Refining the in vitro release test method for a dapivirine-releasing vaginal ring to match in vivo performance.

Previously reported in vitro release test methods for drug-releasing vaginal rings containing poorly water-soluble drugs have described use of water-alcohol systems or surfactant solutions in efforts to maintain sink conditions. Here, as part of efforts to more closely match in vitro and in vivo release for the 25 mg dapivirine matrix-type silicone elastomer vaginal ring for HIV prevention, we have investigated alternatives to the 1:1 v/v water/isopropanol medium described previously. Specifically, we evaluated dapivirine release from rings into (i) monophasic water/isopropanol mixtures of varying compositions and (ii) biphasic buffer/octanol systems using pH 4.2 and pH 7.0 buffers. The rate and mechanism of dapivirine release were dependent upon the isopropanol concentration in the release medium, in accordance with the observed trend in drug solubility. At 0 and 10% v/v isopropanol concentrations, dapivirine release followed a partition-controlled mechansim. For media containing ≥ 20% v/v isopropanol, in vitro release of dapivirine was significantly increased and obeyed permeation-controlled kinetics. Cumulative release of ~3.5 mg dapivirine over 28 days was obtained using a water isopropanol mixture containing 20% v/v isopropanol, similar to the ~4 mg dapivirine released in vivo. Dapivirine release into the biphasic buffer/octanol system (intended to mimic the fluid/tissue environment in vivo) was constrained by the limited solubility of dapivirine in the buffer component in which the ring resided, such that cumulative dapivirine release was consistently lower than that observed with the 20% v/v isopropanol in water medium. Release into the biphasic system was also pH dependent, in line with dapivirine's pKa and with potential implications for in vivo release and absorption in women with elevated vaginal pH.

Use of simulated vaginal and menstrual fluids to model in vivo discolouration of silicone elastomer vaginal rings.

Vaginal rings releasing antiretrovirals - either alone or in combination with contraceptive progestins - are being developed for prevention of human immunodeficiency virus (HIV) transmission via vaginal sex. Following Phase I trials, significant discolouration was observed on the surface of investigational silicone elastomer antiretroviral-contraceptive matrix-type vaginal rings containing either 25 mg dapivirine or 200 mg dapivirine plus levonorgestrel. In this study, potential causes of the discolouration have been assessed in vitro using simulated vaginal and menstrual fluids (SVF and SMF, respectively) to model in vivo exposure. The fluid compositions also included hydrogen peroxide (H2O2), hydrogen peroxide plus a copper intrauterine device (IUD), or synthetic dyes (representing personal care and household cleaning products). No discolouration was observed for rings exposed to SVF + hydrogen peroxide (with or without an IUD). However, the SVF + dye compositions showed significant ring discolouration, with staining patterns similar to those observed with rings that had been exposed to highly-coloured personal care and household cleaning products during clinical trial use. Exposure of rings to SMF compositions invariably caused yellow surface discolouration, dark spotting and markings, similar to the staining patterns observed following clinical use. The darker marks on the ring surface were identified as blood debris derived from the SMF. The study indicates that surface discolouration of rings in vivo can be attributed to exposure to menstrual fluid or highly coloured personal care or household cleaning products. Discolouration of the rings was not associated with any specific safety risks for the user, though severe discolouration could potentially impact acceptability and adherence.

Drug stability and product performance characteristics of a dapivirine-releasing vaginal ring under simulated real-world conditions.

In two recent Phase III clinical trials, use of a 25 mg dapivirine vaginal ring significantly reduced HIV acquisition rates. Post hoc analysis from one of the trials indicated higher rates of protection among women over the age of 21 years when compared to younger women, most likely due to reduced adherence in the latter group. There is currently no information available on how release of dapivirine from the ring is affected by either its intermittent removal from the vagina or women's cleaning of the ring before re-insertion. Here, in vitro drug stability and product performance characteristics of the dapivirine ring were assessed under simulated conditions of real-world use. The impact of systematic deviations from the 28-day continuous use protocol upon in vitro release performance, was investigated. Also, the effect of ring exposure to a range of common household chemicals - including bath salts, bleach, detergent and personal lubricants - was examined through measurement of dapivirine content and stability. Dapivirine in vitro release under intermittent schedules was similar to that obtained under the normal continuous schedule ignoring the periods of interruption. Ring exposure to various household chemicals had no discernible impact on dapivirine assay value, degradation or stability.

Effect of polyanions on the structure and stability of repifermin (keratinocyte growth factor-2).

The interaction of several of the fibroblast growth factors (FGFs) with polyanions is thought to be of physiological significance and has been exploited to create more stable pharmaceutical formulations of FGF-1 and -2. The extent of such phenomena throughout the 23-member FGF family is, however, unknown. In these studies, we examine the effect of several polyanions on the structure and stability of keratinocyte growth factor 2 (KGF-2, FGF-10), a candidate for use as a wound-healing agent. Employing a variety of methods sensitive to the protein's structure including circular dichroism (CD), intrinsic fluorescence, derivative near-UV absorption spectroscopy, bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5-disulfonic acid) fluorescence, differential scanning calorimetry (DSC), and dynamic light scattering (DLS), we find that a variety of polyanions (e.g., heparin, sucrose octasulfate (SOS), and inositol hexaphosphate (IHP)) stabilize KGF-2 by increasing the thermal-unfolding temperature by approximately 9-15 degrees C. Negatively charged liposomes produce a similar effect, arguing for relatively nonspecific interactions of polyanions with KGF-2. Unlike some other FGFs, no evidence for the presence of a molten globule state is found during thermal perturbation of this growth factor. The generality of this polyanion/protein interaction is discussed as well as its potential role in various cellular events such as protein folding and transport.

Packing Polymorphism of Dapivirine and Its Impact on the Performance of a Dapivirine-Releasing Silicone Elastomer Vaginal Ring.

A silicone elastomer vaginal ring providing sustained release over 28 days of the anti-retroviral microbicide dapivirine has recently completed phase III clinical testing and showed moderate protection against HIV acquisition. In support of the product licensure program, we report the impact of dapivirine packing polymorphism on the thermal and solubility characteristics of dapivirine and on the in vitro performance of the 25 mg dapivirine ring product. This is the first time that polymorphism has been reported for a drug-releasing vaginal ring product. Thermal, particle size, powder X-ray diffraction, and thermodynamic solubility analyses of dapivirine polymorphic forms I and IV, both of which are persistent at room temperature and with form I being the thermodynamically stable form, were conducted for both micronized and non-micronized materials. No significant differences in solubility between DPV forms I and IV were observed in media commonly used for in vitro release testing. Matrix-type silicone elastomer vaginal rings were manufactured and the impact of dapivirine polymorphism on key in vitro parameters (compression and tensile behavior; content assay; in vitro release; residual content assay) was investigated. The data demonstrate that dapivirine packing polymorphism has no significant impact on in vitro performance of the 25 mg dapivirine vaginal ring.

Post-use assay of vaginal rings (VRs) as a potential measure of clinical trial adherence.

Adherence measurement for microbicide use within the clinical trial setting remains a challenge for the HIV prevention field. This paper describes an assay method used for determining residual dapivirine levels in post-use vaginal rings from clinical trials conducted with the Dapivirine Vaginal Matrix Ring-004 developed by the International Partnership for Microbicides to prevent male to female HIV transmission. Post-use assay results from three Ring-004 clinical trials showed that of the 25mg drug load, approximately 4mg of dapivirine is released from the matrix ring over a 28-day use period. Data obtained by both in vitro and in vivo studies indicate that dapivirine is released according to a diffusion mechanism, as determined by conformance of both data sets to the Higuchi equation. This, coupled with the low variability associated with batch production over two manufacturing sites and 20 batches of material, provides evidence that post-use ring analysis can contribute to the assessment of adherence to ring use. Limitations of this method include the potential of intra-participant and inter-participant variability and uncertainty associated with measuring the low amount of dapivirine actually released relative to the drug load. Therefore, residual drug levels should not serve as the only direct measurement for microbicide adherence in vaginal ring clinical trials but should preferably be used as part of a multi-pronged approach towards understanding and assessing adherence to vaginal ring use.

Analysis of protein/ligand interactions with NMR diffusion measurements: the importance of eliminating the protein background.

Pulsed-field gradient nuclear magnetic resonance (PFG-NMR) is a well-established method for the determination of translational diffusion coefficients. Recently, this method has found applicability in the combinatorial arena with the introduction of affinity NMR for characterizing protein/ligand interactions. Although affinity NMR has been reported to be an effective method for the identification of active compounds in a complex mixture, there are limitations of this method. We have developed a simple mathematical model to predict optimum concentration ratios of the ligand and protein in order to observe maximum changes in the ligand diffusion coefficient upon protein binding. The ligand/protein systems of L-tryptophan and ibuprofen binding to human serum albumin were chosen to demonstrate the usefulness of this model. However, even when the conditions of the mathematical model are satisfied, the spectral background arising from the protein in proton-detected experiments can be problematic. To this end, we have employed spectral subtraction of the protein spectrum to yield ligand diffusion coefficients that are in agreement with those predicted by simulation.

Effect of metal cations on the conformation and inactivation of recombinant human factor VIII.

Heavy metals have been implicated in the aggregation of proteins and the pathophysiology of several neurodegenerative diseases. Herein, we describe the interaction of recombinant human factor VIII (rhFVIII) with Al(+3), Tb(+3), Co(+2), and Fe(+3) using a combination of intrinsic fluorescence, circular dichroism, and high-resolution fourth-derivative absorbance analysis. rhFVIII in solution was titrated with the metal cations and the properties of the resulting complexes were examined. rhFVIII has a tendency to aggregate and inactivate slowly over time under physiological conditions, but this aggregation process is greatly accelerated in the presence of metals with Al(+3) being the most efficient. This leads to a complete loss of activity of the protein. Al(+3)-induced conformational changes in the protein were small but detectable with limited changes seen in secondary and tertiary structure. Because rhFVIII is a multidomain protein with subunits linked through divalent metal cations, the small intramolecular changes seen may be attributed to rearrangements of the subunits to an aggregation-competent conformer that is very similar to that of the native form.