Identification and circumvention of bottlenecks in CYP21A2-mediated premedrol production using recombinant Escherichia coli.

Synthetic glucocorticoids such as methylprednisolone are compounds of fundamental interest to the pharmaceutical industry as their modifications within the sterane scaffold lead to higher inflammatory potency and reduced side effects compared with their parent compound cortisol. In methylprednisolone production, the complex chemical hydroxylation of its precursor medrane in position C21 exhibits poor stereo- and regioselectivity making the process unprofitable and unsustainable. By contrast, the use of a recombinant E. coli system has recently shown high suitability and efficiency. In this study, we aim to overcome limitations in this biotechnological medrane conversion yielding the essential methylprednisolone-precursor premedrol by optimizing the CYP21A2-based whole-cell system on a laboratory scale. We successfully improved the whole-cell process in terms of premedrol production by (a) improving the electron supply to CYP21A2; here we use the N-terminally truncated version of the bovine NADPH-dependent cytochrome P450 reductase (bCPR-27 ) and coexpression of microsomal cytochrome b5 ; (b) enhancing substrate access to the heme by modification of the CYP21A2 substrate access channel; and (c) circumventing substrate inhibition which is presumed to be the main limiting factor of the presented system by developing an improved fed-batch protocol. By overcoming the presented limitations in whole-cell biotransformation, we were able to achieve a more than 100% improvement over the next best system under equal conditions resulting in 691 mg·L-1 ·d-1 premedrol.

Residual methylprednisolone suppresses human T-cell responses to spleen, but not islet, extracts from deceased organ donors.

Pancreatic islets, transplanted into recipients with type 1 diabetes, are exposed to allogenic and auto-immune T-cell responses. We set out to develop an assay to measure these responses using PBMC. Our approach was to prepare spleen extract from the islet donors (allo-antigen) and islet extracts (auto-antigen). To our surprise, we found that spleen extracts potently inhibited the proliferation of human T cells driven by antigen (tetanus toxoid) and mitogen (anti-CD3 mAb, OKT3), whereas extracts prepared from pancreatic islets from the same donor did not suppress T-cell proliferation. Suppression mediated by spleen extracts was unaffected by blocking mAbs against the IL-10R, transforming growth factor-ß or CD152 (CTLA-4). It was also unaffected by denaturing the spleen extracts by heating, exposing to reducing agents or protease digestion. Because deceased organ donors are commonly given the immunosuppressive glucocorticoid methylprednisolone prior to death, we hypothesized that suppression was due to residual methylprednisolone in the spleen extracts. Methylprednisolone could be detected by mass spectrometry in spleen extracts at concentrations that suppress T-cell proliferation. Finally, the glucocorticoid receptor antagonist mifepristone completely reversed the suppression caused by the spleen extracts. We conclude that extracts of human spleen, but not islets, from deceased organ donors contain sufficient residual methylprednisolone to suppress the proliferation of T-cells in vitro.

Methylprednisolone Concentrations in Breast Milk and Serum of Patients with Multiple Sclerosis Treated with IV Pulse Methylprednisolone.

BACKGROUND: It is commonly known that women with multiple sclerosis (MS) have an increased risk for relapses during the post-partum period. High-dose IV methylprednisolone is the first-line treatment for acute relapses. Methylprednisolone is administered to lactating women although there is insufficient data as to the levels of concentration in breast milk and serum, and the calculated steroid exposure to infants. OBJECTIVES: The study aimed to measure the transfer of methylprednisolone into breast milk and the correlation of milk and serum methylprednisolone concentrations in breastfeeding MS patients during and after infusion therapy. METHODS: IV methylprednisolone pulse therapy was given to 12 lactating MS patients. Breast milk and maternal serum samples were obtained; before infusion, 30 minutes into the infusion, at the end of infusion and at the 1st, 2nd, 4th, 8th, 12th and 24th hours subsequently. RESULTS: The highest level of methylprednisolone concentration in breast milk (2.09 µg/ml) and serum (6.09 µg/ml) was detected at the end of the infusion. According to the measurements recorded at the 1st, 2nd, 4th, 8th, 12th, and 24th hours after infusion, the concentrations showed a gradual decrease both breast milk and serum. The milk and serum methylprednisolone concentrations were below detection limits just before infusion and at the 24th hour after infusion. A highly significant correlation was found between breast milk and maternal serum levels. The absolute infant dose was calculated to be 69.50 µg/kg/day and the relative infant dose (RID) was 0.50%. CONCLUSION: Results have shown that the transfer of methylprednisolone into breast milk seems to be low. Although, concentration levels may not seem to pose a threat to the infant, mothers can choose to wait 2 to 4 hours to further limit the level of exposure.

Stability and Sterility of Extemporaneously Prepared Nonpreserved Cefazolin, Ceftazidime, Vancomycin, Amphotericin B, and Methylprednisolone Eye Drops.

PURPOSE: To determine in-use stability and sterility of fortified cefazolin, ceftazidime, vancomycin, amphotericin B, and methylprednisolone eye drops in a simulated inpatient setting with and without a mobile refrigerated container (MR). METHODS: Each drug was prepared and divided into 4 groups: 1) simulated patient use with the MR group: stored at 4°C and kept in the MR during drug administration, 2) simulated patient use without the MR (NoMR) group: stored at 4°C and no MR, 3) refrigerated control group: stored at 4°C, and 4) room temperature control group: stored at room temperature. Stability and sterility data were evaluated at days 0, 4, 7, 14, 21, and 28. Linear mixed-effects model and survival analysis were performed. RESULTS: Median time to 10% loss of concentration for in-use medications (MR/NoMR groups) was >28/27.9, 22.2/22.2, 19.4/19.4, 10.18/<4, and >28/>28 days for cefazolin, ceftazidime, vancomycin, amphotericin B, and methylprednisolone, respectively. There was no significant difference in the predicted concentration loss per day among all groups for vancomycin and methylprednisolone (all P > 0.05). For the other study medications, all room temperature control groups, the cefazolin NoMR group, and the ceftazidime NoMR group had significantly greater predicted concentration loss per day compared with the refrigerated control groups (all P ≤ 0.02). Culture results were negative for all drugs throughout the study. CONCLUSIONS: The NoMR group showed that the drug significantly degraded rapidly for cefazolin, ceftazidime, and amphotericin B. Implementation of MR could decrease the predicted loss of concentration per day for cefazolin and ceftazidime. In vitro antimicrobial activity and sterility were retained for 28 days.

A novel and innovative hair test to determine glucocorticoid levels in racing camels for use in assessment of doping, health, and disease.

The aim of this project was to develop and validate a new test for the analysis of glucocorticoids in camel hair and to use the new test to analyse hair samples from a variety of camel breeds in sports and racing applications. These findings could be of importance when evaluating racing camels for suspected doping offenses or for injury and disease control. Camel hair samples were collected from 30 non-racing dromedary camels along with 3 racing camels in Al Ain, UAE and were decontaminated, pulverised, sonicated, and extracted prior to analysis. A liquid chromatographic-mass spectrometric method was employed to determine the levels of glucocorticoids in the hair samples. The 4 drugs of interest, namely hydrocortisone, dexamethasone, flumethasone and methylprednisolone, and an internal standard were quantified in camel hair samples. All 4 of the glucocorticoids were detected in camel hair samples with concentrations ranging between 31 and 935 pg/mg for hydrocortisone, 8-59 pg/mg for dexamethasone, 0.7-1034 pg/mg for flumethasone and 5-66 pg/mg for methylprednisolone in non-racing camels. One of the racing camels displayed high concentrations of hydrocortisone (1130 pg/mg), flumethasone (2576 pg/mg), methylprednisone (1156 pg/mg) and dexamethasone (29 pg/mg). The authors believe this is the first report of a test for corticosteroids in camel hair. The new test has been validated according to Food and Drug Administration (FDA) guidelines. This new hair test could be useful for further studies in doping control, toxicological studies, pharmacological studies and other clinical applications in camel health, injury, and disease.

Concentration of methylprednisolone in the centrodistal joint after administration of methylprednisolone acetate in the tarsometatarsal joint.

REASONS FOR PERFORMING STUDY: The centrodistal (CD) and tarsometatarsal (TMT) joints are often injected individually with a corticosteroid to resolve lameness caused by osteoarthritis (OA). There are no data available regarding diffusion of methylprednisolone (MP) from the TMT joint to the CD joint. HYPOTHESIS: A therapeutic concentration of MP diffuses into the CD joint after methylprednisolone acetate (MPA) is administered into the TMT joint. OBJECTIVE: To measure the concentration of MP in the CD joint after MPA was administered into the TMT joint. METHODS: MPA was administered into a TMT joint of 16 horses. At different times, the ipsilateral CD joint of these horses was injected with a small amount of saline and recovered saline was measured for concentration of MP using high performance liquid chromatography. RESULTS: Six hours after administration of MPA into the TMT joint, a therapeutic concentration of MP was found in all 10 CD joints sampled at this time. CONCLUSIONS: Horses with pain arising from the distal 2 joints of the hock can be treated by administering MPA into the TMT joint alone. POTENTIAL RELEVANCE: Administering MPA into the TMT joint only, to treat OA of the distal 2 hock joints, reduces the difficulties and risks associated with centesis of the CD joint.

Kinetic study of USP blue tetrazolium assay with methylprednisolone, hydrocortisone, and their hemisuccinate esters by high-pressure liquid chromatography.

The reactions of blue tetrazolium (I) with methylprednisolone and hydrocortisone and their hemisuccinate esters at ambient temperature under USP assay conditions in alcohol USP and in absolute alcohol were followed by high-pressure liquid chromatography (HPLC) and spectrophotometry. The disappearance of the ester and the increase and then decrease in the alcohol, as well as the formation of several reaction products with time, were observed by HPLC analysis. The reduction of I was observed spectrophotometrically. A sequential kinetic model was used to describe the overall reaction. The rate constants for the hydrolysis of the hemiester (k1), the reaction with I (k2), and the degradation of the parent steroid (k3) were determined by discrete kinetic experiments using HPLC. The following observations were made: (a) k1 is proportional to [H2O] and is the rate-limiting step, (b) k2 is about 100 times the value of k3 and (c) k2 for methylprednisolone is about the same as for hydrocortisone and appears to be independent of the concentration of water. With these rate constants, simulated time-concentration profiles for the reaction of the esters with I favorable compared with experimental data in alcohol USP and absolute alcohol. This study shows that the USP blue tetrazolium assay with these esters has potential for variability and is not stability indicating.

Carboxyl group catalysis of acyl transfer reactions in corticosteroid 17- and 21-monoesters.

Succinate esters, although frequently employed as water-soluble prodrugs of poorly soluble parent drugs, are not sufficiently stable to allow long-term storage in solution. Intramolecular catalysis of ester hydrolysis by the terminal succinate carboxyl group is a contributing factor to this instability. Methylprednisolone 21-succinate has recently been reported to undergo both hydrolysis and 21 in equilibrium 17 acyl migration in aqueous solutions. Intramolecular catalysis by the terminal carboxyl group is seen in both reactions, but the catalytic mechanisms are not well understood. While acyl migration can only be catalyzed via the carboxyl group acting as a general acid or general base, hydrolysis may undergo either nucleophilic or general acid-base catalysis. To gain further insight into the catalytic mechanism, hydrolysis of methylprednisolone 21-succinate was carried out in aniline buffers to trap any succinic anhydride (as the anilide) that would form if the catalysis were nucleophilic. The nucleophilic mechanism was shown to account for only 15-20% of the overall catalysis. Comparisons of the rates of the intramolecularly catalyzed reactions of methylprednisolone 21- and 17-succinate were made with the same reactions of methylprednisolone 21- and 17-acetate catalyzed intermolecularly by acetate ion. Interestingly, intramolecular catalysis appears to favor acyl migration over hydrolysis. Hence, the hydrolysis of methylprednisolone 21-succinate is faster in basic solutions (pH greater than 7.4), while acyl migration becomes the dominant reaction in the catalyzed region of the pH profile between pH 3.6 and 7.4. Arguments are presented to account for these differences in catalytic efficiency in terms of the transition-state structures for the two reactions.

Two chromatographic methods for the determination of corticosteroids in human biological fluids: pharmacokinetic applications.

Two techniques, high-performance liquid chromatography (HPLC) and quantitative high-performance thin-layer chromatography coupled with densitometry (HPTLC), were developed for the determination of prednisolone (PL), methylprednisolone (MP), and methylprednisolone sodium succinate (MPSS) in human plasma, saliva, and urine. The HPLC and HPTLC methods shared a single and simple step of an organic extraction procedure and separation of steroids using a normal-phase column or HPTLC plate. The methods allow simultaneous measurement of endogenous cortisol in plasma following the administration of PL and MP. The calibration curves of steroids in all biological fluids were linear over a wide range of concentrations of PL and MP in all biological fluids (0.025-4 micrograms/mL). The limit of detection of both assays for PL and MP was 10 ng/mL in plasma and saliva and 25 ng/mL in urine, and of MPSS was 50 ng/mL in plasma. Both methods were reproducible with an inter- and intra-assay coefficient of variation of less than 10% for all steroids over a wide range of concentrations in all biological fluids. No interference from endogenous steroids was found. The presented methods are simple, rapid, specific, sensitive, reproducible, and economical for the pharmacokinetic study of these steroids. The application of these methods for the pharmacokinetic study of both MP and PL in vivo and the in vitro hydrolysis of MPSS is discussed.

Purity evaluation of 6 alpha-methylprednisolone acetate by HPLC.

A high-performance liquid chromatographic method is described for the analysis and identification of related impurities in pharmaceutical-grade 6 alpha-methylprednisolone acetate (MPA). Eight MPA impurities derived from synthesis and/or degradation processes were identified. The method is compared to other analytical procedures recently proposed for the European Pharmacopoeia.

Determination of CMC of sodium glucocorticides hemisuccinates by CE.

The capillary zone electrophoresis (CZE) method was applied to the determination of the critical micelle concentration (CMC) and the anionic mobilities (mu(e)) for sodium glycocorticides hemisuccinates (Urbason solubile forte 1000, Hydrocortison 100 Rotexmedica, Prednisolut 100) in phosphate solution at pH 7.2. The CZE enables an efficient and very fast determination of parameters for characterizing physicochemical properties of micelles.

Simultaneous analysis of dextran-methylprednisolone succinate, methylprednisolone succinate, and methylprednisolone by size-exclusion chromatography.

An analytical HPLC method is reported for simultaneous measurement of low (1.0-100 microg ml(-1)) concentrations of dextran-methylprednisolone succinate (DEX-MPS) and its degradation products methylprednisolone hemisuccinate (MPS) and methylprednisolone (MP). The analytes were detected at 250 nm after resolution using a size exclusion column with a mobile phase of KH2PO4 (10 mM): acetonitrile (3:1) and a flow rate of 1 ml min(-1). The resolution of MP and MPS peaks was substantially affected by the pH of the mobile phase; while MP and MPS co-eluted at pH 3.4, they were baseline-resolved at pH > or = 5. Linear relationships (r > or = 0.997) were found between the detector response and the concentrations of the analytes (1.0-100 microg ml(-1) for MP and MPS and 2.5-100 microg ml(-1) for DEX-MPS). Intra- and inter-run error (< 13%) and precision (CV of < or = 6%) data indicated that the assay could accurately and precisely quantitate all three components in the examined concentration range. The application of the assay to determination of degree of substitution, purity, and stability of DEX-MPS was also demonstrated.

Plasma, urine, and synovial fluid disposition of methylprednisolone acetate and isoflupredone acetate after intra-articular administration in horses.

OBJECTIVE--To document plasma, urine, and synovial fluid disposition of 2 common intra-articularly administered steroid preparations, methylprednisolone acetate (MPA) and isoflupredone acetate (IPA). DESIGN--Descriptive investigation. SAMPLE POPULATION--100 mg of MPA or 4 mg of IPA was administered to 2 groups of 4 healthy sound radiographically normal female horses. PROCEDURE--Blood samples were collected at time 0 (before) and 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, and 96 hours after administration of the designated steroid. Complete urine collection for measurement of designated steroid was accomplished by use of occluding 28-F balloon catheters. Synovial fluid samples were aseptically aspirated from the injected and contralateral uninjected tarsocrural joint at time 0 and 8, 24, 48, 240, and 672 hours after administration of the designated steroid. All samples were screened by ELISA to detect parent drug or metabolite equivalent, with a sensitivity of 2.5 ng/ml for MPA and 0.1 ng/ml for IPA. If drug was detected by ELISA in the plasma or synovial fluid, the samples were further quantified and specified, using HPLC with a lower limit of quantification (10 ng/ml). RESULTS--Between 2 and 12 hours after administration, plasma contained < 10 ng of MPA or IPA/ml (parent drug or metabolite equivalent), as intermittently detected by ELISA. Parent drug or metabolite equivalent was detected in the urine for 24 and 72 hours after injection of IPA and MPA, respectively. Synovial fluid from the contralateral joint contained no detectable MPA or IPA at any sample collection time. Median half-life for MPA, as detected by HPLC, was 10.3 hours (range, 6.1 to 10.6) in the synovial space. Median half-life for methylprednisolone, as detected by HPLC, was 10.4 (range, 9.9 to 32.1) hours. CONCLUSIONS--Both steroids appeared to be rapidly hydrolyzed to their respective ester forms, as detected by HPLC. The ELISA appeared to be a useful screening tool for detection of corticosteroids in this variety of body fluids.

[Evaluation of some thin-layer chromatography procedures for the identification and detection of impurities of hormonal steroids]. / Studio collaborativo per la valutazione di aleune metodiche di cromatografia in strato sottile per i'dentificazione ed il saggio di purezza di steroidi ad attività ormonale

PIP: The thin-layer chromatography methods described in Volume 2 of the "European Pharmacopeia" were tested to determine the most suitable methods for the identification of hormonal steroids and for the detection of impurities in these substances. The methods are based on partition chromatography for identification and on adsorption chromatogr aphy for the detection of impurities. Other adsorption methods developed by the authors were also tested. The study was conducted on 44 steroids, belonging to the groups of androgens, estrogens, progestinic and corticoid steroids. 9 chromatographic methods based on adsorption, 2 in bidimensional succession, and 7 methods based on partit ion were used. The work was carried out in 6 laboratories. The findings are summarized in 18 tables and illustrated in 13 figures. They were also processed by statistical methods in order to assess the reproducibility of the tests.^ieng

A rapid screen for four corticosteroids in equine synovial fluid.

Most antidoping method development in the equine industry has been for plasma and urine, though there has been recent interest in the analysis of synovial fluid for evidence of doping by intra-articular corticosteroid injection. Published methods for corticosteroid analysis in synovial fluid are primarily singleplex methods, do not screen for all corticosteroids of interest and are not adequately sensitive. The purpose of this study is to develop a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) screening method for the detection of four of the most common intra-articularly administered corticosteroids--betamethasone, methylprednisolone, methylprednisolone acetate and triamcinolone acetonide. Sample preparation consisted of protein precipitation followed by a basified liquid-liquid extraction. LC-MS-MS experiments consisted of a six-min isocratic separation using a Phenomenex Polar-RP stationary phase and a mobile phase consisting of 35% acetonitrile, 5 mM ammonium acetate and 0.1% formic acid in nanopure water. The detection system used was a triple quadrupole mass analyzer with thermospray ionization, and compounds were identified using selective reaction monitoring. The method was validated to the ISO/IEC 17025 standard, and real synovial fluid samples were analyzed to demonstrate the application of the method in an antidoping context. The method was highly selective for the four corticosteroids with limits of detection of 1-3 ng/mL. The extraction efficiency was 50-101%, and the matrix effects were 14-31%. These results indicate that the method is a rapid and sensitive screen for the four corticosteroids in equine synovial fluid, fit for purpose for equine antidoping assays.

Pharmaceutical evaluation of steroidal ointments by ATR-IR chemical imaging: distribution of active and inactive pharmaceutical ingredients.

We recently used micro attenuated total reflection infrared (ATR-IR) spectroscopy to conduct imaging analysis of ointments and evaluate the distributions of the active pharmaceutical ingredient (API) and excipients. An alclometasone dipropionate (ALC) ointment was used as a model product. Almeta, a brand-name product, had a domain with absorbance at 1656 cm(-1) attributable to the carbonyl group of ALC, the API. Absorbances at 1040 and 3300 cm(-1) were also noted in this domain, indicating the presence of the solubilizer, propylene glycol. Data also suggested the presence of benzyl alcohol in this domain. More detailed analysis showed the distribution of surfactants and other excipients in the base. Similar results were obtained for Vitra, a generic version of Almeta. Imaging analysis with micro ATR-IR confirmed that both ointments are liquid droplet dispersions with ALC dissolved in propylene glycol and dispersed in a base. However, minor differences in the ingredient distributions of the two ointments were detected and reflect differences in excipient concentrations and type, or manufacturing differences. In summary, we used micro ATR-IR for imaging analysis of an original ointment, Almeta, and its generic form Vitra, and established a method for visually evaluating the distributions of the API and excipients in these ointments.

Determination of MRL regulated corticosteroids in liver from various species using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC).

Dexamethasone, betamethasone, prednisolone and methylprednisolone are corticosteroids widely used in animal husbandry. These compounds are licensed for therapy in veterinary practices while their use for growth promoting practices, mainly in combination with other growth promoters, is prohibited within the European Union. In order to protect the consumer, maximum residue limits (MRLs) have been set by the European Community in liver to 2.0 µg kg(-1) (dexamethasone and betamethasone) and 10.0 µg kg(-1) (prednisolone and methylprednisolone) for different species. The major challenges in the analysis of dexamethasone and betamethasone consist in performing an efficient separation of both isomers and in detecting and identifying all the molecules according to the regulatory requirements fixed in Commission Decision 2002/657/EC. In this context, an UHPLC-MS/MS method with a short runtime (7 min) and using the SRM acquisition mode was developed and validated. An efficient selectivity of the sample preparation combined with a high sensitivity of the measurement system allowed identifying and quantifying the four corticosteroids of interest in this complex biological matrix. Signals obtained were found very repeatable, even at very low concentration levels with an unambiguous identification of the compounds. The performance limits of the method have been validated according to the regulatory requirements and the method has been successfully applied to the confirmation of incurred liver samples.