Rectal pre-exposure prophylaxis (PrEP) will be a critical component of HIV prevention products due to the prevalence of unprotected receptive anal intercourse among men who have sex with men and heterosexual couples. Given the biological considerations of this compartment and the complexity of HIV infection, design of a successful rectal microbicide product faces a number of challenges. Important information is being compiled to begin to address deficits in knowledge toward design of rectal PrEP products for men and women. Aspects of formulation development and preclinical and clinical evaluation of rectal products studied to date are summarized in this review. This article is based on a presentation at the "Product Development Workshop 2013: HIV and Multipurpose Prevention Technologies," held in Arlington, Virginia on February 21–22, 2013. It forms part of a special supplement to Antiviral Research.
Rectal pre-exposure prophylaxis; microbicides; HIV prevention
The purpose of this study was to determine the impact of contraception, menopause and vaginal flora on the physical and biochemical properties of cervicovaginal fluid.
Vaginal swabs, cervicovaginal fluid (CVF) and cervicovaginal lavage (CVL) were collected from a total of 165 healthy asymptomatic women including: post-menopausal women (n=29), women in the proliferative (n=26) or follicular (n=27) phase, and women using the levonogestrel intrauterine device (LNG-IUD) (n=28), depomedroxyprogesterone acetate (DMPA) (n=28) or combined oral contraceptives (OCPs) (n=27). Vaginal smears were evaluated using the Nugent score. The osmolality, viscosity, density and pH of CVL samples were measured.
CVL from postmenopausal women and women with abnormal vaginal flora was less viscous and had higher pH than premenopausal women and women with normal flora, respectively. Women using hormonal contraceptives had more viscous CVL as compared to premenopausal women not using hormonal contraceptives, but this increase in viscosity was mitigated in the presence of bacterial vaginosis. Women using DMPA had less total protein in the CVL as compared to women using the LNG-IUD, and had similar protein content when compared to postmenopausal women.
The differences in CVL protein content between DMPA and LNG-IUD suggest that type of progesterone and route of delivery impact the vaginal environment. Contraceptive hormone users had more viscous CVL than women not using contraceptives. However, the presence of bacterial vaginosis impacted both the pH and viscosity (regardless of hormonal contraceptive use), demonstrating that vaginal flora has a greater impact on the physical properties of cervicovaginal fluid than reproductive hormones.
Contraceptives; cervicovaginal fluid; bacterial vaginosis
The gastrointestinal tract is structurally and functionally different from the vagina. Thus, the paradigm of topical microbicide development and evaluation has evolved to include rectal microbicides (RMs). Our interest was to create unique RM formulations to safely and effectively deliver antiretroviral drugs to mucosal tissue. RMs were designed to include those that spread and coat all surfaces of the rectum and distal colon rapidly (liquid) and those that create a deformable, erodible barrier and remain localized at the administration site (gel). Tenofovir (TFV) (1%) was formulated as an aqueous thermoreversible fluid and a carbopol-based aqueous hydrogel. Lipid-based liquid and gel formulations were prepared for UC781 (0.1%) using isopropyl myristate and GTCC (Caprylic/Capric Triglycerides), respectively. Formulations were characterized for pH, viscosity, osmolality, and drug content. Pre-clinical testing incorporated ex vivo colonic tissue obtained through surgical resections and flexible sigmoidoscopy (flex sig). As this was the first time using tissue from both sources side-by-side, the ability to replicate HIV-1 was compared. Efficacy of the RM formulations was tested by applying the products with HIV-1 directly to polarized colonic tissue and following viral replication. Safety of the formulations was determined by MTT assay and histology. All products had a neutral pH and were isoosmolar. While HIV-1BaL and HIV-1JR-CSF alone and in the presence of semen had similar replication trends between surgically resected and flex sig tissues, the magnitude of viral replication was significantly better in flex sig tissues. Both TFV and UC781 formulations protected the colonic tissue, regardless of tissue source, from HIV-1 and retained tissue viability and architecture. Our in vitro and ex vivo results show successful formulation of unique RMs. Moreover, the results of flex sig and surgically resected tissues were comparable suggesting the incorporation of both in pre-clinical testing algorithms.
Drug combination studies of 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) with FDA-approved drugs were evaluated by two different methods, MacSynergy II and CalcuSyn. Most of the combinations, including the combination of the two adenosine analogs EFdA and tenofovir, were essentially additive, without substantial antagonism or synergism. The combination of EFdA and rilpivirine showed apparent synergism. These studies provide information that may be useful for the design of EFdA combination regimens for initial and salvage therapy assessment.
Vaginal microbicides represent a promising approach for preventing heterosexual HIV transmission. However, preclinical evaluation should be conducted to ensure that microbicides will be safe for human cells and healthy microflora of the female reproductive tract. One microbicide candidate, RC-101, has been effective and well-tolerated in preliminary cell culture and macaque models. However, the effect of RC-101 on primary vaginal tissues and resident vaginal microflora requires further evaluation.
Method of Study
We treated primary vaginal tissues and vaginal bacteria, both pathogenic and commensal, with RC-101 to investigate effects of this microbicide.
RC-101 was well-tolerated by host tissues, and also by commensal vaginal bacteria. Simultaneously, pathogenic vaginal bacteria, which are known to increase susceptibility to HIV acquisition, were inhibited by RC-101.
By establishing vaginal microflora, the specific antibacterial activity of RC-101 may provide a dual mechanism of HIV protection. These findings support advancement of RC-101 to clinical trials.
Bacterial Vaginosis; Lactobacilli; RC-101; Theta-Defensin; Vaginal Microbicide
Microbicides have been evaluated mostly against cell-free HIV-1. Because semen contains both cell-free and cell-associated HIV-1, HIV-1 transmission could occur via either or both sources. Therefore, it is important to examine the antiviral activity of microbicides against cell-associated HIV-1. The cyclic antimicrobial peptide retrocyclin RC-101 has been shown previously to have antiviral activity against cell-free HIV-1, with no associated cellular toxicity. In this article we have examined the antiviral activity of RC-101 against cell-associated HIV-1. The results demonstrate potent antiviral activity of RC-101 against cell–cell HIV-1 transmission in both CD4-dependent and CD4-independent assays against CCR5- and CXCR4-tropic HIV-1, with no cellular toxicity. Furthermore, this antiviral activity was retained in the presence of human seminal plasma. The potent antiviral activity of RC-101 against cell-associated HIV-1 reported here, and the previously reported antiviral activity in cervical tissues, suggest that RC-101 is an excellent and promising microbicide candidate against HIV-1.
Coital use of 1% tenofovir gel was shown to be modestly effective at preventing HIV transmission when applied vaginally in the CAPRISA 004 trial. Because the gel is hyperosmolar, which would reduce the integrity of the epithelium and induce fluid movement into the lumen, rectal use may not be acceptable. This study evaluated the pre-clinical safety and efficacy of a reformulated (reduced osmolality) tenofovir gel product.
Reduced glycerine (RG)-tenofovir gel was compared with the original tenofovir gel for physiochemical characteristics, product safety and anti-HIV-1 activity.
The formulations were similar in all characteristics except for osmolality and spreadability/firmness. The RG-tenofovir gel had a 73% lower osmolality, a 29.6% increase in spreadability and a 27% decrease in firmness as compared with the original tenofovir gel. When applied to epithelial cell monolayers, tenofovir gel showed a transient reduction in the transepithelial resistance while the RG-tenofovir gel did not. Both gels retained ectocervical and colorectal explant viability. However, tenofovir gel treatment resulted in epithelial stripping that was absent after RG-tenofovir gel treatment of the polarized explants. Anti-HIV-1 activity was confirmed by lack of HIV-1 infection in polarized explants treated with either gel as compared with the control explants.
Reducing the osmolality of the tenofovir gel resulted in improved epithelial integrity, which suggests better safety upon rectal use. The improved gel safety did not compromise drug release or anti-HIV-1 activity. These data support the use of this gel as a dual compartment microbicide.
HIV prevention; rectal microbicide; formulation; preclinical testing; safety
Cervical tissue based organ cultures have been used successfully to evaluate microbicides for toxicity and antiviral activity. The antimicrobial peptide retrocyclin RC-101 has been shown to have potent anti-HIV activity in cell culture.
To evaluate RC-101 in organ culture for toxicity and its ability to block HIV-1 transmission across cervical mucosa.
A Cervical tissue based organ culture was used to measure antiviral activity of RC101. Cytotoxicity in tissues was determined by immunostaining of cellular proteins and by measuring inflammatory cytokines using realtime RTPCR and luminex technology.
RC-101 blocked transmission of both R5 and X4 HIV-1 across cervical mucosa in this organ culture model. Furthermore, film-formulated RC-101 exhibited potent antiviral activity in organ culture. Such antiviral activity of RC-101 was retained in the presence of semen and vaginal fluid. RC-101 showed no cytotoxicity in cervical tissue. Furthermore, RC-101 did not induce proinflammatory cytokine response in tissues. RC-101 also did not have any effect on NK activity, cell proliferation of CD4 and CD8 cells, and did not show chemotactic activity.
Therefore, because of strong antiviral activity and low cytotoxicity in cervical tissues, RC-101 should be considered as an excellent microbicide candidate against HIV-1.
retrocyclin; HIV; organ culture; microbicide
CSIC (5-chloro-3-phenylsulfonylindole-2-carboxamide), a non-nucleoside reverse transcriptase inhibitor (NNRTI) has not been advanced as a therapeutic anti-HIV candidate drug due to its low aqueous solubility and poor bioavailability.
The objective of this work was to formulate CSIC into self-emulsifying oil formulations for the purpose of improving its aqueous solubility and evaluating in vitro antiretroviral activity.
CSIC self-emulsifying oil formulations (SEFs) were formulated and evaluated for droplet size, zeta potential, polydispersity index (PDI), viscosity, emulsification time, stability and bioactivity.
Results showed significantly improved solubility of CSIC in the SEFs.The concentration of co-surfactant affected the droplet size, zeta potential and polydispersity index. In vitro bioactivity studies showed that the CSIC SEFs retained full anti-HIV activity.
The in vitro data from this first attempt to formulate CSIC SEFs suggest that improvement on the aqueous solubility of CSIC through this delivery system may accentuate its antiretroviral effectiveness in vivo via bioavailability enhancement. The formulation is therefore intended as an oral anti-HIV agent for prophylactic and therapeutic uses.
Anti-HIV; Poorly soluble; Self-emulsification; CSIC; Bioactivity; Anhydrous emulsion
Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.
An urgent need exists for HIV-1 microbicides. Here, we describe the in vivo testing of lactic acid bacteria bioengineered to secrete cyanovirin-N. We fed pigtail macaques a yogurt formulation that used bioengineered strains as a starter culture. Cyanovirin-N expression could be detected in the rectal vault during and immediately after feeding. Ex vivo viral challenge of rectal tissue biopsies revealed that peak viral burden was significantly lower in tissue obtained from experimental animals compared to control animals. Formulation of candidate compounds in lactic acid bacteria and their oral administration appears to be a feasible strategy for mucosal delivery of microbicides.
HIV-1; cyanovirin-N; C V-N; lactic acid bacteria; microbicide; pigtail macaque
Because lubricants may decrease trauma during coitus, it is hypothesized that they could aid in the prevention of HIV acquisition. Therefore, safety and anti-HIV-1 activity of over-the-counter (OTC) aqueous- (n = 10), lipid- (n = 2), and silicone-based (n = 2) products were tested. The rheological properties of the lipid-based lubricants precluded testing with the exception of explant safety testing. Six aqueous-based gels were hyperosmolar, two were nearly iso-osmolar, and two were hypo-osmolar. Evaluation of the panel of products showed Gynol II (a spermicidal gel containing 2% nonoxynol-9), KY Jelly, and Replens were toxic to Lactobacillus. Two nearly iso-osmolar aqueous- and both silicone-based gels were not toxic toward epithelial cell lines or ectocervical or colorectal explant tissues. Hyperosmolar lubricants demonstrated reduction of tissue viability and epithelial fracture/sloughing while the nearly iso-osmolar and silicon-based lubricants showed no significant changes in tissue viability or epithelial modifications. While most of the lubricants had no measurable anti-HIV-1 activity, three lubricants which retained cell viability did demonstrate modest anti-HIV-1 activity in vitro. To determine if this would result in protection of mucosal tissue or conversely determine if the epithelial damage associated with the hyperosmolar lubricants increased HIV-1 infection ex vivo, ectocervical tissue was exposed to selected lubricants and then challenged with HIV-1. None of the lubricants that had a moderate to high therapeutic index protected the mucosal tissue. These results show hyperosmolar lubricant gels were associated with cellular toxicity and epithelial damage while showing no anti-viral activity. The two iso-osmolar lubricants, Good Clean Love and PRÉ, and both silicone-based lubricants, Female Condom 2 lubricant and Wet Platinum, were the safest in our testing algorithm.
In conjunction with the routine role of delivering the active ingredient, carefully designed drug delivery vehicles can also provide ancillary functions that augment the overall efficacy of the system. Inspired by the ability of the cervicovaginal mucus to impede the movement of HIV virions at acidic pH, we have engineered a pH-responsive synthetic polymer that shows improved barrier properties over the naturally occurring cervicovaginal mucus by inhibiting viral transport at both acidic and neutral pH. The pH-responsive synthetic mucin-like polymer is constructed with phenylboronic acid (PBA) and salicylhydroxamic acid (SHA), each individually copolymerized with a 2-hydroxypropyl methacrylamide (pHPMA) polymer backbone. At pH 4.8, the crosslinked polymers form a transient network with a characteristic relaxation time of 0.9 s and elastic modulus of 11 Pa. On addition of semen, the polymers form a densely crosslinked elastic network with a characteristic relaxation time greater than 60 s and elastic modulus of 1800 Pa. Interactions between the PBA-SHA crosslinked polymers and mucin at acidic pH showed a significant increase in elastic modulus and crosslink lifetime (p < 0.05). A transport assay revealed that migration of HIV and cells was significantly impeded by the polymer network at pH ≥ 4.8 with a diffusion coefficient of 0.160 × 10−3 µm2 /s for HIV. Additionally, these crosslinked polymers did not induce symptoms of toxicity or irritation in either human vaginal explants or a mouse model. In summary the, pH-responsive crosslinked polymer system reported here holds promise as a class of microbicide delivery vehicle that could inhibit the transport of virions from semen to the target tissue and, thereby, contribute to the overall activity of the microbicide formulation.
Pyrimidinediones, a novel class of compounds, have previously been shown to possess antiviral activity at nanomolar concentrations. One member of this class of compounds, IQP-0528, was selected as the lead molecule for formulation development owing to its stability at physiologically relevant conditions, wide therapeutic window, and antiviral activity in the nanomolar range. Here, we report the development of two vaginal gels—3.0% hydroxyethyl cellulose (HEC) formulation and a 0.65% Carbopol formulation—for the sustained delivery of IQP-0528. Stability studies under accelerated conditions confirmed the chemical stability of IQP-0528 and mechanical stability of the gel formulation for 3 months. In vitro release studies revealed that diffusion-controlled release of IQP-0528 occurred over 6 h, with an initial lag time of approximately 1 h. Based on the drug release profile, the 3.0% HEC gel was selected as the lead formulation for safety and activity evaluations. The in vitro and ex vivo safety evaluations showed no significant loss in cell viability or significant inflammatory response after treatment with a 3.0% HEC gel containing 0.25% IQP-0528. In an in vitro HIV-1 entry inhibition assay, the lead formulation showed an 50% effective concentration of 0.14 μg/ml for gel in culture media, which corresponds to ∼0.001 μM IQP-0528. The antiviral activity was further confirmed by using polarized cervical explants, in which the formulation showed complete protection against HIV infection. In summary, these results are encouraging and warrant further evaluation of IQP-0528 gel formulations in in vivo models, as well as the development of alternative formulations for the delivery of IQP-0528 as a microbicide.
RC-101, a cationic peptide retrocyclin analog, has in vitro activity against HIV-1. Peptide drugs are commonly prone to conformational changes, oxidation and hydrolysis when exposed to excipients in a formulation or biological fluids in the body, this can affect product efficacy. We aimed to investigate RC-101 stability under several conditions including the presence of human vaginal fluids (HVF), enabling the efficient design of a safe and effective microbicide product. Stability studies (temperature, pH, and oxidation) were performed by HPLC, Circular Dichroism, and Mass Spectrometry (LC-MS/MS). Additionally, the effect of HVF on formulated RC-101 was evaluated with fluids collected from healthy volunteers, or from subjects with bacterial vaginosis (BV). RC-101 was monitored by LC-MS/MS for up to 72 h.
RC-101 was stable at pH 3, 4, and 7, at 25 and 37°C. High concentrations of hydrogen peroxide resulted in less than 10% RC-101 reduction over 24 h. RC-101 was detected 48 h after incubation with normal HVF; however, not following incubation with HVF from BV subjects.
Our results emphasize the importance of preformulation evaluations and highlight the impact of HVF on microbicide product stability and efficacy. RC-101 was stable in normal HVF for at least 48 h, indicating that it is a promising candidate for microbicide product development. However, RC-101 stability appears compromised in individuals with BV, requiring more advanced formulation strategies for stabilization in this environment.
To identify and apply core competencies for training students enrolled in the clinical pharmaceutical scientist PhD training program at the University of Pittsburgh School of Pharmacy.
Faculty members reached consensus on the required core competencies for the program and mapped them to curricular and experiential requirements.
A rubric was created based on core competencies spanning 8 major categories of proficiency, and competencies of clinical versus traditional PhD training were delineated. A retrospective evaluation of the written comprehensive examinations of 12 former students was conducted using the rubric. Students scored above satisfactory in 11 out of 14 comprehensive examination metrics, with a mean score greater than 3.8 on a 5-point scale.
The core competencies identified will provide an essential foundation for informed decision-making and assessment of PhD training in the clinical pharmaceutical sciences.
graduate education; competencies; clinical research; pharmaceutical sciences; doctor of philosophy degree
The objective of this study is to identify the critical formulation parameters controlling distribution and function for the rectal administration of microbicides in humans. Four placebo formulations were designed with a wide range of hydrophilic characteristics (aqueous to lipid) and rheological properties (Newtonian, shear thinning, thermal sensitive and thixotropic). Aqueous formulations using typical polymers to control viscosity were iso-osmotic and buffered to pH 7. Lipid formulations were developed from lipid solvent/lipid gelling agent binary mixtures. Testing included pharmaceutical function and stability as well as in vitro and in vivo toxicity.
The aqueous fluid placebo, based on poloxamer, was fluid at room temperature, thickened and became shear thinning at 37°C. The aqueous gel placebo used carbopol as the gelling agent, was shear thinning at room temperature and showed a typical decrease in viscosity with an increase in temperature. The lipid fluid placebo, myristyl myristate in isopropyl myristate, was relatively thin and temperature independent. The lipid gel placebo, glyceryl stearate and PEG-75 stearate in caprylic/capric triglycerides, was also shear thinning at both room temperature and 37°C but with significant time dependency or thixotropy. All formulations showed no rectal irritation in rabbits and were non-toxic using an ex vivo rectal explant model.
Four placebo formulations ranging from fluid to gel in aqueous and lipid formats with a range of rheological properties were developed, tested, scaled-up, manufactured under cGMP conditions and enrolled in a formal stability program. Clinical testing of these formulations as placebos will serve as the basis for further microbicide formulation development with drug-containing products.
RC-101 is a congener of the antiretroviral peptide retrocyclin, which we and others have reported is active against clinical HIV-1 isolates from all major clades, does not hemagglutinate, and is non-toxic and non-inflammatory in cervicovaginal cell culture. Herein, film-formulated RC-101 was assessed for its antiviral activity in vitro, safety in vivo, retention in the cervix and vagina, and ability to remain active against HIV-1 and SHIV after intravaginal application in macaques.
RC-101 was formulated as a quick-dissolving film (2000 µg/film), retained complete activity in vitro as compared to unformulated peptide, and was applied intravaginally in six pigtailed macaques daily for four days. At one and four days following the final application, the presence of RC-101 was assessed in peripheral blood, cervicovaginal lavage, cytobrushed cervicovaginal cells, and biopsied cervical and vaginal tissues by quantitative western blots. One day following the last film application, cervical biopsies from RC-101-exposed and placebo-controlled macaques were collected and were subjected to challenge with RT-SHIV in an ex vivo organ culture model. RC-101 peptide was detected primarily in the cytobrush and biopsied cervical and vaginal tissues, with little to no peptide detected in lavage samples, suggesting that the peptide was associated with the cervicovaginal epithelia. RC-101 remained in the tissues and cytobrush samples up to four days post-application, yet was not detected in any sera or plasma samples. RC-101, extracted from cytobrushes obtained one day post-application, remained active against HIV-1 BaL. Importantly, cervical biopsies from RC-101-treated animals reduced RT-SHIV replication in ex vivo organ culture as compared to placebo-treated animals.
Formulated RC-101 was stable in vivo and was retained in the mucosa. The presence of antivirally active RC-101 after five days in vivo suggests that RC-101 would be an important molecule to develop further as a topical microbicide to prevent HIV-1 transmission.
Tenofovir gel has entered into clinical trials for use as a topical microbicide to prevent HIV-1 infection but has no published data regarding pre-clinical testing using in vitro and ex vivo models. To validate our findings with on-going clinical trial results, we evaluated topical tenofovir gel for safety and efficacy. We also modeled systemic application of tenofovir for efficacy.
Methods and Findings
Formulation assessment of tenofovir gel included osmolality, viscosity, in vitro release, and permeability testing. Safety was evaluated by measuring the effect on the viability of vaginal flora, PBMCs, epithelial cells, and ectocervical and colorectal explant tissues. For efficacy testing, PBMCs were cultured with tenofovir or vehicle control gels and HIV-1 representing subtypes A, B, and C. Additionally, polarized ectocervical and colorectal explant cultures were treated apically with either gel. Tenofovir was added basolaterally to simulate systemic application. All tissues were challenged with HIV-1 applied apically. Infection was assessed by measuring p24 by ELISA on collected supernatants and immunohistochemistry for ectocervical explants. Formulation testing showed the tenofovir and vehicle control gels were >10 times isosmolar. Permeability through ectocervical tissue was variable but in all cases the receptor compartment drug concentration reached levels that inhibit HIV-1 infection in vitro. The gels were non-toxic toward vaginal flora, PBMCs, or epithelial cells. A transient reduction in epithelial monolayer integrity and epithelial fracture for ectocervical and colorectal explants was noted and likely due to the hyperosmolar nature of the formulation. Tenofovir gel prevented HIV-1 infection of PBMCs regardless of HIV-1 subtype. Topical and systemic tenofovir were effective at preventing HIV-1 infection of explant cultures.
These studies provide a mechanism for pre-clinical prediction of safety and efficacy of formulated microbicides. Tenofovir was effective against HIV-1 infection in our algorithm. These data support the use of tenofovir for pre-exposure prophylaxis.
The highly potent anti-HIV agent UC781 is being evaluated for use in topical microbicides to prevent HIV transmission. However, UC781 is extremely hydrophobic with poor water solubility, a property that may complicate appropriate formulation of the drug. In this study, we examined the ability of several cyclodextrins, beta-cyclodextrin (βCD), methyl-beta-cyclodextrin (MβCD), and 2-hydroxylpropyl-beta-cyclodextrin (HPβCD), to enhance the aqueous solubility of UC781. Each of the cyclodextrins provided dramatic increases in UC781 aqueous solubility, the order being MβCD>HPβCD>βCD. The complexation constants (K1:1) of the inclusion complexes were determined via a phase solubility technique using high-performance liquid chromatography and showed that UC781 solubility increased linearly as a function of cyclodextrin concentration. Ultraviolet spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and 2D 1H ROESY NMR spectroscopy were used to further characterize these UC781/cyclodextrin complexes. The inhibitory potency of UC781 and its HPβCD inclusion complex were evaluated using an in vitro HIV-1 reverse transcriptase inhibition assay The inhibitory potency of the UC781/HPβCD complex was 30-fold greater than that of UC781 alone, showing that the complexed drug is able to provide substantial inhibition of its target. The enhancement of UC781 aqueous solubility is essential for the development of a useful vaginal microbicide dosage form, and our data suggest that UC781/cyclodextrin inclusion complexes may be useful in this context.
beta-cyclodextrin inclusion complex; DSC; FTIR; HIV-1; NMR; non-nucleoside reverse transcriptase inhibitor; solubility; UC781
N-Hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine (HET0016) is a potent inhibitor of 20-hydroxyeicosatetraenoic acid (20-HETE) formation by specific cytochrome P450 (CYP) isoforms. Previous studies have demonstrated that administration of HET0016 inhibits brain formation of 20-HETE and reduces brain damage in a rat model of thromboembolic stroke. Delineation of the dose, concentration, neuroprotective effect relationship of HET0016 has been hampered by the relative insolubility of HET0016 in aqueous solutions and the lack of information concerning the mechanism and duration of HET0016 inhibition of brain 20-HETE formation. Therefore, it was the purpose of this study to develop a water soluble formulation of HET0016 suitable for intravenous (iv) administration and to determine the time course and mechanism of brain 20-HETE inhibition after in vivo dosing. In this study we report that HET0016 is a non-competitive inhibitor of rat brain 20-HETE formation, which demonstrates a tissue concentration range for brain inhibition. In addition, we demonstrate that complexation of HET0016 with hydroxypropyl-β-cyclodextrin (HPβCD) results in increased aqueous solubility of HET0016 from 34.2 ± 31.2 μg/mL to 452.7 ± 63.3 μg/mL. Administration of the complex containing formulation as a single HET0016 iv dose (1 mg/kg) rapidly reduced rat brain 20-HETE concentrations from 289 pmol/g to 91pmol/g. Collectively, these data demonstrate that the iv formulation of HET0016 rapidly penetrates the rat brain and significantly inhibits 20-HETE tissue concentrations. These results will enable future studies to determine biopharmaceutics of HET0016 for inhibition of 20-HETE after cerebral ischemia.