IN VITRO LIPOSOMAL MYOTOXICITY STUDIES
5. IN VITRO LIPOSOMAL MYOTOXICITY STUDIES
The optimization process during formulation development must include studies early on in the process that test the tis- sue-damaging potential of the individual components in the formulation. If this process is employed, the formulator is able to select those excipients or formulation factors that will mini- mize tissue damage in the final formulation. Prior to develop- ing any liposomal drug formulations, it is essential to determine the influence of the selected liposomal composition and characteristics, in the absence of drug, on muscle damage (14). Three specific factors were investigated in these studies: liposome charge (positive or negative), size (large, ranging from 1.5 to 2.0 mm and small, ranging from 0.2 to 0.5 mm) and fluidity. The ratio of the lipid components of the various lipid formulations is discussed below.
Liposomes were prepared using the standard thin film hydration method (3,14). The two sizes of liposomes (1.5–2.0 and 0.2–0.5 mm) were obtained using extrusion through poly- carbonate membranes (3,14) followed by size determination using laser light scattering. Negatively charged liposomes were prepared using phosphatidylcholine and phosphatidyl- glycerol (PC–PG) (7:3 M). Positive liposomes were prepared using phosphatidylcholine and stearylamine (PC–SA) (9:1). The fluidity of the membranes was changed by adding choles- terol to the liposomes. The ratio of phosphatidylcholine: stearylamine:cholesterol (PC–SA–CH) was 7:1:2 M for the positively charged liposomes, while for the negatively charged liposomes the ratio of phosphatidylcholine:phosphatidylgly-
Case Study: Optimization of Liposomal Formulation 533
cerol:cholesterol (PC–PG–CH) was 4:3:3 M. The total concen- tration of lipids in these formulations was 25 mg =mL. Since we injected 15 ml of each formulation, a total of 0.375 mg of lipid was injected into each muscle in these in vitro studies.
Since it will often be necessary to conduct tissue damage or pharmacokinetic studies in separate groups of animals with a given formulation over a period of 1–2 weeks, the investigator must ensure that the prepared liposomes do not change their size with time. The size of all the tested formula- tions was stable over 6–11 days, with the coefficient of varia- tion of size as a function of time ranging from 3 to 10% (3).
The myotoxicities of these selected formulations are shown in Fig. 3. For comparison, the two negative controls, saline and untreated muscles, and the two positive control formulations, Phenytoin (Dilantin Õ , a commercially available formulation at a concentration of 50 mg =mL with 40% propy- lene glycol, 10% ethanol at pH 12) and a muscle sliced in half, are provided as reference points. All of the eight liposomal for- mulations were determined to be equal to the normal saline negative control in creatine kinase released, but significantly lower than the two positive controls. It could be concluded
Figure 3 In vitro myotoxicity of empty liposomal formulations.
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that these selected liposomal formulations did not result in muscle damage following injection compared to normal saline. Furthermore, the extent of muscle damage in these selected formulations appeared to be independent of liposome size, charge, or fluidity. These findings provided further evidence to demonstrate the compatibility of these dispersed systems containing phosphatidylcholine, phosphatidylglycerol steary- lamine, and cholesterol with muscle tissue.
Another important consideration is the pH of the final formulation. In separate studies using buffer solutions, it was demonstrated that muscle myotoxicity was related to pH with more acidic preparations causing more myotoxicity relative to formulations with pH values near physiological or muscle pH (6.0). Furthermore, myotoxicity was related to proton concentration and buffer capacity. It was found that myotoxicity was greater in those formulations at low pH with higher buffer capacity compared to those with lower buffer capacity (15).