Common Deficiencies with Bioequivalence Submissions in Abbreviated New Drug Applications Assessed by FDA

Purpose

A generic product must meet the standards established by the Food and Drug Administration (FDA) to be approved for marketing in the USA. FDA approves a generic product for marketing if it is proved to be therapeutically equivalent to the reference product. Bioequivalence (BE) between a proposed generic product and its corresponding reference product is one of the major components of therapeutic equivalence. These approvals may be delayed if the BE portion of the submission is determined to be deficient. Many of these BE deficiencies recur commonly and can be avoided.

Method

We conducted a survey of the BE submissions to abbreviated new drug applications (ANDAs) over years 2001 to 2008 to identify the most commonly occurring BE deficiencies.

Results

Recurring deficiencies are found in a majority of the ANDAs reviewed by FDA’s Division of Bioequivalence. The most common deficiencies were the two deficiencies related to dissolution (method and specifications) found in 23.3% of the applications and analytical method validation and/or report found in 16.5% of the applications. The approval of generic drugs would be greatly accelerated if these deficiencies could be avoided.KEY WORDS: ANDA, bioequivalence, common deficiency, FDA     

Regulation of the H4 tail binding and folding landscapes via Lys-16 acetylation

Intrinsically disordered proteins (IDP) are a broad class of proteins with relatively flat energy landscapes showing a high level of functional promiscuity, which are frequently regulated through posttranslational covalent modifications. Histone tails, which are the terminal segments of the histone proteins, are prominent IDPs that are implicated in a variety of signaling processes, which control chromatin organization and dynamics. Although a large body of work has been done on elucidating the roles of posttranslational modifications in functional regulation of IDPs, molecular mechanisms behind the observed behaviors are not fully understood. Using extensive atomistic molecular dynamics simulations, we found in this work that H4 tail mono-acetylation at LYS-16, which is a key covalent modification, induces a significant reorganization of the tail’s conformational landscape, inducing partial ordering and enhancing the propensity for alpha-helical segments. Furthermore, our calculations of the potentials of mean force between the H4 tail and a DNA fragment indicate that contrary to the expectations based on simple electrostatic reasoning, the Lys-16 mono-acetylated H4 tail binds to DNA stronger than the unacetylated protein. Based on these results, we propose a molecular mechanism for the way Lys-16 acetylation might lead to experimentally observed disruption of compact chromatin fibers.     

On the dephasing of genetic oscillators

The digital nature of genes combined with the associated low copy numbers of proteins regulating them is a significant source of stochasticity, which affects the phase of biochemical oscillations. We show that unlike ordinary chemical oscillators, the dichotomic molecular noise of gene state switching in gene oscillators affects the stochastic dephasing in a way that may not always be captured by phenomenological limit cycle-based models. Through simulations of a realistic model of the NFκB/IκB network, we also illustrate the dephasing phenomena that are important for reconciling single-cell and population-based experiments on gene oscillators.Cyclic rhythms are a common feature of many self-organized systems (1) manifesting themselves in myriad forms in biology, ranging from subcellular biochemical oscillations to cell division and on to the familiar predator–prey cycles of ecology. An oscillatory response of a gene regulatory circuit, whether transient or self-sustained, can provide several advantages over a temporally monotonic response (2, 3). The ability of copies of a system to synchronize may lead to dramatic noise reduction and greater precision for timing in assemblies. On the subcellular level, rhythmic dynamics span time scales from a few seconds, as in the calcium oscillations, to days, as in the circadian rhythms, or years for cicada cycles (1, 4–6). Ultradian genetic oscillations, which take place on an intermediate scale from minutes to a few hours, are medically important. A singularly important case is the Nuclear Factor Kappa B (NFκB) gene network, which organizes the mammalian cell’s response to various types of external stress and plays a role in regulating inflammation levels in populations of cells. The response of the NFκB circuit to continuous external stimulation has been studied by Hoffmann et al. (7) who observed damped oscillatory dynamics for NFκB, which has been linked to the presence or absence of particular isoforms of the inhibitor IκB. On the other hand, experiments carried out on individual cells have detected more sustained NFκB/IκB oscillations that either are completely self-sustained (8, 9) or damp at a much slower rate (9) than found for the population, depending on the duration of external stimulation. It follows that some type of averaging takes place, but the physical mechanisms and the stochastic aspects of this population averaging are not fully understood. Many aspects of the NFκB oscillatory dynamics may be rationalized using deterministic mass action rate equations (10, 11), but how stochastic self-sustained oscillations average out at a cell population level remains unclear.In this work, we provide a conceptual framework for understanding stochastic averaging as a result of “dephasing” of genetic oscillators. By dephasing, one essentially means the loss of common phase or coherence of oscillations in populations of mRNA or protein by-products of gene activation, caused by the stochastic molecular events in the course of an oscillator’s operation that occur at different times in different cells. When the phases of genetic oscillators are not controlled by external coupling to other cells or by exogenous signals, the random character of molecular events induces phase diffusion. In the absence of synchronizing forces, an ensemble of genetic oscillators in the long time limit inevitably will be dephased completely as part of the entropic drive to randomize the phase distribution across the ensemble. In an extreme deterministic limit of ”newtonian” genetic oscillators, there would be no dephasing and the oscillators would preserve the memories of their phases forever. The oscillators of the cell, on the other hand, are driven by inherently fluctuating molecular entities and will get out of phase and eventually lose memory of their initial phase in a rather modest time (12, 13).We explore a particularly simple yet realistic model of the NFκB/IκB circuit (Fig. 1) and demonstrate how self-sustained stochastic single-cell oscillatory dynamics yield damped oscillations at the population level, as observed in the experiments of Hoffmann et al. (7). Another related but more fundamental question is how the single-molecule nature of the gene contributes to the stochasticity of gene oscillator networks. In our model, we explicitly account for the highly non-Gaussian noise that comes from a single gene switching on and off and investigate how the time scale of gene-state fluctuation affects the noisiness of the oscillations.Open in a separate windowFig. 1.A minimalist model of an NFκB/IκB genetic oscillator. Bold arrows indicate binding (k_on) and unbinding (k_off) of NFκB to the gene. Once bound, mRNA is produced, which initiates the synthesis (k_tl) of the IκB. The copies of mRNA also are constantly degraded. The IκB inhibits the NFκB by binding to it and preventing the activation of the gene. The IKK drives the irreversible degradation of IκB in the complex and prevents the full deactivation of NFκB.     

Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation

The activation of initiator protein tissue factor (TF) is likely to be a crucial step in the blood coagulation process, which leads to fibrin formation. The stimuli responsible for inducing TF activation are largely undefined. Here we show that the oxidoreductase protein disulfide isomerase (PDI) directly promotes TF-dependent fibrin production during thrombus formation in vivo. After endothelial denudation of mouse carotid arteries, PDI was released at the injury site from adherent platelets and disrupted vessel wall cells. Inhibition of PDI decreased TF-triggered fibrin formation in different in vivo murine models of thrombus formation, as determined by intravital fluorescence microscopy. PDI infusion increased - and, under conditions of decreased platelet adhesion, PDI inhibition reduced - fibrin generation at the injury site, indicating that PDI can directly initiate blood coagulation. In vitro, human platelet-secreted PDI contributed to the activation of cryptic TF on microvesicles (microparticles). Mass spectrometry analyses indicated that part of the extracellular cysteine 209 of TF was constitutively glutathionylated. Mixed disulfide formation contributed to maintaining TF in a state of low functionality. We propose that reduced PDI activates TF by isomerization of a mixed disulfide and a free thiol to an intramolecular disulfide. Our findings suggest that disulfide isomerases can act as injury response signals that trigger the activation of fibrin formation following vessel injury.