Appella, Ettore

Butera, Sal

Hayashi, Ryo

Lanier, Nattawan

Malcolm, Karl

McConville, Christopher

Otten, Ronald

Schito, Marco

Smith, James M.

Woolfson, David

Background: Small molecule inhibitors of the zinc finger domain (ZFI) in the nucleocapsid protein (NCp7) of HIV-1 are potent inhibitors of HIV and SIVreplication and may have utility as topical products to prevent infection. Furthermore, intravaginal rings (IVRs) were developed as coitally-independent,sustained release devices which could be used for administration of HIV microbicides. The aims of these studies were to demonstrate that IVRs sized formacaques are practical and compatible with the current generation of thioester-based NCp7 inhibitors.Methods: Non-medicated silicone elastomer vaginal rings of various sizes thought to be applicable for macaques were prepared and tested for vaginal fit in Pigtailed and Chinese Rhesus macaques. Macaques were monitored for 8 weeks for mucosal disruption by colposcopy and proinflammatory cytokine markers in cervical vaginal lavages (CVL) using Luminex bead-based technology. Three different ZFIs (compounds 52, 89 and 122, each derived from an N-substituted S-acyl-2-mercaptobenzamide thioester scaffold) were loaded at 50 mg into an optimal matrix-type ring design. In vitro continuous release studies were then conducted over 28 days and analyzed by HPLC. Rate of release was determined by linear regression analysis.Results: Qualitative evaluation at the time of ring insertion suggested that the 25 mm ring provided optimal fit in both macaque species. All rings remained inplace during the study period (2 to 4 weeks), and the animals did not attempt to remove the rings. No tissue irritation was observed, and no signs of physicaldiscomfort were noted. Also, no significant induction of cervicovaginal proinflammatory markers was observed during the 8-week period during and following ring insertion. One Pigtailed macaque showed elevated IL-8 levels in the CVL during the period when the ring was in place; however, these levels were comparable to those observed in two control macaques. In vitro release of the ZFIs peaked at day 1 and then continually declined to near steady-state rates between 20-30 mcg/day. The percent release after 14 days was 2.9, 2.0 and 0.9 for ZFI 89, 52 and 122, respectively.Conclusions: IVRs of 25mm diameter, determined to be the optimal size for macaques, were well tolerated and did not induce inflammation. Release of all ZFI compounds followed t 0.5 kinetics. These findings suggest that efficacy testing in primate models is warranted to fully evaluate the potential to preventtransmission

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Size-fitting of Intravaginal Rings for Macaques and in vitroRelease Kinetics of Zinc Finger InhibitorsMalcolm, K., Smith, J. M., Appella, E., Schito, M., Hayashi, R., Lanier, N., ... Woolfson, D. (2008). Size-fitting ofIntravaginal Rings for Macaques and in vitro Release Kinetics of Zinc Finger Inhibitors. Poster session presentedat 15th Conference on Retroviruses and Opportunistic Infections, Boston, United States.Document Version:Early version, also known as pre-printQueen's University Belfast - Research Portal:Link to publication record in Queen's University Belfast Research PortalGeneral rightsCopyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or othercopyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associatedwith these rights.Take down policyThe Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made toensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in theResearch Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk.Download date:16. Feb. 2017Size-fitting of Intravaginal Rings for Macaques and in vitro Release Kinetics of Zinc Finger InhibitorsJames M. Smith1, Karl Malcolm2, Ettore Appella3, Chris McConville2, Marco Schito4, David Woolfson2, Ryo Hayashi3, Nattawan Lanier1, Ronald Otten1, Salvatore Butera11Centers for Disease Control and Prevention, Atlanta, GA  USA; 2School of Pharmacy, Queen's Univ. of Belfast, Northern Ireland, UK; 3National Cancer Institute, NIH, Bethesda, MD, USA; 4NIAID, NIH, Bethesda, MD, USA Table 3 Study design for intravaginal ring size-fitting in pig-tailed and Chinese rhesus macaques Size-fitting IVR for Macaques ring observed to be in place (colposcopy) and no adverse effects or behavioral issues/problems noted, based on daily monitoring    CVL - cervicovaginal lavage      P-Blood collected for plasma Figure 2  IVRs do not induce expression of pro-inflammatory markers   A) Pro-inflammatory cytokines are plotted for each individual animal during the course of the study  B) Scatter plots for each cytokine were plotted for all 4 macaques before IVR was inserted (pre); in the presence of the IVR (IVR); and after removal of the IVR (post).   Results?As assessed by physical examination, colposcopy and behavioral observation, the 25mm rings were well tolerated by both Chinese rhesus and pig-tailed macaques.?Levels of pro-inflammatory cytokine markers from cervicovaginal lavages remained stable throughout the study period of 8 weeks   Conclusions?Silicone-based rings of 25mm diameter are easily inserted into the vaginal vault of both Chinese Rhesus and pig-tailed macaques and their presence does not alter the behavior of the animals or induce inflammation. These results warrant further studies utilizing IVRs loaded with antiviral drugs in the non-human primate model.   AB A1.E-011.E+001.E+011.E+021.E+031.E+041.E+05Pre IVR PostIL-1β IL-6 IL-8TNF-αPre IVR Post Pre IVR Post Pre IVR Post1.0E-011.0E+001.0E+011.0E+021.0E+031.0E+041.0E+050 1 2 3 4 5 6 7 81.0E-011.0E+001.0E+011.0E+021.0E+031.0E+041.0E+050 1 2 3 4 5 6 7 81.0E-011.0E+001.0E+011.0E+021.0E+031.0E+041.0E+050 1 2 3 4 5 6 7 81.0E-011.0E+001.0E+011.0E+021.0E+031.0E+041.0E+050 1 2 3 4 5 6 7 8WEEKS WEEKSIL-8 IL-1β TNF- α IL-6IVR present IVR absentpg / mLpg / mL96PO25 96PO58RO5232RQ5155pg / mLpg / mLCVLPCVLPCVLPCVLPCVLCVLCVLCVLPRQ5155/20 x 5mmCVLPCVLPCVLPCVLPCVLCVLCVLCVLPRO5232/25 x 5mmChinese Rhesus Ring sizeCVLPCVLPCVLPCVLPCVLCVLCVLCVLP96P058/25 x 5mmCVLPCVLPCVLPCVLPCVLCVLCVLCVLP96P025/30 x 5mmPig-tailed macaqueRing sizeWk 8Wk 7Wk 6Wk 5Wk 4Wk 3Wk 2Wk 1Wk 0IVR IN PLACE1 Viral replication in PBMC and monocytes/macrophage cultures were determined on day 7 post-infection by measuring supernatant reverse transcriptase (RT) activity or p24 antigen expression by ELISA. Units are in µM.     EC50 - high antiviral activity (low EC50), IC50 - low cellular toxicity (high IC50), TC50 – low cellular toxicity (high TC50), TI – Therapeutic index = IC50 or TC50 / EC50Table 5  Comparison of ZFI release rates and physicochemical properties; compounds presented in order of decreasing release rate.0.1320.46 mg / 0.9%5.1167.11.02374.41220.2530.99 mg / 2.0%4.7197.82.27364.4520.4271.45mg / 2.9%5.7166.51.31418.589Release Rate*(2ASDC)0.5Cumulative and % release after 14 daysHPLC retention time (min)DSC Melting Point (oC)log PMW(g/mol)ZFICompoundFigure 4   Matrix-type IVRs were loaded with 50mg of ZFI 52, 89 or 122. Release was determined by HPLC analysis of drug levels in media under sink conditions. A) daily release-versus-time profiles  B) mean cumulative release-versus-time profiles   C) mean cumulative release-versus-root time* equal to the gradient of cumulative release versus root time profile, where A is the drug loading per unit volume, S is the surface area of the ring, D is the apparent diffusion coefficient, and C is the silicone elastomer solubility per unit volume of the active agent.0.350.300.250.200.150.100.050.000 2 4 6 8 10 12 14ZFI 89ZFI 52ZFI 122Time (Days)Daily Release (mg)Daily Release (mg)Time (Days)2.52.01.01.50.50.0Cumulative Release (mg)0 4 8 12 2416 20 28ZFI 89ZFI 52ZFI 122Cumulative Release (mg)1 2 3 4 5 60Root Time (Days 0.5)Cumulative Release (mg) 2.01.81.61.41.21.00.80.60.40.20.0y = 0.3994x – 0.0848R 2 = 0.9957y = 0.2482x + 0.0432R 2 = 0.9976y = 0.1448x - .0702R 2 = 0.9955ZFI 89ZFI 52ZFI 122Cumulative Release (mg)B CResults?The daily and cumulative release profiles (Fig 4) are typical of diffusion-controlled matrix-type devices, showing an initial Day 1 burst followed by a declining daily release rate on subsequent days. Daily release rates range from 20-300 mcg/day. ?Compound 89 released at the fastest rate, followed by Compound 52, and Compound 122 the slowest?The almost perfect straight-line plots of the cumulative release-versus-root time are indicative of t 0.5 kinetics, confirming diffusion-controlled release from a matrix delivery systemConclusionsDisclaimer The findings and conclusions in this presentation have not been formally disseminated by [the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry] and should not be construed to represent any agency determination or policy.In several independent studies, the utility and acceptance of intravaginal rings as a delivery device for hormones and contraception has been well documented.  The controlled sustained release of inhibitors from IVRs for pre-exposure prophylaxis has many of the desired characteristics of an ideal vaginal microbicide.  This study was launched to adapt and expand this versatile delivery device to the non-human primate model of HIV prevention.  The fact that IVR did not cause inflammation and the zinc finger inhibitor was successfully released makes this combination, as well as other classes of HIV inhibitors, an excellent candidate for preclinical evaluation in the non-human primate repeat challenge model.In Vitro Release of Zinc Finger InhibitorsFigure 3  Zinc finger inhibitors are thioester-based compounds that interfere with the interaction of the nucleocapsid protein (NCp7) of SIV and HIV with zinc molecules. Table 4 In vitro inhibition of replication against SIV and HIV-1 with ZFI compounds (Adapted from: Srivastava, P. et. al. Optimization of unique, uncharged thioesters as inhibitors of HIV replication. Bioorg Med Chem 2004, 12, (24), 6437-50)SONHONH2OSO OMeOMeNHH2NOOSOOMeNHH2NOOH3CHOMeOMeSO FFNHONH2O8952 122RThioester scaffoldJames Smith Centers for Disease Control and Prevention                  Laboratory Branch, Div. of HIV/AIDS Prevention      1600 Clifton Rd.    Mailstop A-25 Atlanta, GA 30333 tel: 404-639-1312fax: 404-639-1174email:ajo9@cdc.govBackground: Small molecule inhibitors of the zinc finger domain (ZFI) in the nucleocapsid protein (NCp7) of HIV-1 are potent inhibitors of HIV and SIV replication and may have utility as topical products to prevent infection. Furthermore, intravaginal rings (IVRs) were developed as coitally-independent, sustained release devices which could be used for administration of HIV microbicides. The aims of these studies were to demonstrate that IVRs sized for macaques are practical and compatible with the current generation of thioester-based NCp7 inhibitors. Methods: Non-medicated silicone elastomer vaginal rings of various sizes thought to be applicable for macaques were prepared and tested for vaginal fit in Pigtailed and Chinese Rhesus macaques. Macaques were monitored for 8 weeks for mucosal disruption by colposcopy and proinflammatory cytokine markers in cervical vaginal lavages (CVL) using Luminex bead-based technology.  Three different ZFIs (compounds 52, 89 and 122, each derived from an N-substituted S-acyl-2-mercaptobenzamide thioester scaffold) were loaded at 50 mg into an optimal matrix-type ring design. In vitro continuous release studies were then conducted over 28 days and analyzed by HPLC. Rate of release was determined by linear regression analysis.Results: Qualitative evaluation at the time of ring insertion suggested that the 25 mm ring provided optimal fit in both macaque species. All rings remained in place during the study period (2 to 4 weeks), and the animals did not attempt to remove the rings. No tissue irritation was observed, and no signs of physical discomfort were noted. Also, no significant induction of cervicovaginal proinflammatory markers was observed during the 8-week period during and following ring insertion. One Pigtailed macaque showed elevated IL-8 levels in the CVL during the period when the ring was in place; however, these levels were comparable to those observed in two control macaques. In vitro release of the ZFIs peaked at day 1 and then continually declined to near steady-state rates between 20-30 mcg/day. The percent release after 14 days was 2.9, 2.0 and 0.9 for ZFI 89, 52 and 122, respectively.Conclusions: IVRs of 25mm diameter, determined to be the optimal size for macaques, were well tolerated and did not induce inflammation. Release of all ZFI compounds followed t 0.5 kinetics. These findings suggest that efficacy testing in primate models is warranted to fully evaluate the potential to prevent transmission.AbstractIntroductionThe HIV pandemic can arguably be best slowed and eventually stopped by an effective vaccine. Although great strides have been made towards that end, an effective vaccine is realistically still many years away, providing a compelling argument for the exploration of other avenues of HIV prevention.  Currently, there are a handful of effective microbicides that are designed to prevent HIV transmission by targeting specific viral proteins and hindering viral replication.  Many of these compounds can potentially inhibit replication of HIV at the site of exposure and are, therefore, especially important to female sex workers and women in heterosexual relationships.NCp7 is involved in both the early phase (facilitating reverse transcription and mediating undefined effects on integration) and the late phase (incorporation of the viral RNA genome into newly budding particles) of virus replication. HIV NCp7 contains two highly conserved zinc finger motifs that form an ordered and unique structure. Single amino acid mutations introduced within chelating (CCHC) or specific non-chelating residues of the zinc fingers result in the production of noninfectious virus particles. The potential virucidal nature of ZFI and the ability to inhibit cell-cell transmission together with the large number of conserved residues within the structure among HIV strains makes the NCp7 target highly desirable for microbicide applications.The characteristics of an ideal vaginal microbicide are summarized in Table 1.  Although many of the available antiviral drugs and compounds are highly effective, delivery and retention of the microbicide at the mucosal surface is challenging. Currently, the major challenges include the need to constantly reapply the microbicide gels and that gels are not universally received by all cultures. Therefore, there is a great need for an effective microbicide delivery device that will afford protection, is easy to use and socially and culturally acceptable.Intravaginal rings (IVRs) are widely accepted and currently being used as contraceptive devices for the long-term, controlled release of hormones (Table 2). Adapting IVRs for sustained delivery of microbicides against HIV seems a logical progression of this technology since they possess many of the desirable attributes listed in Table 1. We describe the adaptation of this technology to the non-human primate model for studying HIV transmission, and report the initial in vitro release of a class of investigative drugs, zinc finger inhibitors.559a  Abstract Y-165Table 2  Vaginal rings either marketed or in development [Adapted from Woolfson et. al. Potential Use of Vaginal Rings for Prevention of Heterosexual Transmission of HIV Am J Drug Deliv 2006; 4 (1)]Table 1   Characteristics of an ideal vaginal HIV microbicide product [Woolfson et. al. Potential Use of Vaginal Rings for Prevention of Heterosexual Transmission of HIV Am J Drug Deliv 2006; 4 (1)] 1 HRT – Hormone replacement therapyFigure 1  Types of silicone-based intravaginal rings  IVRs can be manufactured to release drug at constant levels (reservoir) or at a high initial level with a decreasing release rate over time (matrix). Reservoir MatrixCross-sectionDrug ReleaseProfileTimedrugTimedrugAbility to be used without knowledge of male partnerRetain activity in presence of semenLong duration of activityTake account of physiological changes that occur during  intercourseNonteratogenicGood vaginal distributionOdorlessGood vaginal retentionTastelessNon-messyAvailability of contraceptive and noncontraceptive formsMaintain the integrity of the vaginal tissueHigh user acceptabilityCause no local irritancyNegligible systemic absorptionLong shelf-lifeCompatible with condomsImmediate protection after applicationActivity against other sexually transmitted pathogensCause no physical discomfortMaintain normal vaginal ecologyEase of applicationRetain activity over broad pH rangeInexpensive to manufactureNATMC120SiliconeHIVInternational Partnership for MicrobicidesNA10 mg/day (1 year)ProgesteroneSiliconeContraceptivePopulation Council/CONRADProgering50 or 100 µg/day estradiol (3 months)Estradiol acetate (12.4 or 24.8mg)SiliconeHRTWarner ChilcottFemring120 + 15 µg/day(3 weeks)Etonogestrel + ethinylestradiol(11.7/2.7mg)Poly(ethylene-co-vinylacetate)ContraceptionOrganonNuvaring7.5 µg/day (3months)Estradiol (2mg)SiliconeHRT 1Pharmacia & UpjohnEstringRelease rate(period of use)Active ingredients (loading)ElastomerdescriptionIndicationCompany Product name>105>5000>192TI>100>100>100IC5010.020.5EC50PBMC 1HIV-1,  B subtype70.48.656.3TI56.340.456.3TC500.84.71EC50PBMC 1 SIVMac 2511228952ZFIpg/ / mL

Size-fitting of Intravaginal Rings for Macaques and in vitro Release Kinetics of Zinc Finger Inhibitors

Size-fitting of Intravaginal Rings for Macaques and in vitro Release Kinetics of Zinc Finger Inhibitors

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