Isolation and identification of the C6-hydroxy and C20-hydroxy metabolites and glucuronide conjugate of methylprednisolone by preparative high-performance liquid chromatography from urine of patients receiving high-dose pulse therapy.

In the present study metabolites of methylprednisolone were detected using gradient elution high-performance liquid chromatography. Separation was performed by a Cp Spherisorb ODS 5 microm (250 mmx4.6 mm I.D.) column, connected to a guard column, packed with pellicular reversed phase. The mobile phase was a mixture of acetonitrile and 1% acetic acid in water. At t = 0, this phase consisted of 2% acetonitrile and 98% acetic acid 1% in water (v/v). During the following 35 min the phase changed linearly until it attained a composition of acetonitrile-buffer (50:50, v/v). At 40 min (t = 40) the mobile phase was changed over 5 min to the initial composition, followed by equilibration during 2 min. The flow-rate was 1.5 ml/min. UV detection was achieved at 248 nm. We have isolated the respective compounds with the most abundant concentration and suggested their chemical structure based on NMR, IR, UV, MS, retention behaviour and melting points. The c/, stereochemistry could not be solved in this study. The overall picture of the metabolic pathways of methylprednisolone is apparently simple: reduction of the C20 carbonyl group and further oxidation of the C20-C21 side chain (into C21-COOH and C20-COOH), in competition with or additional to the oxidation at the C6-position.

Prediction of the in vivo biological activity of human recombinant follicle stimulating hormone using quantitative isoelectric focusing.

Currently, the in vivo biopotency of commercial preparations containing the glycoprotein follicle-stimulating hormone (FSH) is declared on the basis of the ovarian weight augmentation assay as described in the British, European and United States Pharmacopoeias. Human FSH contains approximately 35% (W/W) carbohydrate which introduces considerable microheterogeneity (isohormones). Analysis of isohormones of recombinant FSH has revealed a relation between the isoelectric point (pl) and the in vivo bioactivity. Isohormones in the range of pl 3.5 are 100- to 200-fold more potent in the in vivo bioassay than isohormones with a pl of 5.5-6.0. These data suggest that quantification of the isohormone profile should enable us to predict the in vivo bioactivity. Thus, isohormones of recFSH samples were separated by isoelectric focusing (IEF), visualized by Coomassie brilliant blue G250 staining and quantified using densitometry. The data from 21 samples were compared with the in vivo bioassay data using partial least square (PLS) regression. A close correlation was found using a model with 2 PLS factors (correlation coefficient (r)=0.95, standard error of estimation SD 1. 02 lU/microg protein). In addition, ordinary least square (OLS) regression revealed a similar correlation between the fraction of isohormones between pl 3.9 and 4.9 and the in vivo bioactivity: r=0. 95, sd=1.4 lU/microg protein. Thus, an increase in the acidic isohormone fraction results in an increase in the in vivo bioactivity. The reverse is true for the amount of isohormones focusing between pl 5.1 and 5.7. An increase of this fraction results in a decrease of the in vivo bioactivity. These data are consistent with what might be expected from the in vivo bioassay data of the isohormones. The OLS model was subsequently validated using the guidelines of the European Centre for Validation of Alternative Methods (ECVAM) using 10 samples of recFSH that had not been used for the calibration. The relative standard deviation (RSD) of the mean difference between experimental and predicted in vivo bioactivity was approximately 6%. A Student's t-test performed on the experimental and predicted bioactivity data indicated that the predicted bioactivities do not deviate significantly from the experimental in vivo bioactivity data (P<0.05). These results demonstrate that the IEF scanning data can be used to predict the in vivo bioactivity with reasonable accuracy. This may be the first step towards replacing the in vivo bioassay for highly purified FSH by a physicochemical alternative. In general, quantitative charge-based separation methods like chromatofocusing, high performance capillary electrophoresis and ion exchange chromatography may also be considered as alternatives. Finally, quantitative charge profiling may prove to be as important for the estimation of the potency of other therapeutic glycoproteins like luteinizing hormone (LH), chorionic gonadotropin (CG), thyroid-stimulating hormone (TSH) and erythropoietin (EPO).

Recombinant rat luteinizing hormone; production by Chinese hamster ovary cells, purification and functional characterization.

Rat recombinant (rec) luteinizing hormone (LH) was produced in Chinese hamster ovary (CHO) cells, to enable studies on LH physiology in this species with homologous hormone. The synthesized hormone was purified, and characterized physico-chemically and biologically in comparison with highly purified preparations of rat pituitary (pit) LH (NIDDK-rLH-I-7 and I-9) and to highly purified urinary (NIH, CR-121) and rec forms of human chorionic gonadotropin (hCG). The 33 kD molecular mass of rat recLH, as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, was comparable with the 32 kD size of pitLH. In chromatofocusing, the isoforms of rat recLH distributed in the pI range 6.5-7.8, similar to rat pitLH. In receptor binding assays using rat testicular membranes, and physiologic salt concentration, rat recLH displayed a 5-10-fold higher affinity than rat pitLH, but about 100-fold lower affinity than hCG. In contrast, in low salt concentrations the affinities of rat recLH and rechCG to rat LH receptor were rather similar. The differences in potency in the mouse Leydig cell in vitro bioassay were in agreement with the receptor binding data at physiologic salt concentration. Neither rat recLH nor pitLH stimulated cAMP production or bound specifically to HEK 293 cells expressing the rec human LH receptor. When injected subcutaneously on four consecutive days to male rats (8.4-33.7 microg/rat/day) rat recLH did not induce seminal vesicle growth in comparison with a significant effect of human menopausal gonadotropin (hMG; 12.5-50 IU/rat/day). In contrast, ovulation was induced in 5/6 and 6/6 female rats following single injections of 3.75 and 7.5 microg of rat recLH, respectively, after pretreatment with 10 microg/kg of a GnRH-antagonist (Org 30850). In conclusion, rat recLH displays clearly lower in vivo and in vitro bioactivity than hCG. Nevertheless, it binds effectively to the rat LH receptor (with affinity dependent on salt concentration) and is bioactive in the mouse Leydig cell bioassay. This newly synthesized recombinant hormone provides a useful tool for further studies on the physiology of LH action in the rat, the most common animal model in reproduction research.

Recombinant rat follicle-stimulating hormone; production by Chinese hamster ovary cells, purification and functional characterization.

In order to obtain homologous follicle-stimulating hormone (FSH) for in vivo and in vitro studies in the rat, rat recombinant (rec) FSH was produced in Chinese Hamster Ovary (CHO) cells. The synthesized rat recFSH was purified and subjected to physico-chemical and biological characterization. including a comparison with two rat pituitary (pit) and reference preparations (NIDDK-rFSH I-8 and NIDDK-rFSH-RP2) as well as with human recFSH (Org 32489). The molecular masses of rat recFSH and human recFSH were determined by SDS-polyacrylamide (SDS-PAGE) and were found to be similar, about 40 kD. The pI distribution of rat recFSH is similar to rat pitFSH, and slightly more acidic than human recFSH (3.6-5.6 vs 3.9-5.5, respectively) as determined by isoelectric focussing in immobilized pH gradients. Rat recFSH displayed dose-response curves parallel and in the same dose range as the rat pitFSH in receptor binding and in vitro bioassays. However, the in vivo activities of rat recFSH and rat pitFSH were 8824 and 3051 IU/mg, respectively, determined by the Steelman Pohley assay. Rat (pit and rec) and human FSH are very different. Human recFSH bound to both calf testicular membranes and CHO cells expressing the human FSH receptor (CHO hFSH-R) with about 10-fold higher affinity (Ka) than pituitary and recombinant rat FSH. In in vitro bioassays with immature rat Sertoli cells and CHO hFSH-R cells human recFSH was also about 10-fold more potent than the rat FSH preparations. In the in vitro bioassays with immature rat granulosa cells the difference was about 5-10-fold. These studies indicate that the receptor binding and in vitro activities of rat pitFSH and rat recFSH are similar. The differences in in vivo activity are probably due to the differences in glycosylation. The biological behaviour of rat FSH (pit and rec) is different from that of human FSH. Therefore, if the rat is used as a model for physiology of gonadotropic action, the results may be greatly influenced by the type (species) of hormone preparation used. The availability of homologous hormone preparations is therefore crucial.

Isolation, identification and determination of sulfadiazine and its hydroxy metabolites and conjugates from man and rhesus monkey by high-performance liquid chromatography.

The following metabolites of sulfadiazine (S) were isolated from monkey urine by preparative HPLC: 5-hydroxysulfadiazine (5OH), 4-hydroxysulfadiazine (4OH) and the glucuronide (5OHgluc) and sulfate conjugate of 5OH (5OHsulf). The compounds were identified by NMR, mass and infrared spectrometry and hydrolysis by beta-glucuronidase. The analysis of S, the hydroxymetabolites (4OH, 5OH) and conjugates N4-acetylsulfadiazine (N4), 5OHgluc and 5OHsulf in human and monkey plasma and urine samples was performed using reversed-phase gradient HPLC with UV detection. In plasma, S and N4 could be detected in high concentrations, whereas the other metabolites were present in only minute concentrations. In urine, S, the metabolites and conjugates were present. The limit of quantification of the compounds in plasma varies between 0.2 and 0.6 microgram/ml (S 0.31, N4 0.40, 4OH 0.20, 5OH 0.37, 5OHgluc 0.33 and 5OHsulf 0.57 microgram/ml). In urine it varies between 0.6 and 1.1 micrograms/ml (S 0.75, N4 0.80, 4OH 0.60, 5OH 0.80, 5OHgluc 0.80 and 5OHsulf 1.1 micrograms/ml). The method was applied to studies with healthy human subjects and Rhesus monkeys. The metabolites 5OH, 5OHgluc and 5OHsulf were present in Rhesus monkey and not in man. Preliminary results of studies of metabolism and pharmacokinetics in Rhesus monkey and man are presented.

Isolation, identification and determination of sulfamethoxazole and its known metabolites in human plasma and urine by high-performance liquid chromatography.

From human urine the following metabolites of sulfamethoxazole (S) were isolated by preparative HPLC: 5-methylhydroxysulfamethoxazole (SOH), N4-acetyl-5-methylhydroxysulfamethoxazole (N4SOH) and sulfamethoxazole-N1-glucuronide (Sgluc). The compounds were identified by NMR, mass spectrometry, infrared spectrometry, hydrolysis by beta-glucuronidase and ratio of capacity factors. The analysis of S and the metabolites N4-acetylsulfamethoxazole (N4), SOH, N4-hydroxysulfamethoxazole (N4OH), N4SOH, and Sgluc in human plasma and urine samples was performed with reversed-phase gradient HPLC with UV detection. In plasma, S and N4 could be detected in high concentrations, while the other metabolites were present in only minute concentrations. In urine, S and the metabolites and conjugates were present. The quantitation limit of the compounds in plasma are respectively: S and N4 0.10 micrograms/ml; N4SOH 0.13 micrograms/ml; N4OH 0.18 micrograms/ml; SOH 0.20 micrograms/ml; and Sgluc 0.39 microgram/ml. In urine the quantitation limits are: N4 and N4OH 1.4 micrograms/ml; S 1.5 micrograms/ml; N4SOH 1.9 micrograms/ml; SOH 3.5 micrograms/ml; and Sgluc 4.1 micrograms/ml. The method was applied to studies with healthy subjects and HIV positive patients.