Thermal stability of a long-acting analogue of luteinizing hormone releasing hormone (D-trp6-pro9-NEt-LHRH).

ABSTRACT

PIP: The stability of solutions of a long acting analogue of luteinizing hormone releasing hormone (D-trp6-pro9-NEt-LHRH [LHRHa]) after heating to 60 degrees Centigrade for 5 days, after repeated freezing and thawing, and after refrigeration at 4 degrees Centrigrade for 8 days was examined. The treatments administered to solutions 2 (60 degrees Centigrade x 5 days), 4 (7 freeze thaw cycles), and 5 (4 degrees Centigrade x 8 days) did not cause any change in the retention time or absorbance (at 210, 230, and 271 nm) of their HPLC profiles when compared to the control solutions 1 and 3. Control solutions degraded with sodium hydroxide (6) showed no LHRHa peak. A 1:1 mixture of the degraded and control solutions (7) gave a peak height of LHRHa that was 51.2% of control. Each of the 5 solutions showed similar biological activity. No effect was seen at a dose of 8 ng, whereas 24 ng caused a significant LH rise (218-277% of the basal level) for each solution. The parallel line potency estimate for solution 2 (60 degrees Centigrade x 5 days) relative to solution 1 was 0.77 (95% confidence limits, 0.32-1.48). The potency ratio for solution 4 (7 freeze thaw cycles) relative to solution 3 was 0.85 (95% confidence limits, 0.23-2.26). In sum, the D-trp6-pro9-NEt-LHRH can withstand mild heating, repeated freezing and thawing, and short term refrigeration without apparent change in HPLC profile or biological activity. The results obtained with the HPLC method correlate will with the results from the "in vivo" bioassay.