Model-Informed Development and Registration of a Once-Daily Regimen of Extended-Release Tofacitinib.

Extended-release (XR) formulations enable less frequent dosing vs. conventional (e.g., immediate release (IR)) formulations. Regulatory registration of such formulations typically requires pharmacokinetic (PK) and clinical efficacy data. Here we illustrate a model-informed, exposure-response (E-R) approach to translate controlled trial data from one formulation to another without a phase III trial, using a tofacitinib case study. Tofacitinib is an oral Janus kinase (JAK) inhibitor for the treatment of rheumatoid arthritis (RA). E-R analyses were conducted using validated clinical endpoints from phase II dose-response and nonclinical dose fractionation studies of the IR formulation. Consistent with the delay in clinical response dynamics relative to PK, average concentration was established as the relevant PK parameter for tofacitinib efficacy and supported pharmacodynamic similarity. These evaluations, alongside demonstrated equivalence in total systemic exposure between IR and XR formulations, provided the basis for the regulatory approval of tofacitinib XR once daily by the US Food and Drug Administration.

Design and synthesis of piperazine-based matrix metalloproteinase inhibitors.

A new generation of cyclic matrix metalloproteinase (MMP) inhibitors derived from dl-piperazinecarboxylic acid has been described. The design involves: incorporation of hydroxamic acid as the bidentate chelating agent for catalytic Zn(2+), placement of a sulfonamide group at the 1N-position of the piperazine ring to fill the S1' pocket of the enzyme, and finally attachment of diverse functional groups at the 4N-position to optimize potency and peroral absorption. A unique combination of all three elements produced inhibitor 20 with high affinity for MMPs 1, 3, 9, and 13 (24, 18, 1.9, and 1.3 nM, respectively). X-ray crystallography data obtained for MMP-3 cocrystallized with 20 gave detailed information on key binding interactions defining an overall scaffold geometry for piperazine-based MMP inhibitors.

Development of new hydroxamate matrix metalloproteinase inhibitors derived from functionalized 4-aminoprolines.

A series of hydroxamates was prepared from an aminoproline scaffold and tested for efficacy as matrix metalloproteinase (MMP) inhibitors. Detailed SAR for the series is reported for five enzymes within the MMP family, and a number of inhibitors, such as compound 47, display broad-spectrum activity with sub-nanomolar potency for some enzymes. Modifications of the P1' portion of the molecule played a key role in affecting both potency and selectivity within the MMP family. Longer-chain aliphatic substituents in this region of the molecule tended to increase potency for MMP-3 and decrease potency for MMP-1, as exemplified by compounds 48-50, while aromatic substituents, as in compound 52, generated broad-spectrum inhibition. The data is rationalized based upon X-ray crystal data which is also presented. While the in vitro peroral absorption seemed to be less predictable, it tended to decrease with longer and more hydrophilic substituents. Finally, a rat model of osteoarthritis was used to evaluate the efficacy of these compounds, and a direct link was established between their pharmacokinetics and their in vivo efficacy.

Design, synthesis, and biological evaluation of matrix metalloproteinase inhibitors derived from a modified proline scaffold.

The synthesis and structure-activity relationship (SAR) studies of a series of proline-based matrix metalloproteinase inhibitors are described. The data reveal a remarkable potency enhancement in those compounds that contain an sp(2) center at the C-4 carbon of the ring relative to similar, saturated compounds. This effect was noted in compounds that contained a functionalized oxime moiety or an exomethylene at C-4, and the potencies were typically <10 nM for MMP-3 and <100 nM for MMP-1. Comparisons were then made against compounds with similar functionality where the C-4 carbon was reduced to sp(3) hybridization and the effect was typically an order of magnitude loss in potency. A comparison of compounds 14 and 34 exemplifies this observation. An X-ray structure was obtained for a stromelysin-inhibitor complex which provided insights into the SAR and selectivity trends observed within the series. In vitro intestinal permeability data for many compounds was also accumulated.

Improved prediction of in vivo peroral absorption from in vitro intestinal permeability using an internal standard to control for intra- and inter-rat variability.

PURPOSE: To evaluate the use of an in vitro intestinal permeability model to predict rat and human absorption as well as to evaluate the use of an internal standard to control for intra- and inter-rat variability. METHODS: In vivo peroral absorption and in vitro steady-state intestinal permeability coefficients were determined in the rat for a variety of structurally different compounds with different physicochemical properties including: progesterone, hydrocortisone, salicylic acid, caffeine, clonidine, p-aminoclonidine, UK-14304, oxymetazoline, mannitol, PEG 900, PEG 4000, and a number of novel hydrophilic chemical entities. RESULTS: The intestinal permeability coefficients determined in vitro could be used to predict the peroral absorption of a compound in both the rat and human. Normalizing the permeability of a test compound to an internal standard, e.g. mannitol, greatly improved the prediction of peroral absorption. CONCLUSIONS: The use of an internal standard can aid in the prediction of the peroral absorption of a test compound, in particular, for one that has moderate absorption in the range of 20-80%. Moreover, these methods would appear to be a useful means to improve the prediction of other absorption models as well, such as the Caco-2 cell systems and in-situ perfusion methods.

Plasmid DNA entry into postmitotic nuclei of primary rat myotubes.

These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin- or gold-labeled plasmid and plasmid DNA expression vectors for Escherichia coli beta-galactosidase or firefly luciferase were microinjected into the cytoplasm of primary rat myotubes in culture. Plasmid DNA was expressed in up to 70% of the injected myotubes, which indicates that it entered intact, postmitotic nuclei. The nuclear transport of plasmid DNA occurred through the nuclear pore by a process common to other large karyophilic macromolecules. The majority of the injected plasmid DNA was sequestered by cytoplasmic elements. This understanding of plasmid DNA nuclear transport provides a basis for increasing the efficiency of gene transfer.

Expression of naked plasmids by cultured myotubes and entry of plasmids into T tubules and caveolae of mammalian skeletal muscle.

Plasmid DNA or artificial mRNA injected intramuscularly into skeletal muscle via a 27 g needle expressed transgenes at relatively efficient levels in skeletal myofibers and cardiac cells. In the present study, several approaches were used to determine the mechanism of cellular uptake. After exposure of naked plasmid DNA, primary rat muscle cells in vitro expressed transgenes to a much greater extent than other types of immortalized or primary cells. In vivo light microscope studies showed that intramuscularly injected plasmid DNA was distributed throughout the muscle and was able to diffuse through the extracellular matrix, cross the external lamina, and enter myofibers. Electron microscope studies showed that colloidal gold conjugated to plasmid DNA traversed the external lamina and entered T tubules and caveolae, while gold complexed with polylysine, polyethylene glycol or polyglutamate primarily remained outside of the myofibers. The results indicate that it is highly unlikely that the plasmid DNA enters the myofiber simply by the needle grossly disrupting the sarcolemma. In addition, transient membrane disruptions do not appear to be responsible for the uptake of DNA. Furthermore, no evidence for endocytosis could be found. The possible uptake of plasmid DNA by some type of cell membrane transporter, in particular via potocytosis, is discussed.

Transport of thyrotropin releasing hormone in rabbit buccal mucosa in vitro.

The transport of thyrotropin releasing hormone (TRH) in rabbit buccal mucosa in vitro has been investigated with respect to (a) rate and type of metabolism of TRH on mucosal and serosal sides of buccal mucosa, (b) mechanism of TRH transport including charge effect on its permeability, and (c) pathway and rate-limiting regions of TRH movement. In addition, the integrity of excised buccal mucosa has been evaluated for purposes of in vitro solute diffusion experiments using tissue ATP level data, transmission electron microscopy, and TRH transport kinetic data. The results indicate that excised rabbit buccal mucosa can be used for TRH diffusion studies for approximately 6 hr. In addition, TRH apparently traverses buccal mucosa by simple diffusion with a steady-state permeability of about 10(-7) cm/sec, and this permeability is independent of pH. Moreover, the primary pathway appears to be via the intercellular space in the rate-limiting barrier, i.e., the upper 50 microns of the epithelium. Finally, TRH is degraded predominantly by deamidase activity, which is followed by, to a lesser degree, carboxypeptidase metabolism.