Evaluation of self-dissolving needles containing low molecular weight heparin (LMWH) in rats

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Abstract

Feasibility study of self-dissolving needles containing polysaccharide was performed. Low molecular weight heparin (LMWH) was used as a representative polysaccharide. Using chondroitin, dextran and dextrin as the base, self-dissolving needles (SDN) were prepared. The obtained SDNs were evaluated in rat absorption experiment, where pharmacological availability (PA) was calculated by comparing the plasma anti-Xa activity vs. time curves between SDNs and i.v. solution. After the insertion of SDNs to rats skin where the doses of LMWH were 25, 50 and 100 IU/kg, plasma samples were collected for 6 h and anti-Xa activity was measured as the pharmacological index of LMWH. The anti-Xa level was maintained above 0.2 IU/ml, the therapeutic level, for about 2 h at a dose of 100 IU/kg. Almost the same PAs of LMWH were obtained with dextran and dextrin SDNs, 97.7% and 102.3%, though lower PA was obtained with chondroitin SDN, 81.5%. In vitro dissolution experiment showed that LMWH was released from dextran, dextrin and chondroitin SDNs within 10 min. The T50%s were 0.84 ± 0.06 min for dextran SDN, 1.07 ± 0.12 min for chondroitin SDN and 2.11 ± 0.31 min for dextrin SDN, respectively. Plasma anti-Xa activity vs. time profiles showed good dose-dependency in the 25–100 IU/kg range and high PAs were obtained, 90.0% for 25 IU/kg, 95.4% for 50 IU/kg and 97.7% for 100 IU/kg from dextran SDNs. Stability experiment was performed with dextran SDNs for 3 months. Above 97% of LMWH were remained in SDNs under three different conditions, −80, 4 and 40 °C. These results suggest the usefulness of SDN to polysaccharide drug.

Introduction

Transdermal drug delivery system (TDDS) made a great progress of DDS and several drugs like estrogen, nicotine and nitroglycerin are clinically supplied as patch preparations (Asmussen, 1991). However, the skin has a strong barrier function for the permeation of other drugs, especially macromolecular drugs. To increase the skin permeability, many approaches including chemical enhancers, electric fields, ultrasound and thermal methods have been challenged (Barry and Williams, 2003, Cevc, 2004, Preat and Vanbever, 2004, Doukas, 2004, Mitragotri and Kost, 2004). However, the application of these TDDSs has been limited because of the strong barrier function of the skin. To overcome these problems, microneedles are under investigation actively (Cormier et al., 2004, Martanto et al., 2004, Davis et al., 2005, Park et al., 2006). We have been studying a new delivery system, self-dissolving microneedles, for the percutaneous administration of macromolecular drugs. In our feasibility studies, insulin and erythropoietin (EPO) were used as representatives of peptide/protein drugs (Ito et al., 2006a, Ito et al., 2006b). With insulin, the efficiency was evaluated by measuring the hypoglycemic effect of insulin after administration of insulin self-dissolving microneedles to mice. The pharmacological availability (PA) of SDN was over 90% as compared to intravenously injected insulin solution (Ito et al., 2006a). Also, EPO self-dissolving microneedles showed high bioavailability (BA) of EPO after percutaneous administration to mice where the BA was about 80% (Ito et al., 2006b). Through these proof-of-concept experiments, the usefulness of self-dissolving microneedles was found out with peptide/protein drugs. On the other hand, there are many macromolecular drugs except peptide/proteins. One of the categories is polysaccharide like heparin.

Heparin is an anticoagulant drug for the treatment of deep vein thrombosis and pulmonary embolism (Agnelli and Sonaglia, 2000, Ageno, 2000). Unfractionated heparin (UH) is a naturally occurring glycosaminoglycan that exists as a heterogenous mixture of oligosaccharides composed of alternating chains of d-glucosamine and uronic acid (Hirsh et al., 1992). Heparin is sulfated, highly acidic and has a negative ionic charge. In many countries, low molecular weight heparins (LMWHs) have replaced UH for the prevention and treatment of venous thrombo-embolism (Boneu, 2000) due mainly to a longer half-life and less bleeding for a given antithrombotic effect compared to UH. Furthermore, the frequency of heparin-induced thrombocytopenia is lower with LMWHs and almost non-existent when used in the short-term because of less immunogenic nature (Lane et al., 1984). LMWHs have a molecular weight of approximately 4.5 kDa and, compared to UH (12.0 kDa), show better distribution and less binding to non-anticoagulant-related plasma proteins and platelets (Heit, 1998). However, LMWH is not absorbed from the gastrointestinal tract owing to its high charge density and large molecular size and is only given by injection.

Therefore, LMWH was used as a representative polysaccharide drug and feasibility study of self-dissolving needles (SDN) for the percutaneous administration of LMWH has been studied in this report.

Section snippets

Materials

LMWH (Parnaparin sodium, anti-Xa (aXa) factor activity: 85.4 IU/mg) was obtained from Ajinomoto Co. Ltd. (Tokyo, Japan). Dextrin was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Chondroitin sulfate and dextran was obtained from Nacalai Tesque Co. Ltd. (Kyoto, Japan). Male Wistar rats used in the study were obtained from Nippon SLC Company (Hamamatsu, Japan) and standard solid meal of commercial food (LabDiet®) was purchased from Nippon Nousan Co., Ltd. (Yokohama, Japan).

Results

LMWH loaded SDN was evaluated in the rat absorption experiment and the results are shown in Fig. 2. Self-dissolving needle is composed of drug and polymer base. After SDN was inserted into the skin, both drug and polymer dissolved with the environmental water and thereafter the drug was thought to be absorbed into the systemic circulation. At first, the effect of base polymer on the absorption of LMWH from rat skin was studied. Dextran and chondroitin based SDNs showed almost the same plasma

Discussion

Based on the development of micro-fabrication technology, the use of micron-scale needles showed a dramatical increase of the skin permeability of drugs. Namely, solid microneedles made of stainless steel have been shown to increase the transdermal permeability of drugs (Kim et al., 2004, Chandrasekaran et al., 2003, Griss et al., 2002). Therefore, microneedles of which size is microns dimensions has been given much attention for the transdermal delivery of drugs having poor membrane

Conclusion

A new self-dissolving needles (SDNs) containing LMWH have been prepared for the percutaneous administration of LMWH. After administration of LMWH SDNs, plasma ant-Xa activity vs. time profiles were studied with three thread-forming polymers, i.e., dextrin, chondroitin and dextran. The Cmax were 0.40 ± 0.03, 0.46 ± 0.03 and 0.47 ± 0.06 IU/ml. AUAs were 1.56 ± 0.23, 1.24 ± 0.10 and 1.49 ± 0.08 IU h/ml. Tmax were 1.67, 0.83 and 1.00 h. The physiological availabilities were 102.3%, 81.5% and 97.7%, respectively.

Acknowledgements

This study was supported in part by a Grant-in-Aid for Scientific Research provided by Ministry of Education, Science and Culture of Japan. This research was also supported in part by a “Collaboration with Bio-Venture Companies” Project for Private Universities: matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology, MEXT), 1999–2006 of Japan and Venture SME-University Research Promotion Program of Japan Society for the Promotion of Science.

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