom in der Mehrzeilenspiralcomputertomographie.

PURPOSE: To evaluate the advantages of multiplanar reconstruction and different axial slice thickness in diagnostic of rectal cancer recurrence after operation and radiotherapy. METHOD: We included 83 patients after operation and radiotherapy of rectal cancer in this study. All patients got a minimum of three CT-examinations in their follow-up program. A total of 294 CT-scans were evaluated. Each examination was reviewed by two experienced radiologists in respect to recurrence. Each examination was presented in axial reconstruction with a slice sickness of 8, 5, and 1.25 mm and in multiplanar reconstruction. The sensitivity, specificity, positive predictive value and accuracy were calculated. RESULTS: Multiplanar reconstructions showed better results for the detection of recurrence than axial reconstruction. A reduced slice thickness did not lead to better results in axial reconstruction. Multiplanar reconstruction showed a sensitivity of 0.88, a specificity of 0.98, an accuracy of 0.96 and a positive predictive value of 0,94, for axial reconstruction we calculated: 0.82, 0.97, 0.94 and 0.88, respectively. Sensitivity and accuracy showed a significant increase after the first and second examination. CONCLUSION: Multiplanar reconstructions allow a significant better detection of rectal cancer recurrence when compared to axial reconstructions. Thinner axial slice thickness shows no diagnostic advantage.

[Contrast media kinetic in multi-slice helical CT cannot detect rectal cancer recurrence with certainty]. / Die Kontrastmittelkinetik im Mehrzeilen-Spiral-CT kann das Rektumkarzinomrezidiv nicht sicher detektieren.

PURPOSE: Evaluation of different parameters of contrast media enhancement for the differentiation between scar tissue and local recurrence of rectal cancer. MATERIALS AND METHODS: We included 83 patients after operation and radiotherapy of rectal cancer. In total, 20 local recurrences were diagnosed. After administration of 75 ml Iopromide (370 mg/ml) and a delay of 65 s, the whole abdomen and pelvis were scanned in a collimation of 4 x 2.5 mm and 12.5 mm table feed per rotation. The suspected tissue was marked by the freehand ROI option in every slice and the minimum, average and maximum densities were calculated. A local recurrence was suspected if maximum density was higher than 90 HU after admission of contrast media. In addition we calculated the maximum difference of density as the difference between maximum and minimum density. RESULTS: The minimum and average densities showed no reliable differences for patients with or without local recurrence (minimum density 4 HU ( +/- 12 HU) vs. 13 HU ( +/- 21 HU), P = 0.23, average density 48 HU ( +/- 10 HU), vs. 48 HU ( +/- 17 HU)), P = 0.52. The patients suffering from local recurrence showed higher maximum densities and a higher maximum difference than the patients without recurrence (maximum density 111 HU ( +/- 13 HU) vs. 81 HU ( +/- 24 HU), P = 0.02, maximum difference 103 HU ( +/- 20 HU) vs. 76 HU ( +/- 31 HU), P = 0.06. These differences were not significant. We calculated a sensitivity of 0.6, a specificity of 0.83, a positive predictive value of 0.52 and an accuracy of 0.77. CONCLUSION: It is not possible to diagnose a recurrent rectal cancer by density values alone.

Frontoorbital volume reductions in adult patients with attention deficit hyperactivity disorder.

Attention deficit hyperactivity disorder (ADHD) is a common psychiatric disorder in childhood and adolescence and in a considerable number of patients it persists into adulthood. A network of brain regions have been shown to be abnormal in ADHD. In the present study we used magnetic resonance volumetry to investigate a possible role of the orbitofrontal cortex (OFC). Eight never medicated male patients fulfilling diagnostic criteria for ADHD and 17 male healthy controls were investigated. There was a significant reduction of the volume of the left OFC in patients with ADHD. It remains unknown whether small volumes are a primary deficit or a result of dysfunctional activation during childhood in terms of a residual deficit or a specific type of adult outcome of the disease.

Absorption of angiotensin II antagonists in Ussing chambers, Caco-2, perfused jejunum loop and in vivo: importance of drug ionisation in the in vitro prediction of in vivo absorption.

The aims of this study were (i) to compare the absorption of three closely related inhibitors of angiotensin II, RU60018, RU60079 and HR720, in various in vitro and in vivo models, and (ii) to explain the differences in the results and to assess the importance of drug ionisation to predict absorption. Drug absorption was investigated in Ussing chambers, Caco-2 cell monolayers, perfused rat jejunum loops and in vivo after oral, intraduodenal or intravenous administration. In Ussing chambers, the analogues showed the same site-related absorption profile and a common mechanism involving the paracellular pathway. At pH 7.4 in Ussing chambers, perfused jejunum loop or Caco-2 transport studies, the three compounds exhibited low and comparable permeability values suggesting that a similar level of oral absorption may be expected for all three compounds. However, after oral or intraduodenal administration, only HR720 was significantly absorbed. The in vivo results can be explained by the ionic distribution profile which indicated that only HR720 possessed a significant amount of uncharged species at pH values close to that found in the upper part of intestinal tract. Hence, it is expected that in this part of the intestine, only HR720 absorption is favoured. This is supported by Caco-2 transport studies performed when the pH of the apical medium was lowered from 7.4 to 6.0, in which a dramatic increase in permeability was observed for HR720 compared to those of the other analogues. This study highlights the usefulness of different absorption models for drug screening and demonstrates that ionisation profiles must be carefully considered to avoid rejection of promising compounds.

Comment on the cylindrical capacitor electrospray interface.

Wang and Hackett have recently proposed (Anal. Chem. 1998, 70, 205-212) a new electrospray interface described as a "concentric cylindrical capacitor". Excellent spectra were obtained, especially in the negative ion mode, with lipid A, a 15-mer oligonucleotide, and a series of proteins. We find that similar results can be obtained merely by ensuring a direct electrical contact between the analyte inside a fused-silica capillary and the high-voltage supply. No capacitor effect need be invoked.

Behavioral effects of vigabatrin correlated with whole brain gamma-aminobutyric acid metabolism in audiogenic sensitive rats.

The present study evaluates dose-dependent behavioral effects of acutely or subacutely administered single doses of vigabatrin (gamma-vinyl gamma-aminobutyric acid, gamma-vinyl GABA, CAS 60643-86-9) in audiogenic sensitive rats, in correlation with whole brain GABA metabolism. There was a discrepancy in timing between behaviorally observed maximal antiepileptic protection (4 h after i.p. administration of gamma-vinyl GABA) and on the other hand maximal inhibition of GABA-transaminase activity and maximal increase of whole brain GABA content (24 h after i.p. administration of gamma-vinyl GABA). This suggests that the antiepileptic properties of gamma-vinyl GABA not only depend on GABA-ergic neurotransmission. A possible explanation is a gamma-vinyl GABA-induced decrease of excitatory amino acids or increased glycine concentrations in the brain.

Analysis of 2-difluoromethyl-DL-ornithine in human plasma, cerebrospinal fluid and urine by cation-exchange high-performance liquid chromatography.

An analytical method has been developed based on cation-exchange liquid chromatography for the measurement of 2-difluoromethyl-DT-ornithine (DFMO) in human plasma, cerebrospinal fluid (CSF) and urine. Fluorescence detection at excitation/emission wavelengths of 340/440 nm is followed by postcolumn derivatization with o-phthalaldehyde-2,mercaptoethanol. All calibration ranges yielded linear relationships with correlation coefficients better than 0.999. In each case the limit of quantitation was equal to the lowest value of the standard curve. The variability of the assay, expressed as relative standard deviations, was less than 7.1%, 15.3% and 7.1% for plasma, CSF and urine, respectively. The accuracy of the assay (expressed as relative errors) ranged between 4.3% and 2.0% for plasma analysis, between -0.1% and 14.0% for CSF analysis and between -8.0% and 2.0% for urine analysis. Plasma, CSF and urinary DFMO concentrations were measured in samples obtained from patients undergoing treatment for trypanosomiasis. The method was found to be applicable for the measurement of DFMO levels in human body fluids for the determination of pharmacokinetic parameters in clinical studies.

Evidence for a novel pentyl radical adduct of the cyclic nitrone spin trap MDL 101,002.

3,4-Dihydro-3,3-dimethyl-isoquinoline-2-oxide (MDL 101,002) is a conformationally constrained cyclic analog of the known spin trap alpha-phenyl N-tert-butyl nitrone (PBN). Because of PBN's ability to scavenge free radicals, MDL 101,002 is currently being evaluated in stroke models as a means to ameliorate the oxidative insult associated with reperfusion injury. To augment our understanding of the radical scavenging mechanism of this potential drug, MDL 101,002 was incubated with soybean lipoxygenase in the presence of linoleic acid to study the interaction between MDL 101,002 and free radicals formed during lipid peroxidation. Analysis of the reaction mixture was performed by high performance liquid chromatography using normal phase conditions with detection by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Similar to the work by Iwahashi et al. [Arch. Biochem. Biophys., 1991, 285, 172], who studied the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN), an adduct that suggested the trapping of pentyl radicals by MDL 101,002 was observed. However, the apparent molecular ion for this adduct (246 Da) was 1 Da lower than would be predicted if a pentyl radical had simply added to MDL 101,002. In addition, the adduct exhibited significant absorbance at 304 nm, consistent with the unsaturated nitrone structure of MDL 101,002. To account for these observations, it is postulated that, after the initial capture of a pentyl radical, subsequent abstraction of a hydrogen atom by a neighboring radical occurs to regenerate a nitrone (1-pentyl analog of MDL 101,002). We present evidence for this adduct and offer a mechanism for its formation. These findings indicate that mass spectroscopic analysis of stable nitrone radical adducts may be useful in the identification of radical-dependent damage in vivo and possibly in clinical development of MDL 101,002 as an antioxidant pharmaceutical.

Simultaneous measurement of dolasetron and its major metabolite, MDL 74,156, in human plasma and urine.

A selective and sensitive analytical method for the simultaneous measurement of dolasetron (I) and its major metabolite, MDL 74,156 (II), in human plasma and urine samples has been developed using a structural analogue. MDL 101,858, as internal standard (I.S.). The compounds were extracted from plasma and urine using solvent extraction after the addition of the I.S. Chromatographic separation was carried out on a reversed-phase HPLC column and detection and quantification was by fluorescence with excitation and emission wavelengths of 285 and 345 nm, respectively. Linear responses were obtained over concentration ranges of 5 to 1000 pmol/ml for plasma samples and 20 to 1000 pmol/ml for urine samples with correlation coefficients for the calibration curves exceeding 0.999 in all cases. Intra-day and inter-day reproducibility yielded limits of quantification of 10 pmol/ml for I and 5 pmol/ml for II plasma and 50 pmol/ml for I and II in urine. The method has been applied to the simultaneous analysis of both compounds in plasma and urine samples coming from clinical pharmacokinetic studies.

Pharmacokinetics and pharmacodynamics of the acetylcholinesterase inhibitor 2,2,2-trifluoro-1-(3-trimethylsilylphenyl) ethanone in dog. Potential for transdermal patch delivery.

MDL 73,745 (2,2,2-trifluoro-1-(3-trimethylsilylphenyl) ethanone, CAS 132236(18-1) is a novel tight-binding inhibitor of acetylcholinesterase (AChE), which is in development as a potential therapeutic compound in the symptomatic treatment of Alzheimer's disease. Pharmacokinetics and pharmacodynamics of the compound were studied in the dog after single intravenous (i.v. 2 mg/kg), oral p.o. 10 mg/kg) and sub-cutaneous (s.c., 10 mg/kg) administrations of [14C]-MDL 73,745. Plasma concentrations of total radioactivity were much higher than those of parent drug after i.v., p.o. and s.c. administration, indicating extensive metabolism of the compound, although this was less after, s.c. administration than after p.o. administration. The bioavailability (F) was 34% after s.c. administration, compared with 4% after p.o. administration. The low bioavailability after p.o. administration was not due to poor drug absorption, as over 64% of the dose was absorbed. Pharmacokinetic parameters, calculated after i.v. administration, showed a terminal elimination half-life of 24 h, total body plasma clearance of around 70 ml/min/kg and apparent volume of distribution of 150 l/kg. AChE activity was almost 100% inhibited after i.v. administration, and over 80% inhibited 1 h after p.o. administration. In both cases, AChE activity returned to baseline levels by 12 h. AChE was around 80% inhibited 4 h after s.c. administration, and did not return to baseline levels until 36 h after drug administration. A combined pharmacokinetic-pharmacodynamic (PK-PD) effect model demonstrated that the extent of AChE inhibition could be correlated with plasma levels of the parent compound. As s.c. administration increased F, and led to longer AChE inhibition, transdermal (t.d.) delivery was assessed in the same animals. Patches, corresponding to a dose of 50 mg/kg, were applied to the shaved lateral abdominal skin for a period of 96 h. Sustained plasma concentrations of the parent drug were observed over the 96 h period of t.d. application. Mean (+/- SD) maximum plasma concentrations (Cmax) of 26.9 +/- 4.3 ng/ml were found 3.7 +/- 2.5 h after t.d. patch application und F was around 13%. AChE inhibition reached a maximum of 72% at 6 h after t.d. application and was still 35% at 96 h. The rate of release from the delivery system, per unit surface area, (ko) was calculated to be 7.7 micrograms/cm2. Transdermal delivery of MDL 73,745 thus decreased the important hepatic first-pass effect, and led to sustained plasma concentrations of drug, thus avoiding peaks and troughs which could lead to side-effects or poor efficacy.

Analysis of (R)-4-oxo-5-phosphononorvaline (MDL 100,453) in rat plasma and brain dialysate using liquid chromatography after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

A liquid chromatographic (LC) method with precolumn derivatization and fluorescence detection has been developed for the quantitation of (R)-4-oxo-5-phosphononorvaline (MDL 100,453), which is a selective antagonist of N-methyl-D-aspartate receptor, in rat plasma and brain dialysate. The plasma samples were deproteinized with acetonitrile and then derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The brain dialysis samples were dried in vacuum, reconstituted with borate buffer, and derivatized with AQC. The derivatized MDL 100,453 was analyzed by LC with a Nova-Pak C18 column at 32 degrees C using a gradient mobile phase. Detection was accomplished by fluorescence with excitation at 250 nm and emission at 395 nm. This analytical method was used to follow the time course of drug concentrations in rat plasma and brain dialysate after intravenous (i.v.) bolus injection of MDL 100,453 or a combination of i.v. bolus injection and i.v. infusion.

Photodynamic and radiolytic inactivation of ion channels formed by gramicidin A: oxidation and fragmentation.

Ion channels formed by the peptide gramicidin A in planar lipid membranes have been reported to react very sensitively upon irradiation of the membrane by ionizing radiation (radiolysis), by UV light (photolysis), or by visible light in the presence of appropriate photosensitizers (photodynamic inactivation). In all three cases the effect is due to the presence of the four tryptophan residues of the pentadecapeptide. Modifications of these amino acids--due to an interaction with free radicals formed upon water radiolysis or due to light absorption--have been found to reduce the membrane conductance by many orders of magnitude. The present study was intended to correlate functional changes, observed at the level of single ion channels, with changes of the molecular structure identified by mass spectrometry. About 98% of the inactivated channels showed a single-channel conductance of virtually zero, while about 2% of the channels present before irradiation are converted to a state of reduced conductance (and reduced lifetime). On the structural level, irradiation in the presence of the photosensitizer Rose Bengal was found to produce oxidation and fragmentation of the peptide at the positions of the tryptophan residues. Our results provide evidence that the main effect of radiolysis, or of photodynamic treatment, is the cleavage of the peptide backbone leading to immediate closure of an open ion channel.

Metabolism and pharmacokinetics of the cardiotonic agent piroximone and of its major metabolite in dog.

1. Piroximone was administered orally (p.o.) and intravenously (i.v.) to male Beagle dog. In vitro, piroximone was incubated with dog liver microsomes. 2. Piroximone was metabolized in vivo to five metabolites (1-5) representing approximately 20% of the total administered dose. 3. The parent drug and its metabolites were totally eliminated in urine. 4. Reduced piroximone (piroximole), representing approximately 10% of the administered dose, was identified as the major metabolic product in vivo. 5. In vitro, piroximone was metabolized by dog liver microsomes to isonicotinic acid (1) and piroximole (4), with the same ratio as in vivo (1:4 = 0.2). The Michaelis-Menten parameters were determined for piroximole formation and were: Kmapp = 733 microM and Vmax app = 232 pmol/mg protein/min. 6. Comparison of the pharmacokinetics of piroximone and piroximole revealed that both compounds were very well absorbed (F = 93 +/- 7 and 89 +/- 8% respectively), slightly distributed (Vd app = 0.78 +/- 0.04 and 1.02 +/- 0.09 l/kg p.o., and 0.95 +/- 0.05 and 0.76 +/- 0.13 1/kg i.v. respectively) and excreted into urine to the same extent (UEx = 54.7 +/- 1.2 and 53.2 +/- 12.6% p.o., and 59.1 +/- 5.3 and 51.2 +/- 5.7% i.v. respectively), except that the clearance of piroximone was two-fold higher than that observed for piroximole (ClT = 7.77 +/- 1.35 and 4.12 +/- 0.44 ml/min/kg p.o., and 7.68 +/- 1.25 and 4.06 +/- 0.51 ml/min/kg i.v. respectively).

Selectivity of MDL 72,974A for MAO-B inhibition based on substrate and metabolite concentrations in plasma.

Plasma concentrations of 3,4-dihydroxyphenylethylglycol (DOPEG), noradrenaline (NA), adrenaline (A), 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), dopamine (DA) and phenylethylamine (PEA) were analyzed in samples taken prior to, during and following the administration of single, daily doses of 12 or 24 mg MDL 72,974A to healthy male volunteers. No effects on the concentrations of DOPA, A, DA or DOPAC were seen during the administration of either dose over 10 days. No treatment-related changes in the concentration of NA were evident at either dose. No changes in DOPEG or PEA concentrations were seen with the 12 mg dose; however, small but significant decreases in plasma DOPEG concentrations and a significant increase in PEA were seen during the administration of the 24 mg dose. This would suggest that at the 24 mg dose some intraneuronal inhibition of MAO-A may be occurring although the lack of increases in NA and A concentrations indicates no accompanying change in sympatho-adrenal activity. Plasma PEA concentrations do not provide a more sensitive or functional indication of MAO-B inhibition. The increase in PEA concentrations at the higher dose may suggest that the inhibition of both forms of the enzyme is necessary to increase its plasma concentration.

pNAT and CYP2D6 gene polymorphism in epileptic patients.

Certain anticonvulsant drugs require N-acetylation as a major route of metabolic clearance. Single point mutations of the polymorphic N-acetyltransferase gene (pNAT) are the primary cause for impaired drug acetylation. Pharmacokinetic parameters are altered in slow acetylator phenotypes and this may compromise drug safety. Genetic analysis of allelic frequencies of individual pNAT genotypes point to significant increases in carriers of the S1/wt and S3/wt (P < 0.05) allele and a significant reduction in carriers of the S2/S2 (P < 0.01) allele, when control and epileptic patients are compared. Furthermore, the presumed link between the cytochrome P450 CYP2D6 polymorphism and the pathogenesis of Parkinson's disease led us to investigate, whether a similar relationship can be expected for other CNS disorders. Our findings indicate that poor metabolizers are more frequent (P < 0.05) amongst epileptic patients, when compared with a control population. An estimate of the odds ratio may suggest an increased risk [95% CI (confidence interval) 1.043-4.734] of up to 5-fold in epileptic patients carrying this mutation. This provides further evidence for a potential link between the debrisoquine hydroxylase gene polymorphism and CNS disorder and therefore warrants further study.

Simultaneous measurement of the major metabolites of dolasetron mesilate in human urine using solid-phase extraction and high-performance liquid chromatography.

A method based on solid-phase extraction and high-performance liquid chromatography (HPLC) has been developed for the simultaneous quantitation of the principal active metabolites of dolasetron mesilate [i.e. MDL 74,156 (II), MDL 102,382 (III) and MDL 73,492 (IV)] in human urine. The method has been validated over the concentration range of 200-5000 pmol/ml for all three metabolites. Within-day and day-to-day coefficients of variation were less than 9 and 14%, respectively, for the three metabolites. The method allowed the simultaneous quantitation of III, IV and II and the evaluation of the urinary excretion of these metabolites in human urine following the administration of dolasetron mesilate.

Kinetics and metabolism of p-tyramine during monoamine oxidase inhibition by mofegiline.

The effects of monoamine oxidase B (MAO-B) inhibition by mofegiline on the pharmacokinetics of p-tyramine and its major metabolite, p-hydroxyphenylacetic acid, were investigated in 24 healthy male volunteers. p-Tyramine doses were administered before and after a 14-day treatment period of 1, 12, or 24 mg mofegiline or placebo. Normalized p-tyramine for area under the plasma concentration-time curve after treatment were not significantly different from their respective before-treatment values for any of the dose groups. The relative bioavailability of p-tyramine after treatment was not significantly different from before treatment, although a tendency to a greater bioavailability was seen with the 12 and 24 mg doses. There were no significant differences between pharmacokinetic parameters for p-hydroxyphenylacetic acid. The data suggest that mofegiline maintains its selectivity for MAO-B in the intestine and liver at doses up to and including 24 mg. Therefore these doses would not be expected to be associated with the hypertensive crises normally associated with the "cheese effect."

Novel carbamate metabolites of mofegiline, a primary amine monoamine oxidase B inhibitor, in dogs and humans.

Mofegiline or MDL 72,974A ((E)-4-fluoro-beta-fluoromethylene benzene butanamine hydrochloride) is a selective enzyme-activated irreversible inhibitor of monoamine oxidase B, which is under development for use in the treatment of Parkinson's disease. Male beagle dogs were given single p.o. (20 mg/kg) and i.v. (5 mg/kg) doses of [14C]-Mofegiline. Total radioactivity excreted in urine and feces over 96 hr was, respectively, 75.5 +/- 3.8 and 6.3 +/- 3.4% of the dose after p.o. and 67.9 +/- 0.5 and 3.9 +/- 2.4% after i.v. administration. Unchanged drug in urine represented 3% of the dose after po and less than 1% after i.v. administration. Mofegiline was thus extensively metabolized in dogs, and urinary excretion was the major route of elimination of metabolites. HPLC, with on-line radioactivity detection, showed the presence of four major peaks (M1, M2, M3, and M4), representing respectively 50, 9, 5, and 0.5% of the administered dose excreted in 0-24 hr urine. TSP-LC-MS, FAB-MS, and NMR spectra of the purified metabolites were obtained. M1, the major metabolite in dogs, was shown to have undergone defluorination of the beta-fluoromethylene moiety, and one carbon addition. Its structure was confirmed to be a cyclic carbamate. M2 was a N-carbamoyl O-beta-D-glucuronide conjugate of parent drug. The formation of M1 and M2 is likely to involve initial reversible addition of CO2 to the primary amine function. M3 was a N-succinyl conjugate of the parent drug. M4 had also undergone defluorination to yield a urea adduct of an unsaturated alpha, beta aldehyde. Structures of M1 and M3 were further confirmed by comparing their MS and NMR spectra with those of authentic reference compounds. TSP-LC-MS ion chromatograms of human urine, obtained from two male volunteers after p.o. administration of 24 mg of drug, showed selected molecular ion peaks with the same retention time as the metabolites identified in dogs. In humans, these common metabolites represented a similar percentage of the administered dose to that in dogs. The present study demonstrates that NMR, TSP-LC-MS are complementary analytical techniques, which allow structural identification of unhydrolyzed drug conjugates. The formation of carbamates of amine-containing drugs may be more common than previously reported.