Analysis of the Metabolism of Dextran-Methylprednisolone Succinate Using
SubjectDrugs--Metabolism.; Drugs--Analysis.; Transplantation immunology.; Immunosuppressive agents.; Adrenocortical hormones.
A brief review of past and present immunosuppressive treatments, including corticosteroids,cyclosporine A, and tacrolimus as they relate to liver transplantation is presented. The most common and detrimental side effects are presented for each therapy, and a method to reduce these adverse effects is proposed. As a method of drug delivery targeted to the liver, methylprednisolone is joined to a 70,000 Da dextran molecule through a succinic acid linker to form Dextran-methylprednisolone succinate @exMPS). A newly developed reversed-phase HPLC assay is validated, and is capable of simultaneous measurement of methylprednisolone (MP), methylprednisolone succinate (MPS), methylprednisone (MPN), and corticosterone (CST) in addition to an internal standard (IS), triamcinolone acetonide. Ratios between drug plasma concentration and IS peak area were determined to be linear in the range of 0.1-4 pglml for MP and M P S , and 0.1- 1.0 pg/ml for MPN and CST. Assay intra- and inter-run errors were less than 8%, demonstrating its accuracy and precision. The efficiency of the assay was determined by analysis of samples with and without the extraction steps. All components were at least 80% extracted. Application of the assay to in vitro hydrolysis experiments included DexMPS incubation in rat blood and phosphate buffer, and in rat liver lysosomes and various buffers. The half-life of DexMPS in blood was determined to be approximately 25 hours, and breakdown is most likely caused by chemical hydrolysis. The hydrolysis of MPS to MP is thought to be enzymatic, based on 1 0-fold faster breakdown in blood than in buffer. No significant hydrolysis was observed in isolated lysosomes for DexMPS or MPS. In addition, preliminary studies regarding rat spleen lymphocyte isolation and mitogen stimulation were performed. Optimum conditions for culture of spleen lymphocytes require RPMI-1640 to be supplemented with 2.5% rat serum. Separation of rat liver cells was accomplished by perfbsion of the liver with HEPES buffer containing collagenase, followed by centrihgation through a density gradient of Percoll to yield populations of hepatocytes (93% pure, with 80% viability) and Kupffer cells (98% pure, with 92% viability). Together, these experiments provide a framework for examining the delivery of DexMPS to the liver, and measuring the effect of the DexMPS hydrolysis in vivo.
39 leaves. Advisor: Dean Hoganson.