Development and Validation of Discriminatory In-vitro Release Method for Intramammary Drug Product.

PURPOSE: Intramammary (IMM) formulations are locally acting and delivered intracisternally into the udder. No pharmacopeial in-vitro release method is available to differentiate between the IMM formulations. Our research aim is to develop in-vitro release methods that discriminate different IMM formulations (SPECTRAMAST® LC and in-house formulations). METHODOLOGY: Different in-house formulations were developed to simulate SPECTRAMAST® LC generics. SPECTRAMAST® LC and the in-house formulations were characterized for physicochemical attributes, such as particle size, rheology, drug content, sedimentation rate, and flocculation rate. The in-vitro release method was optimized by evaluating drug release using USP apparatuses 1, 2 (with and without enhancer/customized cells), and 4. Various test parameters, including medium effect (whole homogenized bovine milk versus aqueous buffer), medium volume (200-900 mL), and rotational speed (50-200 rpm) were investigated. RESULTS: Two potential in-vitro systems can be used as discriminatory methods for IMM formulations: USP apparatus 2 with the IMM formulation loaded into two containers a) customized formulation container (83.1 cm in height and 56.4 cm in width) or b) enhancer cells with their top adapted with mesh #40 (rotation speed:125 rpm and 900 mL of whole homogenized bovine milk). The release profile of SPECTRAMAST® LC at 1 h (99.8%) was not significantly different from formulations with similar physicochemical characteristics F-01 (99.1%) and F-02 (100.5%). Formulation with different physicochemical characteristics F-03 (44.3%) and F-04 (57.2%) showed slower release (1 h) than SPECTRAMAST® LC (98.8%). CONCLUSION: The developed in-vitro release methods can be used as a potential tool for in-vitro comparability evaluations for IMM formulations.

Specificity of pyrrolysyl-tRNA synthetase for pyrrolysine and pyrrolysine analogs.

Pyrrolysine, the 22nd amino acid, is encoded by amber (TAG=UAG) codons in certain methanogenic archaea and bacteria. PylS, the pyrrolysyl-tRNA synthetase, ligates pyrrolysine to tRNA(Pyl) for amber decoding as pyrrolysine. PylS and tRNA(Pyl) have potential utility in making tailored recombinant proteins. Here, we probed interactions necessary for recognition of substrates by archaeal PylS via synthesis of close pyrrolysine analogs and testing their reactivity in amino acid activation assays. Replacement of the methylpyrroline ring of pyrrolysine with cyclopentane indicated that solely hydrophobic interactions with the ring-binding pocket of PylS are sufficient for substrate recognition. However, a 100-fold increase in the specificity constant of PylS was observed with an analog, 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid (2Thf-lys), in which tetrahydrofuran replaced the pyrrolysine methylpyrroline ring. Other analogs in which the electronegative atom was moved to different positions suggested PylS preference for a hydrogen-bond-accepting group at the imine nitrogen position in pyrrolysine. 2Thf-lys was a preferred substrate over a commonly employed pyrrolysine analog, but the specificity constant for 2Thf-lys was 10-fold lower than for pyrrolysine itself, largely due to the change in K(m). The in vivo activity of the analogs in supporting UAG suppression in Escherichia coli bearing genes for PylS and tRNA(Pyl) was similar to in vitro results, with L-pyrrolysine and 2Thf-lys supporting the highest amounts of UAG translation. Increasing concentrations of either PylS substrate resulted in a linear increase in UAG suppression, providing a facile method to assay bioactive pyrrolysine analogs. These results illustrate the relative importance of the H-bonding and hydrophobic interactions in the recognition of the methylpyrroline ring of pyrrolysine and provide a promising new series of easily synthesized pyrrolysine analogs that can serve as scaffolds for the introduction of novel functional groups into recombinant proteins.

Direct charging of tRNA(CUA) with pyrrolysine in vitro and in vivo.

Pyrrolysine is the 22nd amino acid. An unresolved question has been how this atypical genetically encoded residue is inserted into proteins, because all previously described naturally occurring aminoacyl-tRNA synthetases are specific for one of the 20 universally distributed amino acids. Here we establish that synthetic L-pyrrolysine is attached as a free molecule to tRNA(CUA) by PylS, an archaeal class II aminoacyl-tRNA synthetase. PylS activates pyrrolysine with ATP and ligates pyrrolysine to tRNA(CUA) in vitro in reactions specific for pyrrolysine. The addition of pyrrolysine to Escherichia coli cells expressing pylT (encoding tRNA(CUA)) and pylS results in the translation of UAG in vivo as a sense codon. This is the first example from nature of direct aminoacylation of a tRNA with a non-canonical amino acid and shows that the genetic code of E. coli can be expanded to include UAG-directed pyrrolysine incorporation into proteins.