Factors Governing the Precision of Subvisible Particle Measurement Methods - A Case Study with a Low-Concentration Therapeutic Protein Product in a Prefilled Syringe.

PURPOSE: The current study was performed to assess the precision of the principal subvisible particle measurement methods available today. Special attention was given to identifying the sources of error and the factors governing analytical performance. METHODS: The performance of individual techniques was evaluated using a commercial biologic drug product in a prefilled syringe container. In control experiments, latex spheres were used as standards and instrument calibration suspensions. RESULTS: The results reported in this manuscript clearly demonstrated that the particle measurement techniques operating in the submicrometer range have much lower precision than the micrometer size-range methods. It was established that the main factor governing the relatively poor precision of submicrometer methods in general and inherently, is their low sampling volume and the corresponding large extrapolation factors for calculating final results. CONCLUSIONS: The variety of new methods for submicrometer particle analysis may in the future support product characterization; however, the performance of the existing methods does not yet allow for their use in routine practice and quality control.

Hepatic reduction of the secondary bile acid 7-oxolithocholic acid is mediated by 11ß-hydroxysteroid dehydrogenase 1.

The oxidized bile acid 7-oxoLCA (7-oxolithocholic acid), formed primarily by gut micro-organisms, is reduced in human liver to CDCA (chenodeoxycholic acid) and, to a lesser extent, UDCA (ursodeoxycholic acid). The enzyme(s) responsible remained unknown. Using human liver microsomes, we observed enhanced 7-oxoLCA reduction in the presence of detergent. The reaction was dependent on NADPH and stimulated by glucose 6-phosphate, suggesting localization of the enzyme in the ER (endoplasmic reticulum) and dependence on NADPH-generating H6PDH (hexose-6-phosphate dehydrogenase). Using recombinant human 11ß-HSD1 (11ß-hydroxysteroid dehydrogenase 1), we demonstrate efficient conversion of 7-oxoLCA into CDCA and, to a lesser extent, UDCA. Unlike the reversible metabolism of glucocorticoids, 11ß-HSD1 mediated solely 7-oxo reduction of 7-oxoLCA and its taurine and glycine conjugates. Furthermore, we investigated the interference of bile acids with 11ß-HSD1-dependent interconversion of glucocorticoids. 7-OxoLCA and its conjugates preferentially inhibited cortisone reduction, and CDCA and its conjugates inhibited cortisol oxidation. Three-dimensional modelling provided an explanation for the binding mode and selectivity of the bile acids studied. The results reveal that 11ß-HSD1 is responsible for 7-oxoLCA reduction in humans, providing a further link between hepatic glucocorticoid activation and bile acid metabolism. These findings also suggest the need for animal and clinical studies to explore whether inhibition of 11ß-HSD1 to reduce cortisol levels would also lead to an accumulation of 7-oxoLCA, thereby potentially affecting bile acid-mediated functions.

Synthesis of new glycyrrhetinic acid derived ring A azepanone, 29-urea and 29-hydroxamic acid derivatives as selective 11ß-hydroxysteroid dehydrogenase 2 inhibitors.

Glycyrrhetinic acid, the metabolite of the natural product glycyrrhizin, is a well known nonselective inhibitor of 11ß-hydroxysteroid dehydrogenase (11ß-HSD) type 1 and type 2. Whereas inhibition of 11ß-HSD1 is currently under consideration for treatment of metabolic diseases, such as obesity and diabetes, 11ß-HSD2 inhibitors may find therapeutic applications in chronic inflammatory diseases and certain forms of cancer. Recently, we published a series of hydroxamic acid derivatives of glycyrrhetinic acid showing high selectivity for 11ß-HSD2. The most potent and selective compound is active against human 11ß-HSD2 in the low nanomolar range with a 350-fold selectivity over human 11ß-HSD1. Starting from the lead compounds glycyrrhetinic acid and the hydroxamic acid derivatives, novel triterpene type derivatives were synthesized and analyzed for their biological activity against overexpressed human 11ß-HSD1 and 11ß-HSD2 in cell lysates. Here we describe novel 29-urea- and 29-hydroxamic acid derivatives of glycyrrhetinic acid as well as derivatives with the Beckman rearrangement of the 3-oxime to a seven-membered ring, and the rearrangement of the C-ring from 11-keto-12-ene to 12-keto-9(11)-ene. The combination of modifications on different positions led to compounds comprising further improved selective inhibition of 11ß-HSD2 in the lower nanomolar range with up to 3600-fold selectivity.

Synthesis of glycyrrhetinic acid derivatives for the treatment of metabolic diseases.

The effect of glycyrrhetinic acid (GA) and GA-derivatives towards 11beta-hydroxysteroid dehydrogenase (11beta-HSD) was investigated. Novel compounds with modifications at positions C-3, C-11 and C-29 of the GA skeleton were prepared. Single crystal X-ray diffraction data of selected substances are reported and discussed.

EMS Access Constraints And Response Time Delays For Deprived Critically Ill Patients Near Paris, France.

Increased emergency medical services (EMS) response times and areas of low socioeconomic status are both associated with poorer outcomes for several time-sensitive medical conditions attended to by medical personnel before a patient is hospitalized. We evaluated the association between EMS response times, area deprivation level, and on-scene access constraints encountered by EMS in a large urban area in France. We conducted a multicenter prospective cohort study of EMS dispatches occurring in the forty-seven cities in a region southeast of Paris. We fit multilevel mixed-effects linear regression models for multivariate assessment of the predictors of EMS response times and then used multivariate logistic regression on outcomes among a subgroup of patients presenting with out-of-hospital cardiac arrest. We found evidence that access constraints were more frequently encountered by EMS in the most deprived areas compared to less deprived ones, and were associated with increased EMS response times until patient contact and with poorer outcomes from cardiac arrest. Strategies to anticipate and overcome access constraints should be implemented to improve outcomes for emergent conditions attended to by prehospital medical teams.

Carbonyl reduction of triadimefon by human and rodent 11ß-hydroxysteroid dehydrogenase 1.

11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) catalyzes the conversion of inactive 11-oxo glucocorticoids (endogenous cortisone, 11-dehydrocorticosterone and synthetic prednisone) to their potent 11ß-hydroxyl forms (cortisol, corticosterone and prednisolone). Besides, 11ß-HSD1 accepts several other substrates. Using rodent liver microsomes and the unspecific inhibitor glycyrrhetinic acid, it has been proposed earlier that 11ß-HSD1 catalyzes the reversible conversion of the fungicide triadimefon to triadimenol. In the present study, recombinant human, rat and mouse enzymes together with a highly selective 11ß-HSD1 inhibitor were applied to assess the role of 11ß-HSD1 in the reduction of triadimefon and to uncover species-specific differences. To further demonstrate the role of 11ß-HSD1 in the carbonyl reduction of triadimefon, microsomes from liver-specific 11ß-HSD1-deficient mice were employed. Molecular docking was applied to investigate substrate binding. The results revealed important species differences and demonstrated the irreversible 11ß-HSD1-dependent reduction of triadimefon. Human liver microsomes showed 4 and 8 times higher activity than rat and mouse liver microsomes. The apparent Vmax/Km of recombinant human 11ß-HSD1 was 5 and 15 times higher than that of mouse and rat 11ß-HSD1, respectively, indicating isoform-specific differences and different expression levels for the three species. Experiments using inhibitors and microsomes from 11ß-HSD1-deficient mice indicated that 11ß-HSD1 is the major if not only enzyme responsible for triadimenol formation. The IC50 values of triadimefon and triadimenol for cortisone reduction suggested that exposure to these xenobiotica unlikely impairs the 11ß-HSD1-dependent glucocorticoid activation. However, elevated glucocorticoids during stress or upon pharmacological administration likely inhibit 11ß-HSD1-dependent metabolism of triadimefon in humans.

Species-specific differences in the inhibition of human and zebrafish 11ß-hydroxysteroid dehydrogenase 2 by thiram and organotins.

Dithiocarbamates and organotins can inhibit enzymes by interacting with functionally essential sulfhydryl groups. Both classes of chemicals were shown to inhibit human 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2), which converts active cortisol into inactive cortisone and has a role in renal and intestinal electrolyte regulation and in the feto-placental barrier to maternal glucocorticoids. In fish, 11ß-HSD2 has a dual role by inactivating glucocorticoids and generating the major androgen 11-ketotestosterone. Inhibition of this enzyme may enhance glucocorticoid and diminish androgen effects in fish. Here, we characterized 11ß-HSD2 activity of the model species zebrafish. A comparison with human and mouse 11ß-HSD2 revealed species-specific substrate preference. Unexpectedly, assessment of the effects of thiram and several organotins on the activity of zebrafish 11ß-HSD2 showed weak inhibition by thiram and no inhibition by any of the organotins tested. Sequence comparison revealed the presence of an alanine at position 253 on zebrafish 11ß-HSD2, corresponding to cysteine-264 in the substrate-binding pocket of the human enzyme. Substitution of alanine-253 by cysteine resulted in a more than 10-fold increased sensitivity of zebrafish 11ß-HSD2 to thiram. Mutating cysteine-264 on human 11ß-HSD2 to serine resulted in 100-fold lower inhibitory activity. Our results demonstrate significant species differences in the sensitivity of human and zebrafish 11ß-HSD2 to inhibition by thiram and organotins. Site-directed mutagenesis revealed a key role of cysteine-264 in the substrate-binding pocket of human 11ß-HSD2 for sensitivity to sulfhydryl modifying agents.

The anabolic androgenic steroid fluoxymesterone inhibits 11ß-hydroxysteroid dehydrogenase 2-dependent glucocorticoid inactivation.

Anabolic androgenic steroids (AAS) are testosterone derivatives used either clinically, in elite sports, or for body shaping with the goal to increase muscle size and strength. Clinically developed compounds and nonclinically tested designer steroids often marketed as food supplements are widely used. Despite the considerable evidence for various adverse effects of AAS use, the underlying molecular mechanisms are insufficiently understood. Here, we investigated whether some AAS, as a result of a lack of target selectivity, might inhibit 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2)-dependent inactivation of glucocorticoids. Using recombinant human 11ß-HSD2, we observed inhibitory effects for several AAS. Whereas oxymetholone, oxymesterone, danazol, and testosterone showed medium inhibitory potential, fluoxymesterone was a potent inhibitor of human 11ß-HSD2 (half-maximal inhibitory concentration [IC(50)] of 60-100nM in cell lysates; IC(50) of 160nM in intact SW-620, and 530nM in MCF-7 cells). Measurements with rat kidney microsomes and lysates of cells expressing recombinant mouse 11ß-HSD2 revealed much weaker inhibition by the AAS tested, indicating that the adverse effects of AAS-dependent 11ß-HSD2 inhibition cannot be investigated in rats and mice. Furthermore, we provide evidence that fluoxymesterone is metabolized to 11-oxofluoxymesterone by human 11ß-HSD2. Structural modeling revealed similar binding modes for fluoxymesterone and cortisol, supporting a competitive mode of inhibition of 11ß-HSD2-dependent cortisol oxidation by this AAS. No direct modulation of mineralocorticoid receptor (MR) function was observed. Thus, 11ß-HSD2 inhibition by fluoxymesterone may cause cortisol-induced MR activation, thereby leading to electrolyte disturbances and contributing to the development of hypertension and cardiovascular disease.

Characterization of activity and binding mode of glycyrrhetinic acid derivatives inhibiting 11ß-hydroxysteroid dehydrogenase type 2.

Modulation of intracellular glucocorticoid availability is considered as a promising strategy to treat glucocorticoid-dependent diseases. 18ß-Glycyrrhetinic acid (GA), the biologically active triterpenoid metabolite of glycyrrhizin, which is contained in the roots and rhizomes of licorice (Glycyrrhiza spp.), represents a well-known but non-selective inhibitor of 11ß-hydroxysteroid dehydrogenases (11ß-HSDs). However, to assess the physiological functions of the respective enzymes and for potential therapeutic applications selective inhibitors are needed. In the present study, we applied bioassays and 3D-structure modeling to characterize nine 11ß-HSD1 and fifteen 11ß-HSD2 inhibiting GA derivatives. Comparison of the GA derivatives in assays using cell lysates revealed that modifications at the 3-hydroxyl and/or the carboxyl led to highly selective and potent 11ß-HSD2 inhibitors. The data generated significantly extends our knowledge on structure-activity relationship of GA derivatives as 11ß-HSD inhibitors. Using recombinant enzymes we found also potent inhibition of mouse 11ß-HSD2, despite significant species-specific differences. The selected GA derivatives potently inhibited 11ß-HSD2 in intact SW-620 colon cancer cells, although the rank order of inhibitory potential differed from that obtained in cell lysates. The biological activity of compounds was further demonstrated in glucocorticoid receptor (GR) transactivation assays in cells coexpressing GR and 11ß-HSD1 or 11ß-HSD2. 3D-structure modeling provides an explanation for the differences in the selectivity and activity of the GA derivatives investigated. The most potent and selective 11ß-HSD2 inhibitors should prove useful as mechanistic tools for further anti-inflammatory and anti-cancer in vitro and in vivo studies. Article from the Special issue on Targeted Inhibitors.

Selective inhibition of 11beta-hydroxysteroid dehydrogenase 1 by 18alpha-glycyrrhetinic acid but not 18beta-glycyrrhetinic acid.

Elevated cortisol concentrations have been associated with metabolic diseases such as diabetes type 2 and obesity. 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1, catalyzing the conversion of inactive 11-ketoglucocorticoids into their active 11beta-hydroxy forms, plays an important role in the regulation of cortisol levels within specific tissues. The selective inhibition of 11beta-HSD1 is currently considered as promising therapeutic strategy for the treatment of metabolic diseases. In recent years, natural compound-derived drug design has gained considerable interest. 18beta-glycyrrhetinic acid (GA), a metabolite of the natural product glycyrrhizin, is not selective and inhibits both 11beta-HSD1 and 11beta-HSD2. Here, we compare the biological activity of 18beta-GA and its diastereomer 18alpha-GA against the two enzymes in lysates of transfected HEK-293 cells and show that 18alpha-GA selectively inhibits 11beta-HSD1 but not 11beta-HSD2. This is in contrast to 18beta-GA, which preferentially inhibits 11beta-HSD2. Using a pharmacophore model based on the crystal structure of the GA-derivative carbenoxolone in complex with human 11beta-HSD1, we provide an explanation for the differences in the activities of 18alpha-GA and 18beta-GA. This model will be used to design novel selective derivatives of GA.