Rapid measures of user’s adherence to vaginal drug products using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and multivariate discriminant techniques

<div><p>Background</p><p>The topical HIV prevention (microbicides) field is in acute need of a method to rapidly and objectively measure adherence to product use in clinical trials. Infrared (IR) spectroscopy has been used in many pharmaceutical and forensic applications but has yet to be applied to adherence monitoring. In this study, we report on efforts to test the feasibility of using IR spectroscopy as a means to measure residual active or placebo vaginal product, semen exposure and vaginal insertion from a single swab.</p><p>Methods</p><p>A portable IR spectrometer equipped with diamond attenuated total reflectance (ATR) was used to capture spectra of unused vs. vaginally-used swabs, vaginal swabs containing semen, and vaginal swabs to which either tenofovir-containing or matching placebo products (vaginal gel or insert) were added. Spectral data obtained from swabs placed directly on the spectrometer were divided into calibration and testing sets for developing and validating discriminant models set up to provide yes/no predictions of: vaginal vs. non-vaginal use, presence vs. no presence of each test product, and presence vs. no presence of semen. Further validation of models was performed using vaginal swabs collected from a clinical study evaluating vaginally administered placebo insert formulations.</p><p>Results</p><p>For each discriminant model developed to predict vaginal vs. non-vaginal use, presence vs. no presence of each test product, and presence vs. no presence of semen, classified validation samples not included in the model development were correctly identified into their respective classes with minimal prediction error. Clinically obtained vaginal swabs collected 15–60 minutes after placebo insert use were also correctly identified, further validating the models.</p><p>Conclusion</p><p>Our findings demonstrate the proof of concept that IR spectroscopy can be a method for rapid detection and characterization of microbicide products and biological fluids present in vaginal swabs. This novel method has potential to support real-time, on-site adherence monitoring in clinical or field settings.</p></div

Representative IR raw (panel A) and Savitzky-Golay first derivative transformed spectra (panel B) of vaginally inserted swabs, control-dry (rayon swabs), control–water (rayon dipped in water) and control-VSF (rayon dipped in vaginal fluid simulant).

<p>Representative IR raw (panel A) and Savitzky-Golay first derivative transformed spectra (panel B) of vaginally inserted swabs, control-dry (rayon swabs), control–water (rayon dipped in water) and control-VSF (rayon dipped in vaginal fluid simulant).</p

Synthesis and Anti-HIV Activities of Glutamate and Peptide Conjugates of Nucleoside Reverse Transcriptase Inhibitors

Mono-, di-, and trinucleoside conjugates of glutamate or peptide scaffolds containing nucleoside reverse transcriptase inhibitors were synthesized. Among dinucleoside glutamate ester derivatives, <i>N</i>-myristoylated derivatives showed significantly higher anti-HIV activity than the corresponding <i>N</i>-acetylated conjugates against cell-free virus. Myristoyl-Glu­(3TC)-FLT (<b>46</b>, EC₅₀ = 0.3–0.6 μM) and myristoyl-Glu­(FTC)-FLT (<b>47</b>, EC₅₀ = 0.1–0.4 μM) derivatives were the most active glutamate–dinucleoside conjugates. A trinucleoside glutamate derivative containing AZT, FLT, and 3TC (<b>34</b>, EC₅₀ = 0.9–1.4 μM) exhibited higher anti-HIV activity than AZT and 3TC against cell-free virus. Compound <b>34</b> also exhibited higher anti-HIV activity against multidrug (IC₅₀ = 5.9 nM) and NNRTI (IC₅₀ = 12.9 nM) resistant viruses than parent nucleosides. The physical mixture containing FLT–succinate, AZT, 3TC, and glutamic acid exhibited 115-fold less activity against cell associated virus (EC₅₀ = 91.9 μM) when compared to <b>34</b> (EC₅₀ = 0.8 μM). Other conjugates showed less or comparable potency to that of the corresponding physical mixtures

Raw spectra(4000–650 cm^-1) of vaginal swabs (baseline, no placebo insert) and vaginal swabs containing placebo insert.

<p>Raw spectra(4000–650 cm^-1) of vaginal swabs (baseline, no placebo insert) and vaginal swabs containing placebo insert.</p

Summary of classification models developed for identification of vaginal swabs containing HEC placebo gel tested at 5% significance level.

<p>Summary of classification models developed for identification of vaginal swabs containing HEC placebo gel tested at 5% significance level.</p

TFV API spectra profile and its corresponding functional peaks in spectra of TFV gel and insert formulations.

<p>TFV API spectra profile and its corresponding functional peaks in spectra of TFV gel and insert formulations.</p

Summary of classification models developed for identification of vaginal swabs.

<p>Summary of classification models developed for identification of vaginal swabs.</p

Hydroxyethylcellulose (HEC) Placebo Gel Does Not Interfere with DNA Biomarker Detection.

<p>A) Different patterns of DNA biomarkers were still detected in the presence of vaginally expelled HEC gel (Lane 1). These patterns matched the corresponding patterns observed from vaginal swab DNA of those same women (Lane 3). DNA from sham applicators manually handled by the women (Lane 2) and No DNA PCR (Lane 4) controls were negative. B) Exposure of HEC gel did not interfere with the DNA biomarkers before (Lane 1) or after semen exposure (Lane 2). No DNA PCR negative control is included (Lane 3).</p

DNA and CK4 Biomarkers on Applicators Stored for 30 days.

<p>After 30 days of storage at room temperature, applicators from two women (#1 and #2) were swabbed and DNA/cells isolated. CK4 expression was positive (A) while amelogenin and bacterial markers were amplified by the multiplex PCR (B) confirming stability of the biomarkers over the course of 30 days. Vaginal swab DNA from woman #1(“VS”) is included as a positive control to confirm that identical amplified bacterial species were found on the applicator #1 (Lane 1). A no DNA PCR control is also included (“neg”); Normal rabbit IgG negative control (inset pictures).</p

Lack of Bacterial Marker DNA Amplification in Semen or Sperm DNA.

<p>30 and 90 ng, respectively of sperm DNA (lanes 1–2) and semen DNA (lanes 3–4) were used as template DNA for the expanded multiplex PCR. No bacterial markers were amplified. Only the Y-chromosomal DNA markers, TSPY4 and SRY, along with the internal control gene, amelogenin, were amplified. Vaginal swab DNA showing <i>L. jensenii</i> amplification was used as a positive control (lane 5). Lane 6 is a PCR negative control.</p