Preparation, Characterization, and in Vivo Evaluation of an Oil Suspension of a Bovine Growth Hormone Releasing Factor Analog LAWRENCE X. YU X ,TODD P. FOSTER,RON W. SARVER, AND WILLIAM M. MOSELEY Received September 18, 1995, from the Pharmacia & Upjohn Inc., 7000 Portage Road, Kalamazoo, MI 49001. Final revised manuscript received January 24, 1996. Accepted for publication January 30, 1996 X . Abstract 0 Pharmacia & Upjohn Inc. has discovered a superior bovine growth hormone releasing factor analog, [Ile 2 ,Ser 8,28 ,Ala 15 ,Leu 27 ,Hse 30 ] bGRF (1-30)-NH-ethyl, acetate salt (U-90699F), for enhancement of animal growth. This report delineates the preparation, characterization, and in vivo evaluation of a U-90699F oil suspension. The oil suspension formulation was selected because of its simplicity, inexpensiveness, and ability to produce sustained U-90699F release. 40% U-90699F monomers were incorporated into Miglyol oil with acceptable injectability. Both reversed-phase-high-pressure liquid chromatography (RP-HPLC) and Fourier transform infrared spectroscopy (FTIR) were utilized to evaluate its chemical and structural stability. This suspension formulation demonstrated no significant changes in concentration as determined by RP-HPLC for 10 weeks at both 25 and 39 °C. The U-90699F dispersed in oil also showed no signs of structural conversion from R-helix to -sheet as monitored by FTIR. However, there was an increase in R-helix/ disordered coil structure after initial drug incorporation. The suspension was subcutaneously administered to Holstein steers. Areas under the serum somatotropin concentration curve were used to determine the duration of action. It was found that the suspension was able to effectively elevate serum somatotropin for at least 14 days. Introduction Bovine growth hormone releasing factor (bGRF) is a 44 amino acid hypothalamic peptide that stimulates the release of somatotropin from the pituitary gland. 1 Somatotropin (ST) directly or indirectly decreases lipogenesis and promotes protein accretion, resulting in enhanced growth. As with GRFs from other species, bGRF (1-29)-NH 2 retains full growth hormone releasing activity. 2 Pharmacia & Upjohn Inc. has discovered a superior bGRF analog, [Ile 2 ,Ser 8,28 ,Ala 15 ,- Leu 27 ,Hse 30 ] bGRF (1-30)-NH-ethyl, acetate salt (U-90699F), for enhancement of animal growth. Native bGRF is rapidly inactivated by blood plasma in vitro and in vivo via cleavage between Ala 2 -Asp 3 peptide by dipeptidylepeptidase IV. 3 To prepare GRF analogs with high activity in vivo, a strategy was undertaken to selectively replace amino acid residues. It was found that an Ile 2 substitution greatly stabilized peptides against proteolysis in plasma. 4 The replacement of Gly 15 with Ala 15 marginally improved in vitro intrinsic growth hormone (GH) releasing activity and enhanced plasma stability. The modifications of Ser 8,28 and Leu 27 ameliorated solution stability and precluded oxidation of native bGRF. The C-terminal Hse 30 -NH-ethyl analogs released more ST in vivo. Overall, these improve- ments resulted in a super-potent bGRF analog, U-90699F, superior to the native bGRF both in vitro and in vivo. However, U-90699F is not orally active and is limited to parenteral delivery. Furthermore, a sustained-release for- mulation is desirable to reduce the frequency of animal handling in animal production facilities and minimize stress to the treated animals. A suspension formulation is clearly a prime choice of dosage forms because of its relative simplic- ity, inexpensiveness, and ability to produce sustained U-90699F release. Unfortunately, U-90699F is prone to gelation and aggregation in an aqueous system, associated with a second- ary structure transition from disordered coil/R-helix to inter- molecular -sheet. 5 Such a conformational transition is accelerated by increases in temperature, peptide concentra- tion, and ionic strength. Table 1 shows a typical estimate of U-90699F secondary structure obtained from analysis of a U-90699F aqueous gel/suspension. The gelation and aggrega- tion result in a dramatic reduction in aqueous solubility of U-90699F to less than 10 µg/mL at pH 7 (unpublished data). Similar phenomena were observed for other peptides, such as glucagon and insulinotropin, and other proteins. 6-9 It is unlikely that such a solubility would be able to sustain effective plasma concentration upon its delivery. Therefore, an oil suspension was explored to reduce -sheet formation and maintain efficient drug release. Injectable oil suspensions generally provide sustained drug release although rapid absorption has sometimes been ob- served. 10,11 The mechanism for sustaining release is a com- bination of the hydrophobic vehicle delaying drug dissolution and the vehicle protecting drugs from water, enzymes, and ions which hasten drug degradation. Various protein, peptide, and low molecular weight drugs have been dispersed in oils to yield sustained-release formulations. 12-21 One of the first oil suspensions was made in 1950 when an injectable depot formulation of penicillin was developed by dispersing small particles of penicillin G procaine in vegetable oils, such as peanut or sesame oils. The dispersion was then gelled with approximately 2% of aluminum monostearate. 12 Intramuscular injection of this formulation produced pro- longed therapeutic blood levels of penicillin. Other examples of long-acting formulations using oils as vehicles for low molecular weight drugs include up to 50% pamoate salt of normorphinones and 40% free base or pamoate salt of ox- azepines in oils gelled with aluminum monostearate. 13,14 Formulations for prolonged release of proteins in oils have been reported. Anschel has prepared suspensions of relaxin (about 2%) in a vegetable oil gelled with aluminum monostear- ate. 15 Brameley et al. 16 prepared a sustained-release injection suspension containing proteins, such as bovine somatotropin, with concentrations from 5% to 45%. The protein was dispersed in an oil together with a fatty acid and an absorption regulator. An oil suspension for prolonged release of biologi- cally active somatotropin was recently patented. 17 This suspension contained about 10-50% somatotropin in an oil with acceptable injectability. It has been shown that 10-15% somatotropin suspensions in sesame or peanut oils provide at least 7 days’ sustained release in vivo. Other oil suspensions include those reported by Geller 18 and Nestor et al., 19 who prepared extremely low concentration oil formulations for corticotropin (0.03-0.1%) and LHRH (0.01- 1%), respectively. A pharmaceutical formulation of monocyclic peptide, especially cyclosporin, in an oil vehicle has been X Abstract published in Advance ACS Abstracts, March 1, 1996. 0022-3549/96/3185-0396$12.00/0 396 / Journal of Pharmaceutical Sciences © 1996, American Chemical Society and Vol. 85, No. 4, April 1996 American Pharmaceutical Association + +