Sustained release of injectable zinc-recombinant hirudin suspensions: development and validation of in vitro release model.

In humans, recombinant hirudin (rHir), an anticoagulant protein, has a relatively short half-life (about 1 h). Therefore, a rHir formulation with sustained biological activity was previously proposed to result from complexing zinc salts and rHir (Zn-rHir). The purpose of this paper is to introduce and validate an in vitro release model for subcutaneous Zn-rHir formulations. In glass vials the formulations were suspended in agarose gel (2%) and coated with an extra layer of protein-free agarose. The agarose layers were covered with receiver solution, either buffered solutions (HEPES or PBS, pH 7.4) or human serum. To validate the release model and to demonstrate its potential to discriminate between different formulations, several commercial insulin and Zn-insulin formulations were also tested. The release profiles were evaluated by statistical moment analysis (mean times). Only in HEPES buffer was good discrimination between the investigated insulin formulations observed. The mean times of in vitro release of the insulin formulations and the proposed duration of their biological activities were in correlation. Low discrimination was found in PBS. For rHir, clear discrimination between the investigated rHir formulations was achieved in HEPES buffer, whereas low discrimination was found in PBS or in serum. The developed release model may be a sensitive in vitro test to assure the quality of subcutaneous insulin and rHir formulations, and may also be applicable to assess other slow-release protein and low molecular weight drug injectables.

Immunoassays in monitoring biotechnological drugs.

For the evaluation and interpretation of pharmacokinetic data reliable quantitative determinations are a requirement that can only be met by well-characterized and fully validated analytical methods. To cope with these requirements a method is being established that is based on an integrated and automated fiber-optic biospecific interaction analysis system (FOBIA) for immunoassays. Performance characteristics of this system used in monitoring of recombinant hirudin (CGP 39 393) are presented. Recombinant hirudin is a highly potent and selective inhibitor of human thrombin. Owing to its size and charge, recombinant hirudin is mainly eliminated by glomerular filtration. But only a fraction of the hirudin dose seems to be reabsorbed at the proximal tubule by luminal endocytosis and hydrolyzed by lysosomal enzymes, leaving approximately 50% of the dose to be extracted in the urine. Thus, renal clearance of recombinant hirudin in the absence of renal insufficiency appears to depend primarily on the glomerular filtration rate. During a 3-month i.v. tolerability study in dogs, some of the dogs developed antibodies against recombinant hirudin. The hirudin-antibody complex accumulated in plasma and apparent hirudin plasma concentrations were therefore much higher than expected from single-dose kinetics. Hirudin captured by antibodies showed an extended half-life and the hirudin-antibody complex is still pharmacologically active, as demonstrated by the observed increase in thrombin time. In conclusion, only appropriate analytical methods allow adequate monitoring and pharmacokinetic characterization of biotechnology drugs in biological materials.

[Gas chromatographic analysis of cannabinoids on tandem columns]. / Kannabinoidok gázkromatográfiás vizsgálata tandem oszlopokon.

A simple, isotherm packed column gas chromatographic method was developed for the quantitative determination of neutral cannabinoids using 4-androstene-3,17-dion as internal standard. In order to achieve the best resolution and to avoid the evaluation of the disturbing hydrocarbon peaks a method was developed using "tandem" column made of 3% OV-1 (90%) and 3% OV-17 (10%) stationary phases. The psychotropic cannabinoids delta 1-tetrahydrocannabinol (delta 1-THC) and delta 1(6)-tetrahydrocannabionol (delta 1(6)-THC), as well as, their main metabolites cannabidiol (CBD) and cannabichromene (CBC) were baseline separated except the cannabigerol (CBG) and cannabinol (CBN) pair, however for these compounds the separation was also satisfactory for the quantitative determinations. The Kováts retention indices were calculated for the most important cannabinoids and established the detection limits, respectively (20-50 ng range). The reproducibility was found excellent cv% = 1.06 for delta 1-THC and the analysis time was 55 minutes. The practical usefulness of the method was demonstrated by the comparative analyses on hashish- and fibre type hemps.

Sandwich immunoassay for the hapten angiotensin II. A novel assay principle based on antibodies against immune complexes.

Immunoassays for haptens such as short peptides or drugs are usually based on the principle of competition for a limited number of binding sites on antibody molecules. Owing to the small size of these antigens it has been thought that two specific antibodies cannot simultaneously bind a hapten. However, antisera containing so called anti-metatypic antibodies have been reported (Voss et al. (1988) Mol. Immunol. 25, 751-759) that bind to hapten-mAb complexes in a reaction where conformational changes on the primary antibody are important. Here, we report on monoclonal antibody pairs able to form ternary complexes with the octapeptide angiotensin II. The first mAb (mAb1) is conventional and binds angiotensin II with high affinity (Kd 10(-11) M). The secondary (anti-metatypic) mAbs (mAbs2s) recognize the immune complex consisting of angiotensin II bound to mAb1, but only poorly recognize mAb1 alone. An immunization technique involving tolerization with uncomplexed mAb1 was used to generate mAb2s. None of the mAbs2s were able to bind angiotensin II by themselves but all efficiently bound the complex of angiotensin II and mAb1. All mAb2s stabilized the angiotensin II-mAb1 complex and one mAb2 distinctly improved the specificity of the assay for angiotensin II. By either labelling mAb1 and immobilizing mAb2 (or vice versa) two-site immunometric assays with detection limits of 1 pg/ml angiotensin II have been established. The kinetics of the complex formation was investigated by fiber optic biospecific interaction analysis (FOBIA), a system allowing real time observation of binding events on the surface of a glass fiber. The association rate towards the liganded conformation of mAb1 was higher than towards the free mAb1. By contrast, the mAb2s dissociated at similar rates from complexed and uncomplexed mAb1.

Conformational effects in the reversed-phase chromatographic behavior of recombinant human growth hormone (rhGH) and N-methionyl recombinant human growth hormone (Met-hGH).

This paper examines the retention behavior of recombinant DNA-derived human growth hormone (rhGH) in reversed-phase chromatography and its separation from the closely related N-methionyl variant (Met-hGH). It is first shown that retention for rhGH decreases with increasing column temperature when 1-propanol (1-PrOH) is used as organic modifier. On the other hand, retention increases with temperature when acetonitrile (CH3CN) is employed. The differences in behavior for the two organic modifiers could be related to conformational changes in the protein as determined by solution and adsorption intrinsic fluorescence spectroscopy. Specifically, desorption and elution of rhGH using 1-PrOH could be correlated with a solvent-induced conformational change, with retention decreasing with increasing temperature due to the increasing ease of structural alteration. On the other hand for CH3CN the increase in retention correlated with temperature rise was related to a partial structural change yielding a more hydrophobic species. In this case, a surface-driven process is suggested. The work then turned to the separation of rhGH and Met-hGH where it was found for both organic modifiers optimum separation occurred at 45 degrees C and pH 6.5. Separate studies revealed that during the conformational change Met-hGH appeared more hydrophobic than rhGH since protein-protein aggregation was observed at a lower 1-PrOH concentration. It is suggested that this hydrophobic difference, which was optimized under the conditions cited above, resulted in the separation. The study demonstrates the importance of conformational changes in retention behavior and separation of protein samples.

Effect of metal ions on the unfolding kinetics of alpha-lactalbumin on weakly hydrophobic surfaces.

The effect of metal ion [Ca2+ and Zn(NH3)4(2+)] on the unfolding kinetics of bovine-alpha-lactalbumin (alpha-LACT) on a weakly hydrophobic chromatographic surface (ethyl polyether phase bonded on porous silica, C2 ether) has been studied using surface intrinsic fluorescence spectroscopy and liquid chromatography. Chromatographic results on the C2 ether phase revealed two peaks for alpha-LACT, the first being the folded and the second an unfolded conformation, as determined by fluorescence spectroscopy. The retention time for the second peak was found to depend on the specific metal additive in the mobile phase. Fluorescence studies showed a slow change in emission maximum from ca. 330 nm to 350 nm and a 5-fold increase in emission intensity for the adsorbed protein in the unfolded state. By following the fluorescence emission intensity at a given wavelength during the unfolding process, biphasic kinetics were observed with the kinetic constants depending on the specific metal-ion additive. In addition, solution refolding rates of the desorbed, unfolded species were measured and found to be consistent with literature refolding rate constants.

Intrinsic fluorescence studies of the kinetic mechanism of unfolding of alpha-lactalbumin on weakly hydrophobic chromatographic surfaces.

The kinetic mechanism of unfolding of calcium depleted bovine alpha-lactalbumin adsorbed on two weakly hydrophobic chromatographic surfaces, methyl- and ethyl-polyether phases bonded to porous silica, with a solution phase of 3 M ammonium sulfate at pH 6.3, has been determined using intrinsic fluorescence and liquid chromatography (LC). The adsorbent has been packed into quartz flow cells which are used for both fluorescence measurements and as a microcolumn for LC. The LC measurements revealed two peaks for alpha-lactalbumin on both phases, the first being folded and the second unfolded. The rate of unfolding was measured to be 1.75.10(-3) min-1 on the Cl-ether and 7.42.10(-3) min-1 on the C2-ether phase. Fluorescence studies revealed a slow change in emission maximum from ca. 330 nm to 350 nm and a 4-fold increase in intensity for the protein adsorbed on the two supports. Variation of fluorescence intensity at a given wavelength revealed biphasic kinetics in which the rate law on the surface was deduced as F in equilibrium X----U, where F is the folded form, U an unfolded form and X an intermediate. The normalized emission spectra of the three species were calculated and it was found that there was approximately a 20-nm-red shift in the position of the maximum from F to U. The emission maximum for X was close to U on both columns; however, the normalized intensity for X was between F and U. Activation enthalpies and entropies were determined from the temperature dependence of the microscopic rate constants. The formation of the intermediate on the C1-ether phase was entropy driven whereas on the C2-ether phase it was enthalpy driven. Finally, the solution refolding rates of U desorbed from the two supports were found to be identical. The differences observed in the surface kinetics of unfolding on the two supports are related to the hydrophobic differences of the adsorbents.

Histochemical detection of hemp trichomes and their correlation with the THC content.

Authors investigated some fiber and some hashish hemp sorts concerning their trichomes on the leaves. Histochemical reactions were developed using the Fast Blue Salt (FBB) reagent applied so far only thin layer chromatography. Glandular hairs were found giving positive reactions due to cannabinoids contained by the cells. The electron microscopic features were studied and the cannabinoid content was measured with GC. A correlation was found between the number of typical glandular hairs and cannabinoid content.

Separation, quantitation and isolation of cannabinoids from Cannabis sativa L. by overpressured layer chromatography.

Two overpressured layer chromatography (OPLC) methods have been developed for the separation of neutral and acidic cannabinoids. The first is an adaptation of Korte's well known method to the OPLC system, which improves its reproducibility. The second one is a new technique based on the phenomenon of chromatographic solvent demixing. The eluent itself is also divided into zones. In the alpha-zone the neutral cannabinoids and in the beta-zone the acidic ones are separated. As a result of the good and reproducible separation, there is a possibility to quantitate cannabinoids by densitometry. The on-line version of OPLC proved suitable for the isolation of hemp constituents.