Collaborative study for the validation of an improved HPLC assay for recombinant IFN-alfa-2.

The current European Pharmacopoeia (Ph. Eur.) texts for Interferon (IFN)-alfa-2 include a nonspecific photometric protein assay using albumin as calibrator and a highly variable cell-based assay for the potency determination of the protective effects. A request was expressed by the Official Medicines Control Laboratories (OMCLs) for improved methods for the batch control of recombinant interferon alfa-2 bulk and market surveillance testing of finished products, including those formulated with Human Serum Albumin (HSA). A HPLC method was developed at the Medical Products Agency (MPA, Sweden) for the testing of IFN-alfa-2 products. An initial collaborative study run under the Biological Standardisation Programme (BSP; study code BSP039) revealed the need for minor changes to improve linearity of the calibration curves, assay reproducibility and robustness. The goal of the collaborative study, coded BSP071, was to transfer and further validate this improved HPLC method. Ten laboratories participated in the study. Four marketed IFN-alfa-2 preparations (one containing HSA) together with the Ph. Eur. Chemical Reference Substance (CRS) for IFN-alfa-2a and IFN-alfa-2b, and in-house reference standards from two manufacturers were used for the quantitative assay. The modified method was successfully transferred to all laboratories despite local variation in equipment. The resolution between the main and the oxidised forms of IFN-alfa-2 was improved compared to the results from the BSP039 study. The improved method even allowed partial resolution of an extra peak after the principal peak. Symmetry of the main IFN peak was acceptable for all samples in all laboratories. Calibration curves established with the Ph. Eur. IFN-alfa-2a and IFN-alfa-2b CRSs showed excellent linearity with intercepts close to the origin and coefficients of determination greater than 0.9995. Assay repeatability, intermediate precision and reproducibility varied with the tested sample within acceptable ranges. Test accuracy estimated by comparing the values obtained by the participants to the declared contents determined by the manufacturers was good despite the absence of a common reference preparation. In conclusion, the present study showed that the new method is suitable, reproducible and transferable. Proposals for the revision of Ph. Eur. texts are presented.

Collaborative study for establishment of an HPLC-method for batch consistency control of recombinant interferon-alfa-2.

For interferon-alfa-2 (IFN-alfa-2) finished products a simple potency assay for batch consistency control or market surveillance studies is lacking. The European Pharmacopoeia monograph "Interferon alfa-2 concentrated solution" applies to bulk product and requires a cell culture potency assay which is neither straightforward nor are its precision and interlaboratory variability satisfying. Thus a project was initiated within the Biological Standardisation Programme to establish an HPLC assay for batch potency testing of recombinant IFN-alfa-2 finished products. In the collaborative study the suitability and transferability of an HPLC method developed in the project leaders' laboratory was checked. Twelve laboratories participated in the study. Based on a standardised method, calibration curves were established using the European Pharmacopoeia reference substances for IFN-alfa-2a and IFN-alfa-2b. The resolution between IFN-alfa and the oxidised forms of IFN-alfa was evaluated. Three marketed IFN-alfa preparations (one containing albumin) with known IFN-alfa content were used for the quantitative assay. The reproducibility of the method was not satisfactory but appears improvable with a slight modification of the method. Most laboratories succeeded in separating oxidised IFN from the main IFN peak. Five laboratories achieved very good linearity of the calibration curves while poor linearity was observed in three, mainly due to flattening of the curve at the lowest concentrations. The intercepts of the calibration curves were negative in all cases which might be due to a substantial degree of adsorption of IFN on glass and plastic surfaces. The robustness of the method concerning the choice of chromatographic column seems to be low. Using a corresponding column from a different manufacturer instead of the one proposed resulted in a loss of resolution between oxidised IFN and the main peak. Even the use of different batches of the prescribed column seemed to influence the result. The repeatability for the four consecutive injections of the three commercial IFN products does not indicate a systematic methodological error. The reproducibility (variability between laboratories) of the originally proposed method was large. Thus, the originally proposed method appears less suitable. However, the values obtained with a modified technique were less variable; in addition, an improvement of peak symmetry was obtained. Based on the results obtained with the original and the adapted method, a follow-up study with a modified technique (e.g. changes in liquid phase, application of system suitability criteria) is proposed.

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. XIII. Enzyme interactions with a series of beta-alkyl-substituted 2-phenylethanamines and corresponding N-hydroxylamines.

The formation of Metabolic Intermediate (MI) complexes from a series of beta-alkylsubstituted 2-phenylethanamines and corresponding N-hydroxylamines is investigated during NADPH-dependent metabolism in liver microsomes from phenobarbital pretreated rats. The beta-alkyl substituents are methyl, dimethyl, ethyl, di-ethyl, n-propyl, di-n-propyl and i-propyl groups. The amines are synthesized by LiAlH4-reduction of the corresponding nitriles, which are prepared through alkylation of the enolate anion of phenylacetonitrile. The hydroxylamines are prepared either by oxidation of the corresponding benzylimines with m-chloroperbenzoic acid and subsequent hydrolysis of the initially formed 3-phenyloxaziridines, or by H2O2-mediated oxidation of the corresponding amines in the presence of catalytic amounts of sodium tungstate, followed by reduction with cyanoborohydride. The amines are found to be completely devoid of complexing activity, while the hydroxylamines form the MI complex at high rates. Complex formation from these substrates thus parallels the known behaviour of 2-phenylethanamine and its corresponding N-hydroxylamine. Since N-oxygenation is known to be a prerequisite for MI complex formation from amines our results suggest that the beta-alkylated 2-phenylethanamines are metabolized exclusively through other pathways. In accordance with this hypothesis, capillary GC-analysis of the incubation mixture of 2-phenylpropanamine shows no formation of N-hydroxylated metabolites; only 2-phenylpropanol, a metabolite formed through the deamination pathway, is found.

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. XII. Enantioselectivity and temperature dependence in microsomes and reconstituted cytochrome P-450 systems from rat liver.

Formation of metabolic intermediate (MI) complexes was studied with the enantiomers of amphetamine, 1-phenyl-2-pentanamine, N-hydroxyamphetamine, and 2-nitroso-1-phenylpropane (the C-nitroso analogue of amphetamine). Three different enzyme systems were used; liver microsomes from phenobarbital pretreated rats and two reconstituted systems containing the P450 2B1 and P450 2C11 forms of cytochrome P-450. Enantioselective complex formation in microsomes was shown for the amines and the nitroso compound, but not for the hydroxylamine. The highly purified P450 2B1 system formed the MI complex with all substrates tested, and the enantioselectivity observed with the microsomal system was reproduced. In the P450 2C11 system the nitroso compounds were completely inactive, whereas the enantiomers of N-hydroxyamphetamine still produced the complex at a high rate. Changes in temperature were shown to affect (R)-2-nitroso-1-phenylpropane more than its enantiomer. Both enantiomers showed biphasic Arrhenius plots for MI complex formation in microsomes (breaks around 22 degrees C), but the activation energies of the (R)-isomer were about five times higher than those of the (S)-isomer. A theory is presented which suggests different modes of interaction with the active site of P-450 to account for the different behaviour of the various substrates.

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. XI. Peroxygenase versus monooxygenase function of cytochrome P-450 in rat liver microsomes.

Cytochrome P-455 nm complex formation in phenobarbital induced rat liver microsomes was investigated using both an NADPH/O2-dependent monooxygenase system and a peroxygenase/peroxidase system where hydrogen peroxide was substituted for NADPH. The substrates tested were the enantiomers of four 1-alkyl-substituted 2-phenylethanamines (unbranched 1-alkyl substituents, comprising one to four carbons), S(+)- and R(-)-N-hydroxyamphetamine and racemic mixtures of N-hydroxy-1-phenyl-2-butanamine and N-hydroxy-3-methyl-1-phenyl-2-butanamine. During NADPH/O2-dependent metabolism the amines showed a positive correlation between extent of complex formation and lipophilicity; furthermore the S(+)-isomers gave rise to larger amounts of complex than the corresponding R(-)-analogues. With the hydroxylamines the ability to form complexes was greater than with any of the amines but no definite difference was seen among the hydroxylamines. In the peroxygenase system the hydroxylamines still gave larger amounts of complex than the amines but the differences seen within the homologous series of chiral amines when using the monooxygenase system were no longer observed. Although the quantitative trends in complex formation seen in the monooxygenase system were non-existent when H2O2 was substituted for NADPH, mere qualitative rules still seemed to apply; substrates which failed to give the complex during NADPH-dependent metabolism (2-phenylethanamine, phentermine, N-hydroxyphentermine and phenylacetone oxime) were inactive also in the peroxygenase system. The results substantiate the notion that the monooxygenase and peroxygenase reaction mechanisms of cyt. P-450 follow similar but not identical pathways.

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. IX. A comparative study with enantiomeric 2-nitroso-1-phenylpropane in microsomes and a reconstituted cytochrome P-450 system from rat liver.

Formation of cytochrome P-455 nm complexes was investigated with enantiomeric 2-nitroso-1-phenylpropane--the C-nitroso analogue of amphetamine--and optically active N-hydroxyamphetamine, in the presence of NADPH. For comparative reasons, three different drug-metabolizing enzyme systems were used, namely microsomes from control and phenobarbital-treated rats, and a reconstituted system containing the main phenobarbital-induced form of cytochrome P-450 from rat liver. In microsomes obtained from phenobarbital-treated rats, pronounced differences in the kinetics of complex formation were observed between the enantiomeric C-nitroso compounds, but not between the isomers of N-hydroxyamphetamine. In the reconstituted enzyme system the S-nitroso compound formed the P-455 nm chromophore at the highest initial rate, while the R analogue was devoid of complexing activity. The rates of complex formation from the N-hydroxylamine enantiomers were high and equal.

Increased levels of several retinoid binding proteins resulting from retinoic acid-induced differentiation of F9 cells.

The embryonal carcinoma cell line F9 is known to differentiate when exposed to retinoic acid. We have examined the quantities of two intracellular retinoid-binding proteins in undifferentiated and differentiated F9 cells. The existence of a cell surface receptor that recognizes the plasma retinol-binding protein was also explored. It was shown that undifferentiated F9 cells contain low concentrations of the two retinoid-binding proteins. The cellular retinoic acid-binding protein was present in approximately 3-fold molar excess over the cellular retinol-binding protein. Upon culture in the presence of retinoic acid, F9 cells display elevated concentrations of both cellular retinol-binding protein and cellular retinoic acid-binding protein. Since the levels of beta 2-microglobulin, a marker of the differentiated state with no known involvement in the metabolism of vitamin A, increased in parallel with the retinoid-binding proteins, it seems unlikely that retinoic acid selectively increased the levels of the two retinoid-binding proteins. The differentiated, in contrast to the undifferentiated cells, can accumulate retinol from plasma retinol-binding protein and display a cell surface receptor for this protein. Despite the fact that retinoic acid-induced differentiation of F9 cells promotes increased levels of several proteins involved in the normal metabolism of vitamin A, no evidence was obtained to suggest that the cells were dependent on retinoids to maintain their differentiated state.

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. 8. Stereoselectivity in metabolic intermediary complex formation with a series of chiral 2-substituted 1-phenyl-2-aminoethanes.

The formation of cytochrome P-450 metabolic intermediary (MI) complexes from the enantiomers of four 2-alkyl-substituted 1-phenyl-2-aminoethanes was investigated during reduced nicotinamide adenine dinucleotide phosphate (NADPH) dependent metabolism in liver microsomes from phenobarbital-pretreated rats. The 2-alkyl substituents were methyl (amphetamine), ethyl, n-propyl, and n-butyl groups. The chiral amines were prepared from the corresponding alkyl benzyl ketones by asymmetric hydrogenolytic transamination. Circular dichroism analysis showed that all the amines possessed the S-(+) and R-(-) configuration. The maximal velocity (Vmax(obsd) ) of complex formation increased with increasing size of the alkyl group, and for each series of enantiomers a good correlation was obtained between log Vmax(obsd) and the logarithm of the octanol/buffer partition coefficient of the substrates. With increasing lipophilicity, the S-(+) enantiomers became more active than the R-(-) isomers in generating the complex. The rates of complex formation for the faster S-(+) enantiomers coincided with those of the previously investigated racemates, indicating that the R-(-) enantiomers do not act as competitive enzyme inhibitors in the rat liver preparations. In agreement with two previous studies, the results from the present investigation establish a stereoselectivity in cytochrome P-450 MI complex formation by 1-phenyl-2-aminoethanes. However, detection of such differences are dependent on the intrinsic activity of the compound.

Structure and tissue distribution of some retinoid-binding proteins.

Vitamin A has, apart from its function in the visual pigments, general effects on several organs. Early signs of vitamin A deficiency include keratinization of epithelia and hyperkeratosis of the skin. To elucidate a generalized function for vitamin A, we have taken the approach of tracing the vitamin from its storage site in the liver via its blood transport by the retinol-binding protein (RBP) to its uptake by susceptible cells. We have also examined the intracellular occurrence of vitamin A as regards its binding to specific receptor proteins. Here we summarize data on the amino acid sequences of several vitamin A-binding proteins. The finding that CRBP and CRABP, the two intracellular proteins, are homologous to each other, to a myelin protein, and to a fatty acid-binding protein may shed light on the functions of these proteins. Retinoic acid, which binds to CRABP but not CRBP, induces differentiation of teratocarcinoma cells. This is accompanied by a lowering of the CRABP concentration, an increase of the CRBP level, and an increase in the uptake of retinol from RBP. The epidermis contains both CRBP and CRABP, and their distributions are rather similar. However, in contrast to CRBP, CRABP is most abundant in cells lining the hair follicles. CRBP occurs in greatest relative amounts in the outer layers of the epidermis. Since techniques have been developed to measure CRBP and CRABP, normal and disease-affected skin may now be explored as to quantity and cellular distribution of the retinoid-binding proteins.

Cytochromes P-450 LM3b and LM4 in biosynthesis of bile acids.

Cytochromes P-450 LM3b and LM4 were prepared from untreated and cholestyramine-treated rabbits. The catalytic properties of these cytochrome P-450 fractions towards substrates in bile acid biosynthesis were studied in reconstituted systems containing NADPH -- cytochrome P-450 reductase and phospholipid. Cytochrome P-450 LM3b showed no hydroxylase activity towards cholesterol and only low activity towards some other C27-steroids whereas it catalyzed efficient hydroxylation of testosterone and demethylation of ethylmorphine. Preparations of cytochrome P-450 LM4 catalyzed hydroxylation of cholesterol and other C27-steroids more efficiently than microsomes. Cytochrome b5 had no stimulatory effect on the C27-steroid hydroxylase activities.