Enhanced efficacy against cervical carcinomas through polymeric micelles physically incorporating the proteasome inhibitor MG132.

Treatment of recurrent or advanced cervical cancer is still limited, and new therapeutic choices are needed for improving prognosis and quality of life of patients. Because human papilloma virus (HPV) infection is critical in cervical carcinogenesis, with the E6 and E7 oncogenes of HPV degrading tumor suppressor proteins through the ubiquitin proteasome system, the inhibition of the ubiquitin proteasome system appears to be an ideal target to suppress the growth of cervical tumors. Herein, we focused on the ubiquitin proteasome inhibitor MG132 (carbobenzoxy-Leu-Leu-leucinal) as an anticancer agent against cervical cancer cells, and physically incorporated it into micellar nanomedicines for achieving selective delivery to solid tumors and improving its in vivo efficacy. These MG132-loaded polymeric micelles (MG132/m) showed strong tumor inhibitory in vivo effect against HPV-positive tumors from HeLa and CaSki cells, and even in HPV-negative tumors from C33A cells. Repeated injection of MG132/m showed no significant toxicity to mice under analysis by weight change or histopathology. Moreover, the tumors treated with MG132/m showed higher levels of tumor suppressing proteins, hScrib and p53, as well as apoptotic degree, than tumors treated with free MG132. This enhanced efficacy of MG132/m was attributed to their prolonged circulation in the bloodstream, which allowed their gradual extravasation and penetration within the tumor tissue, as determined by intravital microscopy. These results support the use of MG132 incorporated into polymeric micelles as a safe and effective therapeutic strategy against cervical tumors.

Neuromuscular recovery after peripheral nerve repair: effects of an orally-administered peptide in a primate model.

Oral delivery of the tripeptide calpain inhibitor, leupeptin, after median nerve transection and epineural nerve repair in primates (Cebus apella) was studied for its potential benefits to neuromuscular recovery. Results of a controlled, dose-response study indicated that leupeptin was absorbed into plasma by the oral route of administration. When plasma leupeptin concentrations were 3 micrograms/ml or greater, morphologic and functional motor recovery were facilitated after nerve repair. Serial testing in hematology, clotting, and serum biochemistry showed that there were no adverse effects, when leupeptin was administered twice daily for 6 months following nerve repair. These data indicate that leupeptin is an effective and safe pharmaceutic adjunct to nerve repair and may have clinical benefits in humans, where the oral route is a much preferred method of delivery.

Enhancement of neuromuscular recovery after nerve repair in primates.

This investigation describes the use of the calcium-activated protease inhibitor, leupeptin, as an adjunctive therapy to the microsurgical repair of median nerves in a primate model. Our results indicate that leupeptin facilitates morphological recovery in denervated thenar muscles and in distal sensory and mixed motor-sensory nerve trunks and functional recovery measured by motor nerve conduction velocity. Toxicological testing of leupeptin showed that, when administered at a dose of 12 mg/kg, intramuscularly, once daily, haematological and clotting profiles were not adversely affected.

Assay of plasma leupeptin using the reversible binding of leupeptin to bovine pancreatic trypsin.

A competitive binding radioassay for leupeptin has been developed utilizing the reversible binding of leupeptin to bovine pancreatic trypsin. An ethanol precipitation step was introduced to separate trypsin-bound leupeptin from its free form. Advantages of this method are simplicity of the procedure and avoidance of the preparation of antiserum. The possible metabolites of leupeptin exhibit no significant inhibitory effect on leupeptin-trypsin binding in this system. This method was applied to the determination of plasma leupeptin levels in dogs after oral administration of the peptide.

High-performance liquid chromatographic method for monitoring leupeptin in mouse serum and muscle by pre-column fluorescence derivatization with benzoin.

A high-performance liquid chromatographic method is described for the determination of leupeptin, a possible therapeutic drug for muscular dystrophy, in mouse serum and muscle. Leupeptin is reduced with sodium borohydride to leupeptinol, and then converted to a fluorescent derivative with benzoin. The derivative is separated on a reversed-phase column (LiChrosorb RP-18) with isocratic elution and determined with fluorescence detection. The detection limits of leupeptin in serum and muscle are 250 pmol/ml (107 ng/ml) and 500 pmol/g (214 ng/g), respectively, corresponding to approximately 150 fmol each in a 100-microliters injection volume. This method is simple and sensitive enough to permit the quantification of leupeptin in biological samples from mice dosed with leupeptin.