Dextran-methylprednisolone succinate as a prodrug of methylprednisolone: dose-dependent pharmacokinetics in rats

The dose-dependency in the pharmacokinetics of a macromolecular prodrug of methylprednisolone (MP), dextran-methylprednisolone succinate (DMP), was investigated in rats. Single doses (MP equivalent) of 2.5, 5.0, 10, 20, and 30 mg/kg of DMP were administered intravenously to rats (n=5/group), and serial blood samples (0-96 h) and spleen and liver tissues (96 h) were collected. The concentrations of DMP in plasma and spleen were analyzed using a size-exclusion chromatographic method. The concentrations of DMP in the liver samples were determined by an indirect method after sequential hydrolysis by dextranase and esterase enzymes, followed by HPLC analysis of MP. The kinetics of DMP were analyzed by non-compartmental methods. The systemic clearance of DMP decreased approximately 5-fold (from 42.1+/-11.0 to 7.72+/-1.84 ml/h per kg) when the dose was increased from 2.5 to 30 mg/kg. The nonlinearity in the clearance of DMP could be adequately described by a Michaelis-Menten type elimination with a maximum velocity of elimination of 1.72 mg/h per kg and a constant of 24.9 microg/ml. Additionally, the percent of the dose of DMP found at 96 h in the liver and spleen, where the prodrug is sequestered and gradually eliminated, significantly decreased with an increase in the dose. It is concluded that the clearance of DMP in rats is modestly dose-dependent in the dosage range of 2.5-30 mg/kg.     

Dextran-methylprednisolone succinate as a prodrug of methylprednisolone: in vitro immunosuppressive effects on rat blood and spleen lymphocytes

The in vitro immunosuppressive activity of a conjugate of methylprednisolone (MP) with dextran 70 kDa (DEX-MPS) was tested using the lymphocyte proliferation assay after stimulation of lymphocytes with concanavalin A (Con-A). Blood and spleen lymphocytes, isolated from drug-free male Sprague-Dawley rats, were used in the assay. First, the optimum concentration of Con-A for stimulation of lymphocytes was determined. The inhibition of the lymphocyte proliferation was then tested in the presence of 0.25,0.5, 1.0,2.5,5.0,10,20, and 50 nM concentrations (MP equivalent) of DEX-MPS or free MP. The maximum stimulation of lymphocytes with Con-A was observed at mitogen concentrations of 2.5 and 10 microg/ml for the spleen and blood lymphocytes, respectively. For free MP, sigmoidal relationships were observed between the effect (% inhibition of lymphocyte proliferation) and the logarithm of MP concentration. Additionally, the maximum inhibitory effect (I(max)) and MP concentration producing half of I(max) (IC(50)) were, respectively, 98% and 1.38 nM for the blood and 86% and 3.1 nM for the spleen lymphocytes. For MP conjugated to dextran, the response-log concentration curves were substantially shifted to the right with IC(50) values of 40 and 52 nM for the blood and spleen lymphocytes, respectively. It is concluded that compared with free MP, the steroid attached to dextran has minimal immunosuppressive activity. Therefore, to be effective in vivo, DEX-MPS should release MP in the body.     

Sulfate-conjugated methylprednisolone: Evaluation as a colon-specific methylprednisolone prodrug and comparison with sulfate-conjugated prednisolone and dexamethasone

Methylprednisolone 21-sulfate sodium (MPS) was prepared and evaluated as a colon-specific methylprednisolone prodrug and its colon-specific property was compared with prednisolone 21-sulfate sodium (PDS) and dexamethasone 21-sulfate sodium (DS), reported previously as colon-specific prodrugs of the glucocorticoids. The synthetic process and yield of MPS was simple and high. The apparent partition coefficient of methylprednisolone (MP) was greatly reduced by sulfate conjugation. The sulfate conjugates MPS, PDS, and DS were (bio)chemically stable in the homogenates of the upper intestine. In marked contrast, the sulfate conjugates were deconjugated to liberate the corresponding glucocorticoids in the cecal contents. Although the rates of deconjugation were not significantly different, the corresponding glucocorticoids were accumulated with distinct profiles depending on the metabolic susceptibility of the unconjugated glucocorticoids to microbial reductase(s). Upon oral administration of the sulfate conjugates to rats, the plasma concentrations of the conjugates were extremely low and the urinary recoveries were less than 5% of the doses. These results suggest that, like PDS and DS, MPS administered orally is delivered efficiently to the large intestine followed by deconjugation to liberate MP and the metabolic susceptibility of the unconjugated glucocorticoids may affect therapeutic availability of the sulfate conjugates.     

Sulfate-conjugated methylprednisolone as a colon-targeted methylprednisolone prodrug with improved therapeutic properties against rat colitis

Methylprednisolone (MP) is one of the most widely used corticosteroids for the treatment of inflammatory bowel disease (IBD). However, systemic adverse effects of MP limit its availability for the disease. In present study, sulfate-conjugated methylprednisolone (MPS) was evaluated in vivo as a colon-targeted prodrug of MP and its therapeutic properties against 2,4,6-trinitrobenzenesulfonic acid–induced rat colitis were investigated. Upon oral administration, a large fraction of MPS reached the large intestine, where MPS was converted to MP implying that MPS would deliver MP effectively to the large intestine. The fecal recovery of MP (after MPS administration) was much greater than that after MP administration and the urinary recovery of MP (after MPS administration) was much less than that after MP administration, suggesting that MPS should exhibit enhanced therapeutic activity and reduced systemic adverse effects. Consistent with this notion, MPS was more effective than MP in ameliorating rat colitis. Moreover, the adverse effects of MPS on adrenal function and thymus were much lower than those of MP. Taken together, MPS may be therapeutically superior to MP in IBD treatment.     

Simultaneous analysis of methylprednisolone, methylprednisolone succinate, and endogenous corticosterone in rat plasma

A reversed-phase HPLC method is reported for simultaneous quantitation of methylprednisolone (MP), MP succinate (MPS), and endogenous corticosterone (CST) in plasma of rats. Additionally, the 11-keto metabolite of MP (methylprednisone, MPN) is resolved from the other analytes. After addition of internal standard (triamcinolone acetonide; IS) and an initial clean up step, the analytes of interest are extracted into methylene chloride. The steroids are then resolved on a reversed-phase polymer column using a mobile phase of 0.1 M acetate buffer (pH 5.7): acetonitrile (77:23) which is pumped at a flow rate of 1.5 ml min⁻¹. Sample detection was accomplished using an UV detector at a wavelength of 250 nm. All the five components (MPS, MP, MPN, CST and IS) were baseline resolved from each other and other components of plasma. Linear relationships were found between the steroids: IS peak area ratios and plasma concentrations in the range of 0.1–4 μg ml⁻¹ for MP and MPS and 0.1–1.0 μg ml⁻¹ for MPN and CST. The assay is accurate as intra- and inter-run error values were <±8% for all the components. Further, the intra- and inter-run CVs of the assay were <16% at all the concentrations and for all the components. The application of the assay was demonstrated after the injection of a single 5 mg kg⁻¹ (MP equivalent) dose of MPS or a macromolecular prodrug of MP to rats.     

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.     

Disposition of methylprednisolone and its sodium succinate prodrug in vivo and in perfused liver of rats: nonlinear and sequential first-pass elimination

The disposition of methylprednisolone (MP) and its prodrug succinate ester, methylprednisolone sodium succinate (MS), were examined both in vivo and in situ (perfused livers) in rats. In vivo studies included iv and oral dosing of 10 or 50 mg/kg of MP in both forms, while liver perfusion involved initial perfusate concentrations of 5 and 25 micrograms/mL of either compound. Steroid concentrations were measured by HPLC. In the intact rat, clearance (CL) values of both compounds were high, twice the hepatic plasma flow, and decreased by one-half after the high dose, indicating nonlinear kinetics. The volumes of distribution of MS and MP were essentially constant with dose. Incomplete availability of MP from iv MS (52-55%) and from the oral dose (10%) was found. Sequential first-pass metabolism was investigated in situ. Extensive hepatic extraction of MP (84%) occurred at the low dose, but decreased to 48% at the high dose, supporting in vivo observations of high CL and nonlinearity. Extraction of MS was also high (83%), but MP availability was slight (8%). The MS and MP data were fitted to a sequential first-pass model yielding an average fraction of MS metabolized-to-MP value of 0.22. The prodrug MS and the active metabolite MP thus demonstrate both systemic and hepatic nonlinearity in rats, and the low availability of MP from iv MS was due, in part, to sequential first-pass elimination. This factor is more extensive in rats than in other species.     

Pharmacokinetics of methylprednisolone, methylprednisolone sodium succinate, and methylprednisolone acetate in dogs

The absolute bioavailability and pharmacokinetic parameters of two methylprednisolone formulations (methylprednisolone sodium succinate and methylprednisolone acetate) were determined in five dogs. Plasma concentrations of methylprednisolone, methylprednisolone sodium succinate, and methylprednisolone acetate were measured by sensitive and specific high-performance liquid chromatographic methods. After intravenous methylprednisolone sodium succinate administration, methylprednisolone was released rapidly but the extent of availability was rather low (43.6%). This has been tentatively explained in terms of its subsequent single-pass metabolism in the liver, i.e., hepatic hydrolysis of methylprednisolone sodium succinate followed by immediate hepatic elimination of the released methylprednisolone. After intramuscular administration of methylprednisolone acetate, its absorption was slow (half-time of absorption, 69.04 h) and the availability of the released methylprednisolone was low (42.7%). Therapeutic implications of these results are discussed, especially those which are relevant to shock therapy.     

P-glycoprotein modulates aldosterone plasma disposition and tissue uptake

Aldosterone plays an important role in the pathophysiology of numerous cardiovascular disorders including heart failure and hypertension. Because aldosterone's actions are primarily mediated by its interaction with an intracellular mineralocorticoid receptor, factors affecting the cellular uptake and distribution of aldosterone may be important determinants of the hormone's activity. P-glycoprotein (P-gp) is an ATP-binding cassette efflux transporter encoded by the ABCB1 (also known as MDR1) gene in humans. P-gp is expressed on the luminal membrane of the capillary endothelial cells of tissues that are targets for aldosterone, including the brain and heart, where it attenuates cellular uptake of substrates. Recent in vitro evidence indicates P-gp transports aldosterone. Therefore, in this study we tested the hypothesis that P-gp modulates the uptake of aldosterone into the brain and heart by comparing the plasma and tissue distribution of [3H]-aldosterone in wild-type and P-gp-deficient [mdr1a/1b (-/-)] mice. Compared with wild-type mice, [3H]-aldosterone activity in the plasma, brain, and heart was significantly (P < 0.05) higher in the mdr1a/1b (-/-) animals. The area under the plasma or tissue concentration-time curves in the mdr1a/1b (-/-) mice was 2.0, 1.6, and 1.6-fold higher in the brain, heart, and plasma, respectively, than in wild-type controls. Our results demonstrate that P-gp plays an important role in aldosterone plasma disposition and modestly limits its uptake into the brain. The increased exposure of the brain and heart to aldosterone in the absence of P-gp suggests P-gp may play a key role in modulating aldosterone's effects in these organs.     

Pharmacokinetics of methylprednisolone sodium succinate and methylprednisolone in patients undergoing cardiopulmonary bypass

The pharmacokinetics of methylprednisolone sodium succinate (MPHS) and methylprednisolone (MP) were determined in six patients undergoing open heart surgery with cardiopulmonary bypass. Plasma concentrations of both compounds were measured by high-performance liquid chromatography after doses of MPHS of 1.7-2.4 g. The prodrug ester MPHS yields MP with an average formation rate constant of 0.70 +/- 0.29 hr-1. Peak concentrations of MP occur around 1-2 hours after loading and additional administration of MPHS. The pharmacokinetic values of the two drugs in patients having cardiopulmonary bypass were compared to those in younger, healthy subjects. The volume of distribution of MPHS was lower in the patients, and that of MP was similar to the value in controls. Total clearances of both agents were reduced by about 5 and 2 times. The elimination half-life of MPHS was increased slightly, whereas that of MP increased more than twice in the patients. Significant alterations in clearances occurred in patients, but concentrations of MP were appreciable and prolonged MP due to the extensive formation of MP from MPHS and reduced clearance of MP.     

Bioavailability and nonlinear disposition of methylprednisolone and methylprednisone in the rat.

Bioavailability of low (10 mg/kg) and high (50 mg/kg) doses of methylprednisolone was determined after oral administration of the free alcohol of methylprednisolone and iv administration of methylprednisolone sodium succinate. Plasma concentrations of methylprednisolone and methylprednisone (reversible metabolite) were measured by HPLC. Methylprednisolone systemic availability (F) was 49-57% after iv administration and approximately 35% after oral administration. Solubilization of steroids with PEG:ethanol had no effect on their disposition. Apparent systemic clearance (CL) of methylprednisolone was 21 mL/min (low dose), approximately twice the liver blood flow. Dose-dependent changes in steady-state volume of distribution (Vdss) and central volume of distribution (Vdc), volumes, and apparent CL were observed. The methylprednisolone-to-methylprednisone AUC ratio decreased with dose due to saturation of methylprednisone formation clearance (CL12), but this is a minor metabolic pathway. The mean residence time (MRT) increased threefold with dose. Graphical estimates of the Michaelis-Menten capacity (Vmax) and affinity (Km) constants were in reasonable agreement with CL values for the low-dose experimental data. Low systemic availability of iv methylprednisolone sodium succinate was in part due to sequential first-pass hepatic metabolism of the methylprednisolone formed. Methylprednisolone disposition is complex in the rat due to extensive first-pass effects, nonlinear elimination, nonlinear distribution, and reversible metabolism.     

Stereoselective hydrolysis of O-acetyl propranolol as prodrug in rat tissue homogenates.

The stereochemical characteristics of the hydrolysis of O-acetyl propranolol were studied using phosphate buffer (pH 7.4), rat plasma, and rat tissue homogenates. In the phosphate buffer, no difference was observed in the hydrolysis rate between the esters of (R)- and (S)-propranolol. In rat plasma and tissue homogenates, hydrolysis of the ester was both accelerated and stereoselective. Hydrolysis of O-acetyl (R)-propranolol was five times faster than that of the (S)-isomer in rat plasma. However, in the liver and intestine homogenates, the (S)-isomer was hydrolyzed faster than the (R)-isomer. Interconversion between the (R)- and (S)-isomers was not observed under the experimental conditions. The same stereoselective hydrolysis was also observed with racemic O-acetyl propranolol. However, observed rate constants for the hydrolysis were lower than those for the pure isomers. These results indicate that enzymatic hydrolysis of O-acetyl propranolol occurred stereoselectively and the selectivity of the plasma enzyme was different from those of liver and intestine enzymes.     

Compatibility of aminophylline and methylprednisolone sodium succinate intravenous admixtures

The stability of aminophylline and methylprednisolone sodium succinate in admixtures containing both drugs was studied. Admixtures containing aminophylline 1.0 mg/mL and methylprednisolone sodium succinate 2.0 and 0.5 mg/mL were prepared in both 5% dextrose injection and 0.9% sodium chloride injection. Each admixture was prepared in triplicate and samples were kept at room temperature in glass. Immediately after admixture and at one, two, and three hours, samples were visually inspected, tested for pH, filtered, and assayed in duplicate by high-performance liquid chromatography for theophylline concentration and for both methylprednisolone sodium succinate and methylprednisolone alcohol. Control solutions containing only one of the two drugs were also tested. No visual changes were observed. The admixtures had higher pH values after aminophylline was added, but pH of the samples did not change significantly. Aminophylline concentrations did not change significantly throughout the study period. In 0.9% sodium chloride admixtures with methylprednisolone sodium succinate 0.5 mg/mL, less than 90% of the initial methylprednisolone concentration remained at two hours at the 2.0 mg/mL initial concentration, less than 90% remained at three hours. However, methylprednisolone alcohol (a pharmacologically active form of methylprednisolone sodium succinate) was detected in increasing concentrations after the first hour. Aminophylline in a final concentration of 1.0 mg/mL or less can be mixed with methylprednisolone sodium succinate in a final concentration of 2.0 mg/mL or less in 5% dextrose injection or 0.9% sodium chloride injection and administered intravenously within three hours after mixing.     

Compatibility of ondansetron hydrochloride and methylprednisolone sodium succinate in multilayer polyolefin containers.

PURPOSE: The compatibility of ondansetron hydrochloride and methylprednisolone sodium succinate in 5% dextrose injection and 0.9% sodium chloride injection was studied. METHODS: Test solutions of ondansetron hydrochloride 0.16 mg/mL and methylprednisolone sodium succinate 2.4 mg/mL were prepared in triplicate and tested in duplicate. Total volumes of 4 and 2 mL of ondansetron hydrochloride solution and methylprednisolone sodium succinate solution, respectively, were added to 50-mL multilayer polyolefin bags containing 5% dextrose injection or 0.9% sodium chloride injection. Bags were stored for 24 hours at 20-25 degrees C and for 48 hours at 4-8 degrees C. Chemical compatibility was measured with high-performance liquid chromatography, and physical compatibility was determined visually. RESULTS: Ondansetron hydrochloride was stable for up to 24 hours at 20-25 degrees C and up to 48 hours at 4-8 degrees C. Methylprednisolone sodium succinate was stable for up to 48 hours at 4-8 degrees C. When stored at 20-25 degrees C, methylprednisolone sodium succinate was stable for up to 7 hours in 5% dextrose injection and up to 24 hours in 0.9% sodium chloride injection. Compatibility data for solutions containing ondansetron hydrochloride plus methylprednisolone sodium succinate revealed that each drug was stable for up to 24 hours at 20-25 degrees C and up to 48 hours at 4-8 degrees C. CONCLUSION: Ondansetron 0.16 mg/mL (as the hydrochloride) and methylprednisolone 2.4 mg/mL (as the sodium succinate) mixed in 50-mL multilayer polyolefin bags were stable in both 5% dextrose injection and 0.9% sodium chloride injection for up to 24 hours at 20-25 degrees C and up to 48 hours at 4-8 degrees C.     

RP-HPLC法测定注射用甲泼尼龙琥珀酸钠的含量

目的:建立反相HPLC法测定注射用甲泼尼龙琥珀酸钠的含量。方法:采用Agilent TC C18(2)(4.6 mm×250 mm,5μm)色谱柱,以水-四氢呋喃-甲酸(650∶350∶1)为流动相,流速:1.0 mL.min-1,检测波长:245 nm,柱温:30℃,进样体积:20μL。结果:琥珀酸甲泼尼龙在0.01～0.69 mg.mL-1浓度范围内呈良好线性关系(r=0.9999);平均回收率(n=9)为98.3%,RSD为0.3%。结论:本法简便、准确、重复性好,可用于注射用甲泼尼龙琥珀酸钠的含量测定。     

Hydrolysis by carboxylesterase and disposition of prodrug with ester moiety

Prodrug is a useful approach for improving the bioavailability of therapeutic agents through increased passive transport. Carboxylesterases (CESs, EC.3.1.1.1.) that show ubiquitous expression profiles play an important role in the biotransformation of ester-containing prodrugs into their therapeutically active forms in the body. High levels of CESs are found in the liver, small intestine and lungs where prodrugs are firstly hydrolyzed before entering the systemic circulation. Rat intestine single-pass perfusion experiments have shown that CES is involved in the intestinal first-pass hydrolysis. Extensive pulmonary first-pass hydrolysis has been observed in accordance to the substrate specificity of CES1 isozyme. Hydrolysis in the human liver and lungs is mainly catalyzed by hCE1 (a human CES1 family isozyme), whereas that in the small intestine is predominantly mediated by hCE2 (a human CES2 family isozyme). hCE2 preferentially hydrolyzes substrates with a small acyl moiety such as CPT-11, due to conformational steric hindrance in its active site. In contrast, hCE1 is able to hydrolyze a variety of substrates due to spacious and flexible substrate binding region in its active site. In addition, hCE1 has been found to catalyze transesterification. Caco-2 cells mainly expresses CES1 isozyme but not CES2 isozyme. Because of the differences in substrate specificity between CES1 and CES2 enzymes, Caco-2 cell monolayer is not suitable for predicting intestinal absorption of prodrugs. These findings indicate that identification of substrate specificity of CES isozymes and development of an in vitro experimental method are essential to support rational design of prodrug.     

Nefazodone inhibits methylprednisolone disposition and enhances its adrenal-suppressant effect

Nefazodone is a potent and selective inhibitor of cytochrome P450 3A4 (CYP3A4), an enzyme pathway responsible for the biotransformation of a number of steroid compounds. The potential therefore exists that nefazodone inhibits the disposition of methylprednisolone. In this open label, repeated measures study, the effect of 9 days of nefazodone administration on the pharmacokinetic disposition of a single 0.6 mg/kg intravenous dose of methylprednisolone was assessed. Additionally the effect of concomitant nefazodone use on duration of cortisol suppression after methylprednisolone administration was assessed. Eight healthy volunteers completed the study. Following nefazodone administration, the mean (+/-SD) area under the methylprednisolone concentration-time curve was significantly higher (1393 +/- 343 vs. 2966 +/- 928 ug*h/L; P < 0.005), apparent clearance was lower (28.7 +/- 7.2 vs. 14.6 +/- 7.8 L/h; P < 0.02) and the terminal elimination half-life was longer (2.28 +/- 0.49 vs. 3.32 +/- 0.95 hours; P < 0.02). The duration of cortisol suppression after methylprednisolone administration was longer (> or =32 vs. 23.3 +/- 3.43 hours) during nefazodone administration.     

Compatibility of premixed theophylline and methylprednisolone sodium succinate intravenous admixtures

The stability of theophylline supplied as a premixed injection and of methylprednisolone sodium succinate in admixtures containing both drugs was studied. Solutions containing theophylline in concentrations of 4.0 mg/mL and 0.4 mg/mL were used. Methylprednisolone sodium succinate was added to each solution to produce a final concentration of 0.5 mg/mL and 2.0 mg/mL of methylprednisolone alcohol, a pharmacologically active form of methylprednisolone sodium succinate. Each admixture was prepared in triplicate, and samples were kept at room temperature in glass containers. Immediately after admixture and at 3, 6, 12, and 24 hours, samples were visually inspected, tested for pH, filtered, and assayed in duplicate by high-performance liquid chromatography for theophylline concentration and for both methylprednisolone sodium succinate and methylprednisolone alcohol content. Control solutions containing only one of the two drugs were also tested. No visual changes were observed. The addition of theophylline in 5% dextrose injection to the methylprednisolone sodium succinate solutions resulted in decreased pH values for all solutions, which did not vary significantly throughout the study period. Theophylline concentrations did not change significantly compared with baseline. In solutions containing theophylline 0.4 mg/mL with either 2.0 or 0.5 mg/mL of methylprednisolone sodium succinate, less than 90% of the initial methylprednisolone sodium succinate concentrations remained at 24 hours. However, within three hours after admixture preparation, methylprednisolone alcohol was detected in those solutions in increasing concentrations. A commercial preparation of premixed theophylline in 5% dextrose injection in a concentration of 4 mg/mL or less can be mixed with methylprednisolone sodium succinate in a final concentration of 2 mg/mL or less and administered intravenously within 24 hours after mixing.