Preparation, Quality Control And Related In Vivo Studies Of Octreotide Acetate Microspheres

Somatostatin (SS) is a polypeptide that inhibits the release of multiple hormones. However, its clinical application has been largely limited due to its extremely short half-life. Octreotide,a somatostatin analogue(SSA), has therefore been artificially synthesized by various methods.Octreotide,an octapeptide, has higher bioactivity, stronger inhibition of hormones secretion yet longer in vivo half-life(approximately 2 hours), than SS. It has been clinically used mainly for the treatment of cirrhotic portal hypertension, alimentary tract hemorrhage, acromegalia and multiple endocrine tumors, etc.. However, frequent injections may cause great inconvenience to the patients as these diseases require long-term treatment and daily administration of two doses is necessary to reach effective plasma concentration. Currently, long-acting Octreotide preparations have been developed by various overseas companies. Long-acting octreotide microsphere (brand name Sandostatin LAR) produced by Novartis has been on Chinese market since 2004.This drug, however, may be financially unaffordable as it is expense as with other import drugs. This paper discusses how the process parameters for bench scale test of octreotide acetate microspheres were verified and determined using biodegradable polylactic acid glycolic acid (PLGA) as the carrier to make octreotide acetate microspheres into microspheres by encapsulation, how the preparation parameters for pilot scale test were assessed and determined, and how the prepared microspheres were subject to quality study and assessed in terms of in vivo safety and efficacy.In this paper, multiple emulsion solvent evaporation is used to prepare octreotide acetate microspheres considering the fact that microspheres of water-soluble agents have such advantages as high drug load, good homogeneity and stable drug release, which makes octreotide acetate particularly suitable since it is extremely soluble. In the bench scale test, single- and multiple-factor tests were conducted to assess the effects of preparation conditions including inner aqueous phase concentration, oil phase concentration, outer aqueous phase concentration, stirring speed of primary and multiple emulsions, etc., on the appearance and release characteristics of octreotide acetate microsphere, which helped to determine the process parameters. In the single-factor test based on the results of in vitro release test and the size of microspheres, the following critical conditions for the preparation of octreotide acetate microsphere were preliminarily determined:1) The amount of octreotide used is 150mg.The ratio between octreotide and PLGA is 1:9(w/w);2) Primary emulsion is prepared under ice-bath cooling. Ultrasonic emulsification is used for bench scale test, and homogenate emulsification for tests on pilot and above scales, the emulsification time being 5 min;3) The volume of outer aqueous phase solution is at least 50 times that of oil phase. Fixed volume of outer aqueous phase solution for bench scale test is 300mL;4) Stirring over 2 hours is used during solution volatilization , with magnetic bar used for small scale preparation and mechanical stirring used for large scale preparation. A seven-factor, three-level orthogonal test for three selected variables was then conducted based no the pre-determined ranges of each parameter, to verify and determine the process parameters for bench scale preparation of octreotide acetate microsphere. The final process parameters for bench scale preparation were determined.Octreotide acetate microspheres prepared according to the above parameters have the encapsulation rate of 70%, yield of 80% and average microsphere size of 55μm; the microsphere has good globulation and smooth surface; in the subsequent in vitro release test, it was proved that the burst ratio was less than 5%, and the microspheres may be slowly released at a certain rate over 4 weeks, resulting in the total release rate at 8-week termination of approximately 80%.Octreotide acetate microspheres under three different multiple emulsion stirring conditions were prepared using the optimized parameters for bench scale tests and some pilot scale process conditions, so as to test the size and encapsulation rate of microspheres; liquid phase assays were used to test the in vitro release of octreotide in 28 days, so as to assess the most appropriate in vitro release test methods that were to be used to determined the in vitro release profile of octreotide microspheres prepared on pilot scale. Microspheres prepared on pilot scale were assessed combing the results of the above tests and the final preparation parameters of microsphere were determined. The results suggest that octreotide acetate microspheres in Group B demonstrated more well-distributed release, with the release time exact 28 days, which fits the efficacy cycle. Furthermore, the three batches of microspheres showed good reproducibility in the pattern of in vitro release with generally consistent test results within a relatively small range.In the quality study of octreotide acetate microsphere, certain property indicators of octreotide acetate microsphere were investigated, including the appearance, change pattern of relevant substances, drug load, shape, grain size and distribution of microspheres, water content, pH, burst, and release ratio, etc.. The results suggest that the three batches of octreotide acetate microspheres were all white or off-white lyophilized powders; times to HPLC peaks were consistently similar to those of the reference; specific rotation of octapeptide in the three batches were 38.5°,38.9°, and 39.2°, respectively; amino acids assays, ultraviolet spectra scans, dichlormethane residue, sterility tests, pyrogen tests, abnormal toxicities, and size, drug load and content of microspheres were all in conformity with the specifications. In addition, the stability of octreotide acetate microspheres was studied in the long-term stability study where octreotide acetate microsphere injections were tested at temperature 6℃+2℃and relative humidity 60+10%., as well as corresponding accelerated test at temperature 25℃+2℃and relative humidity 60+10% as the preparations were sensitive to temperature. Experimental evidence was provided regarding the production, packaging, storage and transportation conditions of the product; the expiry time of octreotide acetate microsphere injection was also determined. The results of stability studies suggest that three consecutive batches of octreotide acetate microspheres, batch numbers 200701, 200702, and 200703, were stable in description, pH, water content, grain size & distribution values, drug load& content, and relevant substances, after storage of either 18 months at temperature 6℃+2℃and relative humidity 60+10%, or 6 months at temperature 25℃+2℃and relative humidity 60+10%, which means octreotide acetate microspheres can be stored for 6 months at temperature 25℃+2℃and relative humidity 60+10% , and for 18 months at temperature 6℃+2℃and relative humidity 60+10%, suggesting the good stability of octreotide acetate microsphere.In a pharmacokinetic study in beagles, the pharmacokinetic variables and bioavailability were compared betweenlong-acting octapeptide Sandostatin LAR and octreotide acetate microsphere. The results suggest that the pharmacokinetics of Sandostatin LAR and octreotide acetate microsphere, compared to that of Sandostatin, demonstrated constant and slow release of octapeptide, which significantly increased the detention time of octapeptide in the body, allowing longer time for concentration to remain above the effective level. Octreotide acetate microsphere will not induce other adverse effects than those commonly seen with Sandostation since the burst phase plasma concentration of octreotide acetate microsphere is no higher than that of Sandostatin. The AUC(0-1200) observed after single dose of octreotide acetate microsphere and AUC(0-720)(720h[30d] as one dosing cycle) observed after multiple doses of octreotide acetate microsphere were not statistically different, suggesting that the preparation does not accumulate in the body. The AUC0-(t0-1200 h) of Sandostation LAR and octreotide acetate microsphere, after injection, were 554.39±15.6ng·h/mL and 555.11±19.074ng·h/mL, respectively. With Sandostation LAR as the reference, the relative bioavailability of octapeptide in octreotide acetate microsphere was 98.12%, suggesting bioequivalency.Octreotide acetate microsphere was suggested to be safe and effective in animals in studies investigating the pharmacokinetics which provides evidence for the clinical application of octreotide acetate microsphere.