LOXAPINE LIPOSOMAL FORMULATIONS AND IN VITRO MYOTOXICITY STUDIES

6. LOXAPINE LIPOSOMAL FORMULATIONS AND IN VITRO MYOTOXICITY STUDIES

Once the dispersed system without the drug has been shown not to cause tissue damage, the investigator must formulate the desired therapeutic agent in this system such that it achieves the requisite concentration (or solubility) and stabi- lity of the active ingredient. In addition, the final formulation itself must be stable for storage during the study period. It becomes critical to ensure that the active drug is stable in the liposomal formulation both in vitro and in vivo. It also becomes critical to have a sensitive and selective assay that can differentiate between the active agent, metabolites and components of the dispersed system.

Loxapine liposomes were formulated using phosphatidyl- choline:phosphatidylglycerol (7:3 M) at a drug to lipid ratio of 1:2 M and a lipid concentration of 100 mg =mL. The aver -age liposome diameter was 1.1 mm and the encapsulation efficiency was 62

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loxapine in the final formulation for all studies was between

12.2 and 12.4 mg =mL. This was the highest concentration that could be achieved in this formulation; however, it was considered that this would be suitable for future studies based upon the loxapine serum levels and the available HPLC assay methodology. In vitro release studies demonstrated that

71 tonic phosphate buffer 7.4 and a pH 6.0 muscle homogenate, respectively (3).

It now becomes important to determine the myotoxicity of the final formulation to determine if the liposome formulation can reduce tissue damage. In the next study, the myotoxicity of loxapine at a concentration of 12.2 mg =mL in phosphatidyl- choline:phospatidylglycerol liposome was compared with: the commercially available loxapine product (50 mg =mL) with pro- pylene glycol and polysorbate 80; the commercial formulation diluted to the same concentration as the liposomal formula- tion; and the drug free loxapine solvent system (70% propylene glycol and 5% polysorbate 80). The last two treatments are important because this will allow a direct comparison with the liposomal formulation and will also indicate the extent to which these two excipients in the commercial formulation con- tribute to the myotoxicity of the final formulation, respec- tively. In any myotoxicity study, it is always recommended to include the solvent controls and to ensure that the concen- trations of all ingredients are the same as myotoxicity is most often concentration dependent. It would also be important to include as a control the blank liposomal formulation, without drug, as a comparison. In earlier studies and as shown in Fig. 3 , the liposomal formulation to be used in these studies,

7.3 M phosphatidylcholine:phosphatidylglycerol and a size of

1.1 mm (classified as a large liposome) was no more myotoxic than normal saline. As such, this liposomal formulation appears to be non-myotoxic.

The results of the myotoxicity studies for these formula- tions are shown in Fig. 4 . Similar to Fig. 3, the positive and negative controls have been provided as reference points. Interestingly, the myotoxicity of the undiluted commercially available loxapine solution was markedly higher than the

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Figure 4 In vitro myotoxicity of liposomal loxapine (12.2 mg =mL), the commercially available loxapine formulation at the same concen- tration (12.2 mg =mL), the commercially available loxapine formula- tion at 50 mg =mL, and the solvent system for the commercially available loxapine formulation. Mean values are shown above each bar graph.

two positive controls. This is not surprising as the phenytoin formulation only contains 40% propylene glycol compared to 70% propylene glycol in the loxapine solution. Furthermore, this formulation does not contain any surfactant. The solvent system for the commercially available formulation appeared to have contributed significantly to the myotoxicity of the final formulation [84.1 (SEM)]. It is often difficult to discriminate between the myo- toxicity caused by the drug and that caused by the solvent sys- tem because of the difficulty in determining the myotoxicity of the drug alone due to limited aqueous solubility. In the present studies, it was also impossible to determine whether loxapine precipitation occurring at the site of injection caused the myotoxicity.

Furthermore, when the original formulation was diluted to the same concentration as the formulated liposomal

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formulation (12.2 ng =mL), the extent of myotoxicity was not remarkably reduced compared to the undiluted solution [139 solution is diluted, the myotoxicity could have been caused by precipitation of the drug at the injection site as both the drug and solvent system concentration in the formulation is reduced. As such, the solvent system at this lower concentra- tion does not have adequate solubilizing power to keep the drug in solution. These findings suggest that simply diluting the commercially available loxapine formulation does not reduce the extent of myotoxicity.

In contrast, loxapine encapsulated in a liposomal formu- lation significantly (p < 0.05) reduced muscle damage by 80% compared to the commercially available formulation (includ- ing drug and the solvent system) diluted to the same concen- tration. However, the tissue damage was approximately seven times higher compared to normal saline. It appeared that this dispersed system formulation was able to signifi- cantly reduce tissue damage associated with loxapine admin- istration. This suggests that this formulation might be useful for intramuscular administration from the perspective of reducing tissue damage. However, it still needs to be deter- mined if the same results are observed following intramuscu- lar administration in an animal model.