IN VIVO MYOTOXICITY OF A LOXAPINE LIPOSOMAL FORMULATION

7. IN VIVO MYOTOXICITY OF A LOXAPINE LIPOSOMAL FORMULATION

The previous in vitro studies indicate the potential of this liposomal formulation to reduce the degree of muscle damage following intramuscular injection. However, this model only looks at the acute short-time effects and specific interactions of the formulation with the muscle tissue. It is therefore criti- cal for the investigator to determine in animal experiments whether the presence of an intact blood flow system and the drug absorption process may alter the findings. It is possible that the toxicity could be enhanced if the dispersed system causes changes in the vascular permeability, thus allowing

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the potential for inflammatory mediators to reach the injec- tion site. Alternatively, it is possible that blood flow causing absorption from the site of injection could reduce the extent of tissue damage as the formulation components could be diluted at the injection site.

The rabbit and rodent have been the primary in vivo models used to assess tissue damage following injection. The markers of tissue damage that have been used primarily are the release of cytosolic enzymes such as creatine kinase or lactate dehydro- genase and histological evaluation. The specific experimental considerations, advantages, and disadvantages of using these two animal models have been previously discussed (16).

The cannulated rodent model may be the preferred model in the testing of dispersed systems for their in vivo myotoxicity because of the ease in injection, handling, and blood sampling.

The volume of formulation needed for the injection proce- dure is smaller than for other animals and myotoxicity can be easily assessed over a relatively short period of time compared to larger animals (usually no longer than 12–36 hr). In these studies, the carotid artery was cannulated for blood sample determination and the test formulations (0.3 mL) were injected into the thigh muscle (musculus rectus femoris). Myotoxicity was assessed by measuring plasma creatine kinase levels over

a 12 hr period and calculating the area under the curve using the trapezoidal rule. In preliminary work, we have found that plasma creatine kinase levels peak at approximately 2 hr post- injection for all formulations and return to baseline serum levels by 12–24 hr.

The formulations investigated in this study were similar to the in vitro studies: phenytoin; normal saline; loxapine

50 mg =mL; loxapine 10.2 mg=mL; and loxapine liposomes

10.2 mg =mL. The hypothesis was to test whether a liposo- mally encapsulated loxapine would be less myotoxic than the commercial formulation or the commercial formulation diluted to the same concentration as the liposomal formula- tion. It was predicted that the liposomal formulation would

be less myotoxic because of the potential to limit the exposure of the muscle tissue to the drug by slowing the release of the drug over time at the site of injection and =or due to the

Case Study: Optimization of Liposomal Formulation 539

absence of the solvent system. Furthermore, in the diluted solution, there could be the potential for the loxapine to pre- cipitate at the site of injection, which could contribute to the myotoxicity. In this study, the concentration of the diluted loxapine solution was matched to the concentration of the lox- apine in the formulated liposomal treatment (10.2 mg =mL) in order to compare the two formulations at the same drug con- centration. The results of these studies are shown in Fig. 5. As in the previous studies, the undiluted commercially available loxapine formulation was more myotoxic, in this case six times, than the positive control phenytoin. The higher myo- toxicity associated with loxapine formulations compared to phenytoin could be a function of differences in the solvent vehicles. The phenytoin (Dilantin) formulation used in these studies contained 40% propylene glycol and 10% ethanol at

a pH of 12. The phenytoin formulation has been shown to have the potential to precipitate at the injection site (17). It

Figure 5 In vivo myotoxicity of liposomal loxapine (10.2 mg =mL), the commercially available loxapine formulation diluted to the same concentration, the commercially available loxapine formulation at

50 mg =mL, phenytoin (50 mg=mL), and normal saline. Mean values are shown above each bar graph.

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is unknown as to whether the loxapine formulation precipi- tated at the site of injection.

The diluted commercially available loxapine formulation was similar in the extent of the myotoxicity to that of pheny- toin. In contrast, there was only a two-fold difference between the in vivo myotoxicity of the liposomal formulation of loxapine compared to normal saline. These findings suggest that the liposomal encapsulation of loxapine causes less myotoxicity compared to the commercially available formulation associated with injection. It is unclear as to the specific mechanism responsible for this reduced myotoxicity. It could be associated with the lack of a solvent system in this formulation. Organic co-solvents have been shown to cause myotoxicity (11–13). Alternatively, it could be due to the slow release of the loxapine from the liposomes, thus minimizing the drug concentration that muscle fibers are exposed to at the injection site and any potential for precipitation.

In summary in vitro and in vivo studies indicated that this liposomal formulation could reduce tissue damage at the injection site. In subsequent pharmacokinetic intravenous and intramuscular studies with this dispersed system, it was suggested that this liposomal formulation could provide sus- tained drug delivery compared to the loxapine solution follow- ing an intramuscular injection (3). Possible mechanisms to explain the reduced myotoxicity include the absence of the co-solvent propylene glycol and the surfactant polysorbate

80 from the formulation and =or the slow release of loxapine from the liposomal formulation, thus minimizing the concen- tration of the drug at the injection site.

8. CONCLUDING REMARKS In this case study, we have demonstrated the importance of

the formulator determining the extent of potential tissue damage caused by the drug and =or other formulation components early during the development process. The type of tissue damage is certainly a function of whether the drug will be administered intravenously, subcutaneously,

Case Study: Optimization of Liposomal Formulation 541

intramuscularly, or via some other parenteral route. The selected route of administration, in turn, will determine what type of in vitro studies should be conducted during the formu- lation development phase to determine the extent of tissue damage caused by the drug and =or other formulation compo- nents (1). The isolated rodent muscle model has been shown to be a useful and rapid system to optimize a given formulation with respect to minimizing tissue damage for numerous drugs and routes of administration. These in vitro studies do not pre- clude studies in animals to further test for tissue damage and the relationship to the drug absorption and pharmacokinetics. However, it should allow the formulator a rational means to determine what would be the most appropriate formulation to utilize in subsequent animal and clinical studies.

REFERENCES 1. Gupta PK, Brazeau GA, eds. Injectable Drug Development.

Techniques to Reduce Pain and Irritation. Denver, CO: Interpharm Press, 1999.

2. Brazeau GA, Cooper BC, Svetic KA, Smith CL, Gupta P. Current perspectives on pain upon injection of drugs. J Pharm Sci 1998; 87:667.

3. Al-Suwayeh SA. Development of an intramuscular liposomal formulation for the antipsychotic drug loxapine. Thesis, University of Florida, 1997.

4. Arrowsmith M, Hadgraft J, Kelleway IW. The in vivo release of cortisone esters from liposomes and the intramuscular clear- ance of liposomes. Int J Pharm 1984; 20:347.

5. Kadir F, Elling WMC, Abrahams D, Zuidema J, Crommelin JA. Tissue reaction after intramuscular injection of lipo- somes in mice. Int J Clin Pharmacol Ther Toxicol 1992; 30:374.

6. Kadir F, Oussoren C, Crommelin DJA. Liposomal formulations to reduce irritation of intramuscularly and subcutaneously administered drugs. In: Gupta PK, Brazeau GA, eds. Inject- able Drug Development. Techniques to Reduce Pain and Irritation. Denver, CO: Interpharm Press, 1999:337.

542 Brazeau 7. American Hospital Formulary Service Drug Information, Amer-

ican Society of Health-System Pharmacists, Inc., Bethesda, MD, 1999:2013.

8. Simpson M, Cooper TB, Lee JH, Young MA. Clinical and plasma level characteristics of intramuscular and oral loxa- pine. Psychopharmacology 1978; 56:225.

9. Munyon WH, Salo R, Briones DF. Cytotoxic effects of neuro- leptic drugs. Psychopharmacology 1987; 91:182.

10. Meltzer Y, Cola PA, Parsa M. Marked elevations of serum creatine kinase activity associated with antipsychotic drug treatment. Neuropsychopharmacology 1996; 15:395.

11. Brazeau GA, Fung H-L. An in vitro model to evaluate muscle damage following intramuscular injections. Pharm Res 1989; 6:167.

12. Brazeau GA, Fung H-L. Use of an in-vitro model for the assess- ment of muscle damage from intramuscular injections: in vitro- in vivo correlation and predictability with mixed solvent systems. Pharm Res 1989; 6:766.

13. Brazeau GA, Fung H-L. The effect of organic cosolvent- induced muscle damage on the bioavailability on intramuscu-

lar 14 C –Diazepam. J Pharm Sci 1990; 79:113. 14. Al-Suwayeh SA, Tebbett IR, Wielbo D, Brazeau GA. In vitro–

in vivo myotoxicity of intramuscular liposomal formulation. Pharm Res 1996; 13:1384.

15. Napaporn, Thomas M, Svetic K, Shahrokh Z, Brazeau GA. Assessment of the myotoxicity of pharmaceutical buffers using an in vitro muscle model: effect of pH, capacity, tonicity and buffer type. Pharm Dev Technol 2000; 5:123–130.

16. Brazeau GA. A primer on in vitro and in vivo cytosolic enzyme release methods. In: Gupta PK, Brazeau GA, eds. Injectable Drug Development Techniques to Reduce Pain and Irritation. Denver, CO: Interpharm Press, 1999:155.

17. Wilensky J, Lowden JA. Inadequate serum levels after intra- muscular administration of diphenylhydantoins. Neurology 1973; 23:318.

Case Study: In Vitro/In Vivo Release from Injectable Microspheres

BRIAN C. CLARK and IAN T. PYRAH PAUL A. DICKINSON

Safety Assessment, AstraZeneca, Pharmaceutical and Analytical R&D,

Macclesfield, U.K. AstraZeneca, Macclesfield, U.K.

1. INTRODUCTION This case study describes in vitro and in vivo characteriza-

tion performed to support manufacturing scale-up, from laboratory to pilot scale, of an experimental microsphere formulation.

The experimental formulation consists of an active pseudo-decapeptide encapsulated in a poly(lactide-co-glycolide) matrix at a target loading of approximately 8% w =w. Micro- spheres were manufactured in a continuous manner by a proprietary process (1) involving the formation of an oil-in-water emulsion, extraction of the organic phase, drying and collec- tion of microspheres in the size range 25–125 mm. Mannitol is

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added to the extraction phase to facilitate subsequent hand- ling. Three formulation variants, based on polymers differing in lactide:glycolide ratio and molecular weight distribution, were investigated in pre-clinical studies and in vivo and in vitro release characteristics were determined. A preliminary in vitro–in vivo correlation was developed for using a rat model, supporting the use of the in vitro release test to select batches for pre-clinical use.

The formulation was designed for parenteral administra- tion by the subcutaneous or intramuscular routes, and hence was required to be sterile. The feasibility of terminal steriliza- tion by g-irradiation using a 25 kGy cycle was investigated, with reference to impurity levels, active agent concentration and long-term stability.

Microspheres were filled into vials for long-term storage. Immediately prior to use, each vial was shaken to break up agglomerated material, an aliquot of suspending medium added, and the vial shaken again to suspend the micro- spheres. A unit dose was then drawn into a hypodermic syr- inge and administered by the chosen route. The short-term stability of the formulation in the sus-pending medium was investigated, with particular emphasis on extraction of active substance into the suspending medium.