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Sustained release of microbicides by newly engineered vaginal rings

Saxena, Brij Ba; Han, Young Aa; Fu, Dingyia,b; Rathnam, Premilaa; Singh, Mukula; Laurence, Jeffreyb; Lerner, Sidneyc

doi: 10.1097/QAD.0b013e32832af57c

Objective: An effective vaginal microbicide against sexual HIV transmission remains elusive, with requirements for adherence to appropriate application of effective, nontoxic products being a major deterrent. We explored methods to enable sustained release of combinations of antiretroviral microbicides, utilizing intravaginal rings composed of biosoluble Acacia gum or nonbiodegradable hydrogel of 2-hydroxyethyl methacrylate and sodium methacrylate, materials approved for use by the US Food and Drug Administration.

Design and methods: The reverse transcriptase inhibitors TMC120, PMPA, 3′-azido-3′-deoxythymidine, and a newly characterized anti-HIV agent, Boc-lysinated betulonic acid, were incorporated into vaginal rings with different combinations. Daily and cumulative release rates of these inhibitors in ring eluates were determined by high-performance liquid chromatography, gas chromatography, or immunoassay. Anti-HIV effects were measured by assessment of p24 Gag antigen in T-cell cultures exposed to HIV-1 isolates.

Results: Drug release rates were sustained at concentrations higher than the minimum effective dose for HIV inhibition. The release was maintained for no less than 15 and 28 days from the Acacia gum and 2-hydroxyethyl methacrylate and sodium methacrylate rings, respectively. Boc-lysinated betulonic acid showed more than 90% inhibition of HIV-1 infection in H9 cells, with little toxicity to normal cells.

Conclusion: The intravaginal rings described here are capable of efficacious drug delivery. Incorporation of several antiretroviral agents, including betulinol derivatives, which act at multiple levels of the HIV life cycle, may provide a synergistic effect to achieve higher efficacy on the inhibition of HIV infection.

aDivision of Reproductive Endocrinology, Department of Obstetrics and Gynecology, USA

bDepartment of Medicine, Weill Medical College of Cornell University, USA

cBioRing, LLC, New York, New York, USA.

Received 28 October, 2008

Revised 12 February, 2009

Accepted 26 February, 2009

Correspondence to Brij B. Saxena, Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, 515 East 71st Street, Room #S-412, New York, NY 10021, USA. Tel: +1 212 746 3067; fax: +1 212 746 4964; e-mail:

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There are 33 million people living with AIDS/HIV infection worldwide, with about five million new infections and three million deaths per year [1]. Apart from use of antiretroviral drugs in postexposure prophylaxis models, the most promising strategy for prevention of heterosexual transmission of HIV is the vaginal microbicide. More than 15 HIV microbicides have been evaluated in a conventional semisolid gel formulation designed to provide a single dose of the microbicidal agent applied immediately before intercourse [2]. Silicone elastomer vaginal rings containing the nonnucleoside reverse transcriptase inhibitor TMC120 (dapivirine) have been tested for in-vitro release over a 71-day period, yielding 136 μg per day, which is several orders of magnitude in excess of reported HIV inhibitory concentrations [3]. Gel formulations of TMC120 have also been tested in phase I and II clinical trials [3].

Recently, we have shown that Boc-lysinated betulonic acid (Boc-LBA), a pentacyclic triterpenoid that is soluble in aqueous medium, had significant anti-HIV effects, including activity against zidovudine (ZDV)-resistant HIV strains [4,5]. This study evaluated the potential for sustained, simultaneous release of appropriate doses of more than one microbicide from intravaginal rings to provide anti-HIV action at multiple sites in the life cycle of the virus and thus be more efficacious.

Sustained intravaginal delivery of combinations of antiretroviral microbicides that are nontoxic to the vaginal mucosae, target multiple steps of the viral life cycle, and show low systemic absorption may have the greatest potential to suppress heterosexual transmission of HIV [6–8]. For example, development of intravaginal rings to deliver the nucleoside reverse transcriptase inhibitor (NRTI) 3′-azido-3′-deoxythymidine (ZDV) for sustained release has recently been described by us [9]. The rings were composed of biosoluble Acacia gum or nonbiodegradable 2-hydroxyethyl methacrylate and sodium methacrylate (HEMA-co-SMA). The daily release rate of ZDV from the rings was maintained for up to 10 and 28 days from the Acacia gum and HEMA rings, respectively.

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Materials and methods


Boc-LBA was synthesized as described [5]. TMC120 (molecular weight 204.3) and PMPA (tenofovir, molecular weight 305.3) were provided by the International Partnership for Microbicides (IPM; Bethlehem, Pennsylvania, USA). ZDV was purchased from Sigma (Steinheim, Germany).

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Ring construction

Acacia gum and HEMA rings of 0.5–0.7 cm rim thickness and 5.0–5.5 cm diameter were constructed utilizing Teflon and steel molds as described [9]. The rings were sterilized by gamma radiation (Nordion GammaCell 1000 Irradiator, Ottawa, Ontario, Canada). The radioactive source was Cs-137, the dose rate was 2.56 Gy/min (256 rad/min), and the irradiation time was 24 h. Five combinations of microbicides were tested: TMC120 and PMPA (5 mg each), TMC120 (5 mg), and Boc-LBA (5 mg) alone were incorporated into Acacia gum rings; TMC120 and PMPA (10 mg each) and ZDV alone (10 mg) were incorporated into HEMA rings [9].

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Eluate collection and detection

Each ring was placed in a Petri dish containing 10 ml of a 1: 1 mixture of isopropanol and water. The Petri dish was closed airtight and placed on a shaker at a low speed in an incubator at 37°C. Eluates were collected at 2-day intervals, at that time each Petri dish was replenished with 10 ml of fresh 1: 1 isopropanol/water. Eluate collection was made continually for more than a month for each ring. Eluates were stored at 4°C until use for detection.

Concentrations of TMC120 and PMPA in the eluates were determined by high-performance liquid chromatography (HPLC) with a BPD-SAV UV-VIS spectrometric detector (Shimadzu LC-600; Shimadzu Corporation, Columbia, Maryland, USA) [2,10,11] according to the conditions described in Table 1. Boc-LBA in the eluates was determined by gas chromatography (Shimadzu GC-14A, Shimadzu Corporation). ZDV in the eluates was determined using an ELISA kit obtained from Neogen (Lexington, Kentucky, USA), as described earlier [9].

Table 1

Table 1

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Anti-HIV evaluation

H9 and MT-2 CD4+ human T-cell lines, the HIV-1 IIIB isolate 964B (8 AO12 G691-6), and a ZDV-resistant HIV-1 isolate were obtained from NCI AIDS Research and Reference Reagents Program [National Institutes of Health (NIH), Bethesda, Maryland, USA]. Anti-HIV activity was tested using an HIV-1 p24 Gag Antigen Capture Assay [12] according to instructions provided by the NCI-Frederick Cancer Research and Development Center, Frederick, Maryland, USA.

H9 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 media containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C and 5% CO2. The cells were infected with the HIV stock 964B at a multiplicity of infection (MOI) of 1.0 for 2 h at 37°C. The cells were then washed three times with phosphate-buffered saline (PBS). Twenty-five thousand cells in 200 μl of culture media were aliquoted into each well of 96-well culture plates (Falcon Becton, Franklin Lakes, New Jersey, USA). Betulonic acid and its derivatives, betulin aldehyde, betulin diacetate, Boc-LBA, as well as ZDV, were added to wells at various concentrations, each in triplicate. The plates were incubated at 37°C. On day 3, half of the media was replaced with fresh media containing the same concentrations of drugs. On day 8, a 100 μl aliquot of culture supernatant from each well was collected and assayed for HIV p24. In terms of infection with an ZDV-resistant HIV-1 isolate, MT-2 cells were used instead of H9 cells, also at an MOI of 1.0.

H9 cells were used to test the drug toxicities in cell viability assays by exclusion method [13]. One lakh fifty thousand H9 cells per well were incubated in 2 ml of RPMI 1640 media and 10% FBS. Agents betulonic acid, Boc-LBA, and ZDV were added at concentrations of 0, 2, 5, 10, and 20 μmol/l. Cells were kept in 24-well Falcon plates at 37°C in an incubator under 5% CO2 for 3 days. Cells incorporating or excluding trypan blue vital dye were assessed.

Results were treated with a Student's t-test to assess the statistical significance. Data were shown from a single representative experiment (n = 3) as mean ± SD of triplicate determinations.

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Anti-HIV activity of betulonic acid derivatives

Chemicals betulonic acid, betulone aldehyde, and betulin diacetate dissolved in 1% dimethyl sulfoxide (DMSO) showed slightly higher inhibition of HIV-1 replication in H9 cells than ZDV, all at a 1 μmol/l concentration (Fig. 1a). However, when betulonic acid was combined with betulin diacetate or betulone aldehyde, or when betulin diacetate and betulone aldehyde were assayed together, the inhibition rose in the range of 80–85% (Fig. 1b).

Fig. 1

Fig. 1

The cellular toxicity of Boc-LBA was compared with that of betulonic acid and ZDV (Fig. 1c). Boc-LBA showed little toxicity to normal cells up to a concentration of 20 μmol/l. In contrast, there was an increasing toxicity paralleling the increasing doses of betulonic acid and ZDV. At 20 μmol/l of betulonic acid and ZDV, there were only approximately 15 and 10% cell survival, respectively.

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Release of microbicides in the eluates of the rings

As shown in Fig. 2(a), the release of TMC120 in the eluates of an Acacia ring was in the range of 19.5–40.8 μg/ml from days 1 to 15, with an average of 27.7 μg/ml. The cumulative release for the same period reached 193.8 μg/ml. The release rate of PMPA from an Acacia ring ranged from 31.0 to 103 μg/ml during the same period, with an average of 78.0 μg/ml. The cumulative release reached 545.9 μg/ml (Fig. 2b). These release rates were sustained at a significantly higher concentration than the minimum effective HIV inhibitory dose [14,15]. The release of Boc-LBA in the eluates of an Acacia ring was in the range of 9.5–70.8 μg/ml from days 1 to 24, with an average of 55.1 μg/ml. The cumulative release for the same period rose to 257.6 μg/ml (Fig. 2c). Thus, Acacia rings would be expected to provide release of drug at effective anti-HIV concentrations for at least 15 days.

Fig. 2

Fig. 2

The daily and cumulative release of TMC120 from a HEMA ring for the 28-day period ranged from 7.91 to 38.9 μg/ml, and the cumulative release reached to 143.3 μg/ml on day 28 (Fig. 3a). The daily release of PMPA for the same period ranged from 10.4 to 210 μg/ml with an average of 32.4 μg/ml, and the cumulative release was 587.9 μg/ml on day 28 (Fig. 3b). The release of ZDV was in the range of 0.35–17.4 μg/ml until day 28, and the cumulative release on day 28 approached 318 μg/ml (Fig. 3c). Thus, HEMA rings provided effective release of anti-HIV agents for no less than 28 days.

Fig. 3

Fig. 3

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Two antiretroviral microbicides employed in our studies are ideal candidates to include as part of a local, sustained release strategy to block HIV transmission. TMC 120 is now being developed as a vaginal microbicide based on its advantage of low solubility and thus prolonged release into the aqueous phase [16]. PMPA is also being tested, alone and in combination with other anti-HIV agents, as a microbicide. It has a median effective dose of 0.47 nmol/l, with low toxicity when used systemically [14]. ZDV is a potent NRTI widely used in treating HIV infection in clinic. It has a minimum effective concentration of 0.02 μmol/l with low toxicity and high solubility in aqueous medium [15]. The emerging group of betulin derivatives with high anti-HIV activity in vitro may represent an important addition to a combination chemotherapy approach to microbicide development.

The anti-HIV activity of certain betulin derivatives has been compared favorably to ZDV in published reports [17–20] and in our own studies. Betulonic acid, betulone aldehyde, and betulone diacetate showed 81, 77, and 76% inhibition of an HIV-1 isolate in H9 cells, which was not significantly greater than ZDV in this system (Fig. 1a and b). A combination of betulonic acid with its biacetate and aldehyde derivatives showed significantly higher inhibition than betulonic acid alone. Betulonic acid also showed significantly higher inhibition on an ZDV-resistant virus than ZDV in MT-2 cells (unpublished data). In addition, the newest betulonic acid derivative, Boc-LBA that is soluble in aqueous medium, had the highest anti-HIV effect among them, which may be attributed to higher drug bioavailability, with little cellular toxicity. Although our report involved only chemokine (CXC motif) receptor 4 (CXCR4) coreceptor utilizing viral isolates, others have shown equivalent efficacy against chemokine (C–C motif) receptor 5 (CCR5) coreceptor utilizing strains with these compounds [21–23]. This is important, as the primary type of virus transmitted sexually is CCR5 restricted.

The mechanism of action of betulin derivatives involves inhibition of entry [21], fusion [22,23], maturation, and replication [24–27] steps in the HIV life cycle. It is proposed that betulin derivatives inhibit HIV-1 fusion by altering the molecular signals between gp120 and gp41 that are required for triggering receptor-induced conformational changes in gp41. In addition to the inhibitory effects of triterpenoids on the HIV-1 entry stage, betulin derivatives with C-3 substitutions have been shown to specifically inhibit viral assembly and maturation steps that include the final cleavage event required for proper condensation of the viral core Gag protein and the final molecular editorial envelope assembly events to prevent the new viruses from being born for release from the invaded cells [28]. The betulin derivatives containing modifications to the carboxylic acid group at position C-28 appear also to inhibit HIV-1 entry into cells [25]. Recently, a bifunctional betulonic acid derivative containing alterations in the side chains both at C-3 and C-28 was reported, which inhibits the entry, fusion, and maturation stages of the HIV-1 cycle [23]. Most of the observations on the anti-HIV activity of betulin derivatives were determined by in-vitro cell line assays in DMSO as the solvent. In contrast, Boc-LBA, soluble in aqueous medium [5], may provide a more effective candidate for combination therapies in vivo to provide a synergistic effect and allow the use of lower doses of drugs to minimize the emergence of drug resistance via systemic absorption.

Intravaginal rings composed of US Food and Drug Administration approved materials, which are capable of delivering a sustained and efficacious release of TMC120, together with PMPA or ZDV, as well as Boc-LBA alone are described here. Daily release rates of both TMC120 and PMPA from both Acacia and HEMA rings were significantly higher than the minimum effective dose [14,16]. In an earlier study [9], daily and cumulative release of ZDV from Acacia and HEMA rings was shown to be efficacious for 15 and 28 days and possibly longer periods from the HEMA ring. Daily release in the range of 10–50 μg/ml is several order folds higher than the concentration of ZDV that inhibits viral replication by 50% and was lower than the concentration of ZDV that is cytotoxic to 50% of the cells [9,15].

These rings would deliver a combination of antiretroviral microbicides that may inhibit multiple sites in the life cycle of HIV and would provide effective and compliant therapy in prevention of HIV infection possibly with minimum off-target adverse effects. These rings are effective for drug delivery for 10 days from Acacia gum and 28 days from HEMA rings or as and when desired for protection against HIV infection to which women are exposed at any stage and any age.

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The present work was supported by the IPM and BioRing LLC to B.B.S., and research support from NIH grants HL55646 and DK65511 to J.L.

Dr B.B.S. and Dr M.S. were the principal investigator and co-principal investigator, respectively, and made the overall project design. Y.A.H. designed and constructed the rings. D.F. evaluated the antiretroviral microbicides of their anti-HIV activity and cellular toxicity. P.R. contributed in the synthesis of betulinol derivatives and their structural characterization. J.L. provided laboratory facilities for HIV assays and guidance. S.L. provided financial support and useful discussion on the project. Mr Shyam Amin and Ms Pooja Gupta assisted in the HPLC and gas chromatography analyses. Ms Anna Palterman and Ms Kathryn Lohotsky assisted in the preparation of the manuscript and figures.

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anti-HIV drug; controlled release; prevention; therapy; vaginal rings

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