Plasma Pharmacokinetics and Tissue Disposition of Novel Dextran- Methylprednisolone Conjugates with Peptide Linkers in Rats

The plasma and tissue disposition of two novel dextran prodrugs of methylprednisolone (MP) containing one (DMP-1) or five (DMP-5) amino acids as linkers were studied in rats. Single 5-mg/kg doses (MP equivalent) of each prodrug or MP were administered intravenously, and blood and tissue samples were collected. Prodrug and drug concentrations were quantitated using HPLC, and noncompartmental pharmacokinetic parameters were estimated. Whereas conjugation of MP with dextran in both prodrugs substantially decreased the clearance of the drug by ∼200-fold, the accumulations of the drug in the liver, spleen, and kidneys were significantly increased by conjugation. However, the extent of accumulation of DMP-1 in these tissues was substantially greater than that for DMP-5. Substantial amounts of MP were regenerated from both prodrugs in the liver and spleen, with the rate of release from DMP-5 being twice as fast as that from DMP-1. However, the AUCs of MP regenerated from DMP-1 in the liver and spleen were substantially higher than those after DMP-5. In contrast, in the kidneys, the AUC of MP regenerated from DMP-5 was higher than that after DMP-1 administration. These data suggest that DMP-1 may be more suitable than DMP-5 for targeting immunosuppression to the liver and spleen

Synthesis and in Vitro Characterization of Novel Dextran–Methylprednisolone Conjugates with Peptide Linkers: Effects of Linker Length on Hydrolytic and Enzymatic Release of Methylprednisolone and its Peptidyl Intermediates

To control the rate of release of methylprednisolone (MP) in lysosomes, new dextran–MP conjugates with peptide linkers were synthesized and characterized. Methylprednisolone succinate (MPS) was attached to dextran 25 kDa using linkers with 1–5 Gly residues. The release characteristics of the conjugates in pH 4.0 and 7.4 buffers, blood, liver lysosomes, and various lysosomal proteinases were determined using a size-exclusion and/or a newly developed reversed-phase HPLC method capable of simultaneous quantitation of MP, MPS, and all five possible MPS-peptidyl intermediates. We synthesized conjugates with ≥90% purity and 6.9–9.5% (w/w) degree of MP substitution. The conjugates were stable at pH 4.0, but released MP and intact MPS-peptidyl intermediates in the pH 7.4 buffer and rat blood, with faster degradation rates for longer linkers. Rat lysosomal fractions degraded the conjugates to MP and all the possible intermediates also at a rate directly proportional to the length of the peptide. Whereas the degradation of the conjugates by cysteine peptidases (papain or cathepsin B) was relatively substantial, no degradation was observed in the presence of aspartic (cathepsin D) or serine (trypsin) proteinases, which do not cleave peptide bonds with Gly. These newly developed dextran conjugates of MP show promise for controlled delivery of MP in lysosomes

Liquid Chromatography-Tandem Mass Spectrometry for the Determination of Methylprednisolone in Rat Plasma and Liver After Intravenous Administration of Its Liver-Targeted Dextran Prodrug

A specific and sensitive liquid chromatography (LC)-tandem mass spectrometric method for quantitative determination of methylprednisolone (MP) in rat plasma and liver was developed and validated using triamcinolone acetonide as an internal standard. Liquid-liquid extraction using tert-butyl methyl ether was used to extract the drug and the internal standard from plasma and liver. The separation of MP was performed on a C(18) column with a mobile phase of acetonitrile:0.5% formic acid aqueous solution (85:15, v/v) over 4 min. The assay was based on the selected reaction monitoring transitions at m/z 375 -\u3e 161 for MP in plasma, 375 -\u3e 357 for MP in liver, and 435 -\u3e 415 for internal standard in both plasma and liver. The lower limit of quantification was 20 ng/mL based on 100 mu L of plasma or liver homogenate. Intra- and inter-clay assay variations wer

Role of glutamate in lead-induced toxicity: protection by N-acetylcysteine amide (NACA), a novel thiol antioxidant

Despite several efforts to reduce lead levels in the environment, lead exposure continues to be a major public health problem, in the US today. Toxic effect of lead are mainly manifested in central nervous system (CNS) because brain acts as a reservoir for lead deposition. Lead-induced cell damage include glutamatergic component (excitotoxic cell damage arising from impaired clearance of the released glutamate), interference with calcium-mediated cellular processes, and activation of protein kinase C (PKC) resulting in oxidative stress. Chelation therapy has been a choice to reduce the body burden of lead; however chelation does not eliminate the neurotoxicity of lead...In this study we propose to evaluate the involvement of glutamatergic component in lead-induced toxicity and also to investigate a potential compound which counteracts the entire toxic phenomenon of lead --Abstract, page iii

Comparative evaluation of N-acetylcysteine (NAC) and N-acetylcysteine amide (NACA) on glutamate and lead-induced toxicity in CD-1 mice

Recent studies indicate that there is interaction between the glutamatergic neurotransmitters system and lead neurotoxicity. Previously, we have demonstrated the potential effects of glutamate in lead-induced cell death in PC12 cells and the protective role of the novel thiol antioxidant, N-acetylcysteine amide (NACA). The current study (1) investigated the potential effects of glutamate on lead exposed CD-1 mice, (2) evaluated the protective effects of NACA against glutamate and lead toxicity in CD-1 mice, and (3) compared the results with N-aceytylcysteine (a well-known thiol antioxidant). Oxidative stress parameters, including glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG, and malondialdehyde (MDA) levels, were evaluated. Blood and tissue lead levels, glutamate/glutamine (Glu/Gln) ratios, GS activity, and phospholipase-A2 (PLA2) were also analyzed. Results indicated that lead and glutamate decreased GSH levels in the red blood cells, brains, livers, and kidneys. Exposure to glutamate and lead elevated the MDA levels and PLA2 activity. NACA and N-acetylcysteine (NAC) provided protection against the detrimental effects of lead by decreasing the blood and tissue lead levels, restoring intracellular GSH levels, and decreasing the MDA levels. NACA and NAC also increased the GS activity thereby decreasing Glu/Gln levels. However, NACA appeared to have better chelating and antioxidant properties than NAC, due to its higher liphophilicity and its ability to cross the blood-brain barrier

Liquid Chromatography Analysis of N-(2-mercaptopropionyl)-glycine in Biological Samples by ThioGlo™ 3 Derivatization

N-(2-Mercaptopropionyl)-glycine (MPG) is a synthetic aminothiol antioxidant that is used in the treatment of cystinuria, rheumatoid arthritis, liver and skin disorders. Recent studies have shown that MPG can function as a chelating, cardioprotecting and a radioprotecting agent. Several other studies have shown that it may also act as a free radical scavenger because of its thiol group. Thiol-containing compounds have been detected in biological samples by various analytical methods such as spectrophotometric and colorimetric methods. However, these methods require several milliliters of a sample, time-consuming procedures and complicated derivatization steps, as well as having high detection limits. the present study describes a rapid, sensitive and relatively simple method for detecting MPG in biological tissues by using reverse-phase HPLC. with ThioGlo™ 3 [3H-Naphto[2,1-b] pyran, 9-acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl) phenyl-3-oxo-)] as the reagent, highly fluorescent derivatives of thiols can be obtained that are suitable for HPLC. MPG is derivatized with ThioGlo™ 3 and is then detected flourimetrically by reverse phase HPLC using a C18 column as the stationary phase. Acetonitrile: Water (75:25) with acetic acid and phosphoric acid (1 mL/L) is used as the mobile phase (excitation wavelength, 365 nm; emission wavelength, 445 nm). the calibration curve for MPG is linear over a range of 10-2500 nM (r=0.999) and the coefficients of the variation of within-run and between-run precision were found to be 0.3 and 2.1%, respectively. the detection limit was 5.07 nM per 20 μL injection volume. Quantitative relative recovery of MPG in the biological samples (plasma, lung, liver, kidney and brain) ranged from 90.5±5.3 to 106.7±9.3%. Based on these results, we have concluded that this method is suitable for determining MPG in biological samples