(T1030-08-51) Development and Validation of Discriminatory Release Method for Intramammary Drug Products as a Part of In Vitro Quality Metrics

Purpose: Intramammary infusion formulations are products intended for the delivery of therapeutic moieties directly into the udder through the teat canal, thereby maximizing drug exposure at the site of action, the mammary gland, with little to no systemic drug absorption. Currently, no pharmacopeial release method is available to differentiate between the product formulations (i.e., to support in vitro assessments of product bioequivalence) or for quality control testing. Therefore, as a first step in efforts to develop a discriminative method, four in-house test formulations containing ceftiofur hydrochloride were selected in this study for the development and validation of the release method. The FDA-approved intramammary formulation, SpectraMast LC™, was selected as the reference product against which the test formulations were compared.

Methods: Composition of the in-house formulations (F1, F2, F3, and F4) were qualitatively similar to SpectraMast LC™. Formulation F1 and F2 were composed of 7% Wax-408 (microcrystalline wax), and 5% Labrafil M 1944CS. F2 additionally had aluminum monostearate (1%) as a suspending agent. F3 and F4 consisted of 10 and 7% Wax-408, and 5 and 7% Labrafil M 1944CS, respectively. SpectraMast LC™ and in-house formulations were characterized for physicochemical attributes such as particle size distribution (dynamic light scattering and microscopy), rheology, drug content, sedimentation rate, flocculation rate, and weight variation. Release of one gram of each formulation was performed using USP apparatuses 1, 2 (with and without enhancer cells) and 4. Various test parameters were investigated including the effect of medium (milk vs buffer), medium volume (200, 500, and 900 ml), albumin concentration, and paddle/basket speed (50-200 rpm). Enhancer cells were used in different modes, i.e., hanging over the paddle at 45° angle or submerged in the vessel.

Results: The average particle sizes (d₅₀) of SpectraMast LC™ and the in-house formulations F1 and F2 were 13.9±3.9 and 14±4.06 µm, respectively. On the other hand, the average particle sizes of F3 and F4 were 34.08±4.02 and 19.57±4.16 µm, respectively. The flocculation rate of SpectraMast LC™ and formulations F1, F2, and F3 were very similar (1.02-1.03) while it was 3.5 for F4. Spectramast LC^TM and the four in-house formulations exhibited non-Newtonian shear thinning flow rheograms (share rate: 1-100 1/s). The viscosity of SpectraMast LC™, F1, F2, and F3 were 26.74, 27.41, 27.31, and 36.10 Pas, respectively. The solubility of ceftiofur hydrochloride in phosphate buffer (pH 6.4) and milk was 0.097 and 0.839 mg/ml, respectively. The drug release was incomplete ( < 10% in 2 h) in phosphate buffer (pH: 6.4) therefore the buffer media was not further used as a media of choice. USP method 4 was not pursued further after initial trials due to clogging of the lines by the milk. USP apparatuses 1 and 2 were non-discriminatory as release profiles did not change proportionally with changing speed, volume, basket mesh size, etc. USP apparatus 2 with submerged enhancer cell was discriminatory, and showed clear effect of paddle speed, and volume, etc. Metallic mesh of various sizes was used to retain the formulation in the enhancer cell instead of membrane. The release from the enhancer cells using mesh #20 dissolved 100% of the drug within 10 min. Mesh #120 and #400 dissolved < 1% after 120 minutes. Mesh #40 dissolved drug gradually over a period of 2 h. Using the latter mesh size, the drug dissolved from SpectraMast LC™ after 2 hrs at paddle speed of 75, 100, 125, and 150 rpm were 20.9, 24.7, 90.9, and 96.3% respectively (Figure 1). The volume of the release medium affected both the rate and extent of drug release. using mesh #40 and a paddle speed of 125 rpm, medium volumes of 200, 500, and 900 ml resulted in SpectraMast LC^TM % dissolved of 18.6, 38.0, and 100.7, respectively, after 2 h of testing. Release profiles of SpectraMast LC™ (95.1%) was similar to F1 (99.03%) and F2 (99.4%) formulations while it was different from F3 (39.5%) and F4 (55.7%) formulations (Figure 2). The results from the hanging enhancer cells were similar to those from the submerged one (mesh number: 40, rotation speed: 125 rpm and 900 ml milk).

Conclusion: The developed discriminatory release method can differentiate between formulations having different composition or prepared by various manufacturing methods. The method can be used as a part of in-vitro quality metrics for product quality comparison and as a surrogate for in-vivo performance since intramammary products act locally.

Acknowledgements: The opinions expressed in this article are those of the authors and should not be interpreted as the position of the US Food and Drug Administration.

Figure 1: Cumulative percent concentration of Ceftiofur HCl released from SpectraMast™ LC at different rotation speeds.

Figure 2: Cumulative percent concentration of Ceftiofur HCl released from SpectraMast™ LC and the in-house formulations.