Nanotech-derived topical microbicides for HIV prevention: the road to clinical development.

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Contents lists available at ScienceDirect

Antiviral Research journal homepage: www.elsevier.com/locate/antiviral

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Review

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Nanotech-derived topical microbicides for HIV prevention: The road to clinical development

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Javier Sánchez-Rodríguez a,b,1, Enrique Vacas-Córdoba a,b,1, Rafael Gómez c,b, F. Javier De La Mata c,b, Ma Ángeles Muñoz-Fernández a,b,⇑ a b

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Laboratorio InmunoBiología Molecular, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain Dendrimers for Biomedical Applications Group (BioInDen), University of Alcalá, Alcalá de Henares, Madrid, Spain

a r t i c l e

i n f o

Article history: Received 16 September 2014 Revised 20 October 2014 Accepted 29 October 2014 Available online xxxx Keywords: Microbicides HIV Nanotechnology Nanomedicine

a b s t r a c t More than three decades since its discovery, HIV infection remains one of the most aggressive epidemics worldwide, with more than 35 million people infected. In sub-Saharan Africa, heterosexual transmissions represent nearly 80% of new infections, with 50% of these occurring in women. In an effort to stop the dramatic spread of the HIV epidemic, new preventive treatments, such as microbicides, have been developed. Nanotechnology has revolutionized this field by designing and engineering novel highly effective nano-sized materials as microbicide candidates. This review illustrates the most recent advances in nanotech-derived HIV prevention strategies, as well as the main steps required to translate promising in vitro results into clinical trials. Ó 2014 Published by Elsevier B.V.

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Contents 1. 2. 3. 4.

HIV prevention strategies: an urgent necessity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . History of the microbicide pipeline. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nanotechnology: a new field in the design of microbicides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Moving towards pre-clinical development of nanotech-derived microbicides: the main stages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Identification of novel candidates: in vitro screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Characteristics of ideal nanotech-derived microbicide candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Antiviral assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3. Safety and biocompatibility of nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.4. HIV-transmission inhibition assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: AIDS, acquired immunodeficiency syndrome; ARV, antiretroviral; ASPIRE, a study to prevent infection with a ring for extended use; BLT, bone marrow liver thymic mice; CAP, cellulose acetate phthalate; CAPRISA, centre for the AIDS programme of research in South Africa; CBA, carbohydrate-binding agents; CCR5, C–C chemokine receptor type 5; CD34, cluster of differentiation 34 marker; CD4, cluster of differentiation 4 marker; CXCR4, C-X-C chemokine receptor type 4; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; EC50, half maximal effective concentration; ELISA, enzyme-linked immunosorbent assay; HAART, highly active antiretroviral therapy; HIV, human immunodeficiency virus; HSV, herpes simplex virus; IL, interleukin; IPM, international partnership for microbicides; LDH, lactate dehydrogenase; LFA, leukocyte function-associated antigen; MIP, macrophage inflammatory protein; MTN, microbicide trials network; MTS, (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); N-9, nonoxynol-9; NNRTI, nonnucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; PAMAM, poly(amido amine); PBMC, peripheral blood mononuclear cell; PEG, poly(ethylene glycol); PI, protease inhibitor; PLGA, poly-lactic-co-glycolic acid; PrEP, pre-exposure prophylaxis; RANTES, regulated on activation, normal T cell expressed and secreted protein, also known as CCL5 (Chemokine (C–C motif) ligand 5); SHIV, simian–human immunodeficiency virus; STD, sexually transmitted disease; TC50, toxic concentration 50; TEER, trans epithelial electric resistance; TFV, tenofovir; TNF, tumor necrosis factor; UNAIDS, joint United Nations programme on HIV and AIDS; VOICE, vaginal and oral interventions to control the epidemic; XTT, (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide); 7AAD, 7-aminoactinomycin D. ⇑ Corresponding author at: Laboratorio InmunoBiología Molecular, Hospital General Universitario Gregorio Marañón, CIBER BBN, C/Dr. Esquerdo 46, 28007 Madrid, Spain. Tel.: +34 915868018; fax: +34 915868565. E-mail addresses: [email protected], [email protected] (Ma Ángeles Muñoz-Fernández). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.antiviral.2014.10.014 0166-3542/Ó 2014 Published by Elsevier B.V.

Please cite this article in press as: Sánchez-Rodríguez, J., et al. Nanotech-derived topical microbicides for HIV prevention: The road to clinical development. Antiviral Res. (2014), http://dx.doi.org/10.1016/j.antiviral.2014.10.014

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4.1.5. Investigation of nanoparticle inhibitory mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.6. Combination assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.7. Broad spectrum activity of topical microbicides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Ex vivo and in vivo evaluation: a proof-of-concept of compound efficacy prior to human clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future perspectives, obstacles and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. HIV prevention strategies: an urgent necessity

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As announced in the 2013 UNAIDS report, approximately 35.3 million people are living with HIV worldwide (UNAIDS, 2013). Although during the last few years international and national health programs have achieved a progressive reduction in new HIV infections, 2.5 million new infections and 1.7 million AIDSrelated deaths still occurred worldwide. HIV heterosexual transmission is responsible for approximately 80% of these new infections and is one of the most difficult routes of transmission to control. During transmission, HIV-1 must enter or cross the stratified epithelium of the vagina or the columnar epithelium of the uterine cervix. It is unknown whether infectious virions penetrate mucosal tissues far enough to reach epithelial or subepithelial target cells, which cells are initially infected in addition to CD4+ T cells and to what extent trans-presentation by non-infected dendritic cells contributes to HIV-1 dissemination (Fackler et al., 2014). Women constitute over 50% of the HIV infected population, although in some countries, such as South Africa, this percentage rises to approximately 60% (Carter et al., 2013). The problem is even more alarming in sub-Saharan Africa, where more than six out of every ten infected individuals is female, and young women aged 15–24 years are twice as likely to be infected with HIV compared to men of the same age. Furthermore, HIV/AIDS has become the main cause of death among women aged 15–44 years. In many cases, men and women do not share the responsibility of reducing the risk of HIV transmission because women are excluded from sex-related decision-making, do not receive in-depth sex education

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or have unequal access to preventive methods. That translates into 3000 new infections in women every day. For over 20 years, the scientific community has been working hard to develop new preventive strategies to decrease HIV infection rates, especially in women. To date, despite a great deal of effort, there are no effective vaccines against HIV infection (Munier et al., 2011). Thus, it is urgent and imperative to develop prevention systems capable of stopping the spread of HIV in the most disadvantaged and vulnerable populations, especially during sexual intercourse (Anton, 2012; Stephenson, 2011). It is known that behavioral and structural interventions, such as the use of condoms or circumcision, are effective methods in the prevention of HIV sexual transmission (Padian et al., 2008; Quinn, 2007). Other pre-exposure prophylaxis (PrEP) HIV-preventive strategies, such as the oral administration of antiretroviral (ARV) drugs or topical microbicides, also seem to be feasible and effective approaches against the spread of HIV (Abdool Karim and Baxter, 2014; Balzarini and Van Damme, 2005; Buckheit et al., 2010; Granich et al., 2010). Microbicides are compounds that when applied topically to the vagina and/or rectum can reduce the risk of HIV infection. Microbicides are advantageous over other prevention methods, such as condoms, because they are easier to use and provide users, especially women, with the ability to decide on their utilization. Microbicides are extremely advantageous to highly vulnerable groups, such as sex workers or women who suffer from sexual aggression. Due to their great potential, much effort in the past and present has been put into developing vaginal or rectal microbicides, including gels, rings and other formulations, capable of reducing the risk of HIV sexual transmission (Tables 1–3).

Table 1 Ongoing and planned Phase III and Phase II/IIb clinical trials of microbicide candidates. The only candidate to show efficacy to date is the antiretroviral drug tenofovir formulated in 1% gel form. One trial has shown benefit; another has not. Confirmatory research is ongoing in different trials. A new formulation of tenofovir gel is also being evaluated for rectal used, as a possible HIV prevention tool for anal sex. Other leading candidate in large efficacy trials is dapivirine ring. Currently, there are two ongoing efficacy trials of the dapivirine ring recruiting women from communities across several countries in East and Southern Africa. TDF – Tenofovir disoproxil fumarate; TFV – Tenofovir; FTC – emtricitabine; MSM – men who have sex with men. (Adapted from http://www.avac.org/). Trial name

Phase

Start date

Phase III (safety and effectiveness) FACTS 001 Phase III October 2011

Locations

Population

Candidate(s)

Status/expected completion

South Africa

2900 women

1% TFV gel 4-week vaginal dapivirine ring 4-week vaginal dapivirine ring 1% TFV gel

Ongoing/December 2014 Ongoing/August 2015

IPM 027 (The Ring Study) MTN 020 (ASPIRE)

Phase III April 2012

Rwanda, South Africa, Malawi

1650 women

Phase III July 2012

3476 women

CAPRISA 008

Phase III, October 2012 II

Malawi, South Africa, Uganda, Zimbabwe South Africa

Phase II, IIb (safety, adherence, acceptability, feasibility, effectiveness) MTN 017 Phase II September Peru, South Africa, Thailand, 2013 United States, Puerto Rico MTN 019 MTN 023/IPM 030

Phase II Phase II

July 2011 March 2014

MTN 024/IPM 031

Phase II

FACTS 002

Phase II

December 2013 To be determined

United States United States South Africa

700 women

186 transgender, MSM

Daily oral TDF/FTC, Reformulated rectal 1% TFV gel 1% TFV gel Dapivirine vaginal ring

Ongoing/December 2014 Ongoing/February 2015 Ongoing/June 2016

384 women HIV-negative adolescent females 96 postmenopausal women Dapivirine vaginal ring

Ongoing/August, 2015 Ongoing

60 young women (16– 17 years)

Ongoing

Vaginal TFV 1% gel

Ongoing/May 2014


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Table 2 Current Phase I microbicide clinical trials. The majority of microbicide candidates currently in clinical trials are formulated with ARV drugs and most of them have been gels. However, there are several other candidates in early phases of development. In addition, it may be useful to develop non-ARV based candidates and candidates delivered in other ways (e.g., a film or fast dissolving tablets). Trial name

Phase

Start date

Locations

Population

Phase I (safety, adherence, acceptability, feasibility) CONRAD A10-114 Phase I March 2012 United States, Dominican Republic CONRAD 122 Phase I To be United States determined CONRAD 118 Phase I December United States 2013 CONRAD 117 Phase I February United States 2013 CONRAD 114 Phase I March 2012 Dominican Republic, United States Project Gel Phase I March 2014 United States, Puerto Rico

NNRTI Microbicide Phase I Gel MTN-014 Phase I IPM-034 Phase I Population Council Phase I #558 MTN-011 Phase I

Candidate(s)

Status/expected completion

March 2014

United States

April 2014 November 2013 March 2014

United States Belgium

Ongoing/January 2014 40 men and women SILCS diaphragm and 1% TFV gel Protocol in development 54 women, HIV negative 1% TFV gel, Contraceptive Vaginal Ring, Ongoing/November Metronidazole, Terconazole 2014 48 women, HIV negative TFV/FTC and TFV only fast dissolve Ongoing/January vaginal tablets 2014 72 women 1% TFV gel, Depo-provera, Oral Ongoing contraceptive 240 MSM 1% TFV rectal gel once daily for 7 days Ongoing/March 2014 and placebo rectal gel prior to rectal intercourse 30 women, HIV negative PC-1005 gel, HEC gel Ongoing/March 2015 28 women 1% tenofovir reduced-glycerin gel Ongoing 40 women Dapivirine Ring Ongoing/May 2014

United States

women

PC-1005 gel

United States

80 women, men, HIV negative women

1% TFV gel

90 women

TFV and TFV/LNG vaginal ring

CONRAD 123

Phase I

November 2012 2014

CONRAD 128

Phase I

Q4 2013

72 women, HIV negative 1% TFV gel, DMPA

Ongoing/March 2015 Ongoing/April 2015

TFV gel/SILCS Diaphragm

Protocol in development Pending

Table 3 Open label and other clinical trials of microbicide candidates. Various other clinical studies and sub-studies are currently ongoing to better understand, for instance, the adherence and use of the study products or the sexual behavior of users during their participation in the trials. Trial name

Open label MTN 025

Phase

Start date

Locations

Population

Vaginal dapivirine ring

Planned

N/A

Ongoing/ May 2013

MTN 016 (EMBRANCE) IPM-33

Open label

August 2013

IPM-29

Open label

February 2013

United States

Pre-clinical

Q4 2013

To be determined Eastern Virginia Medical School/To be determined

TFV

Planned 2014

South Africa

N/A

Primary analysis completed Primary analysis completed Ongoing/ March 2014

MTN 003B

MTN 015

MTN 003 (VOICE) July 2010 community sub-study MTN 003 (VOICE) bone mineral density sub-study Seroconverter protocol

November Uganda, Zimbabwe 2009 August 2008

140 women/men

275 Participants (includes women in VOICE, their male partners, CAB members and community stakeholders) 518 women

Malawi, South Africa, 500 HIV-positive women Uganda, Zambia, Zimbabwe

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2. History of the microbicide pipeline

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Many agents with antiviral activity have been evaluated as microbicidal candidates. The first tested microbicides were

120

Status/ expected completion

Open label/phase IIIb ASPIRE follow on Open label/pregnancy October exposure registry 2009

Other Effects of Tenofovir on CAPRISA samples MTN 003C

Malawi, South Africa, Uganda, Zimbabwe South Africa, Uganda, 800 women, men, HIV negative United States, Zimbabwe United States 160 women/men

Candidate(s)

Ongoing No ring, condom only/ placebo vaginal ring + female condom used Silicone elastomer vaginal Ongoing/ ring October 2013

Oral TDF; oral TDF/FTC

N/A

surfactants, which are compounds that act as detergents and are able to destroy the integrity of lipid membranes. The most well known is the non-specific microbicide nonoxynol-9 (N-9) (Roddy et al., 1998; Van Damme et al., 2002). This compound was able


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to destroy the virus by damaging the viral envelope (Bourinbaiar and Fruhstorfer, 1996). However, clinical trials showed that N-9 increased the risk of HIV infection (Roddy et al., 1998). Another surfactant, C31G (SAVVY), which was shown to be safe in vitro and exhibited broad-spectrum activity against bacteria and herpes simplex virus-2 (HSV-2), was analyzed against HIV in three independent clinical trials in Ghana and Nigeria (Ballagh et al., 2002; Bax et al., 2002; Feldblum et al., 2008; Krebs et al., 1999; Mauck et al., 2004; Thompson et al., 1996). These trials were suspended because the rates of infection were higher in the surfactant-treated group compared to the placebo group. Currently, the surfactant candidate sodium lauryl sulfate (Invisible Condom; Université Laval, Quebec, Canada) is being investigated (Bestman-Smith et al., 2001; Mbopi-Keou et al., 2010; Piret et al., 2002). It works as an ‘invisible condom’ that could cover the vaginal wall as a liquid at room temperature and subsequently transform into a gel at body temperature, inhibiting the transmission of HIV-1 and other sexual transmitted diseases (STD). The results from a randomized, double blind, placebo-controlled Phase II study in Cameroonian women revealed that sodium lauryl sulfate was well tolerated and acceptable (Mbopi-Keou et al., 2010). Further phases of clinical development are planned (Gupta, 2013). Together with surfactants, many polyanionic compounds have been analyzed as microbicides (Baranova et al., 2011; Pirrone et al., 2011). The anti-HIV activity of these compounds is associated with the establishment of electrostatic interactions between the viral envelope proteins and the anionic functional groups of the compounds. This interaction prevents the binding of the virus to the target cell, blocking viral entry. Three of these polyanionic compounds, Carrageenan (Carraguard), cellulose sulfate and PRO2000, were effective in vitro and in preclinical trials, although none showed any efficacy in clinical trials in humans (Gibson and Arts, 2012; Huskens et al., 2009; Keller et al., 2010; Pirrone et al., 2011; Reina et al., 2010). Surprisingly, in some cases, such as with cellulose sulfate, higher rates of HIV infection were found in the treated group compared to the placebo group (Tao et al., 2008). Several rationales were proposed to explain the failure of these polyanionic compounds, including their lower inhibitory effects in comparison to other antivirals, poor absorption by the mucosa, reduced efficacy in the presence of seminal plasma, or the induction of local inflammation and changes in vaginal microflora, all of which could facilitate HIV-1 infection (Pirrone et al., 2011). Currently, the leading candidates in the microbicide pipeline are ARV (Chirenje et al., 2010; Granich et al., 2010; Klasse et al., 2008; Omar and Bergeron, 2011; Shattock and Rosenberg, 2012). These compounds exhibit potent and specific anti-HIV activity and are safe, as demonstrated by their continuous use in highly active antiretroviral therapy (HAART). Currently, more than thirty ARV targeting different steps of the viral cycle have been approved by the U.S. Food and Drug Administration (FDA) for use in HAART (Fig. 1). HIV inhibitors, including entry inhibitors, nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTI and NNRTI, respectively) or integrase and protease inhibitors have been used as potential microbicide candidates. The only candidate to show high efficacy to date is the NRTI Tenofovir (TFV) (Abdool Karim et al., 2010; Gengiah et al., 2012; Mertenskoetter and Kaptur, 2011). TFV formulated as a 1% gel was tested in the CAPRISA 004 trial in 889 South African women, resulting in a 39% overall reduction in the women’s risk of contracting HIV infection via vaginal sex (Network, 2010). However, other clinical trials failed, such as VOICE, a five-armed proof-of-concept clinical trial that enrolled more than 5000 women in South Africa, Uganda and Zimbabwe (Friend and Kiser, 2013). The VOICE study was prematurely stopped in 2011 due to lack of efficacy. It concluded that none of the interventions tested (daily oral TFV, daily oral TFV/emtricita-

bine or daily TFV gel) reduced the risk of HIV transmission. Some reasons have been provided to explain the apparent lack of efficacy, particularly the low adherence. Most participants did not use the tested interventions daily as recommended. Thus, confirmatory research is ongoing, and trials with other formulations of TFV are planned (Network, 2013a). For instance, CAPRISA 008 will assess the feasibility and effectiveness of providing the TFV gel in a clinic setting. A new reduced glycerin formulation of the TFV gel is being evaluated for rectal use in the MTN 017 trial as an HIV prevention system for rectal transmission (Network, 2013b). This is particularly relevant to homosexual communities and other men who have sex with men, especially when the risk associated with HIV transmission through unprotected receptive anal intercourse is 1.7% per act (versus 0.08% in case of unprotected vaginal intercourse). This is due to anatomical differences between vaginal and rectal tissues. The rectal epithelium is formed by a single layer of cells as opposed to the multilayer squamous epithelium found in the vagina and ectocervix. In addition, the gastrointestinal tract is populated with a large number of HIV-1 infectable cell subsets (van Marle et al., 2007). All these factors make the rectal route a more susceptible route for HIV infection. Several rectal microbicide clinical trials testing others antiretrovirals such as UC781 have been conducted, as deeply reviewed by Rohan et al. (2013). Another candidate in large-scale efficacy trials is the NNRTI dapivirine. It was formulated as a vaginal ring, and currently there are two ongoing phase III clinical trials (ASPIRE and IPM 027) in Africa (Microbicides, 2012; Network, 2012). Another phase I clinical trial, MTN 013/IPM 026, has studied the effectiveness of the combination of dapivirine and maraviroc as a microbicidal vaginal ring (Fetherston et al., 2012). Results were presented this year at the 21st Conference on Retroviruses and Opportunistic Infections (CROI). MTN-013/IPM 026 found that the ring was safe in women. In addition, dapivirine was able to block HIV infection, though levels of maraviroc were not sufficient to have a similar effect (Chen et al., 2014, (CROI)). Because CCR5-tropic viral strains are dominant during HIV-1 sexual transmission, entry inhibitors, such as CCR5 antagonists, have also been evaluated as microbicide candidates (Maeda et al., 2012). CCR5 antagonist CMPD167, a cyclopentane-based compound used as a vaginal gel that has provided protection from vaginal SHIV challenge in macaques, and maraviroc, a small molecule that binds to the CCR5 co-receptor and blocks HIV-1 entry into cells, are being tested in humans. A proof-of-concept study in macaques showed that maraviroc could play a role in protecting women from HIV-1 infection if developed as a vaginal microbicide (Veazey et al., 2010). A major challenge in considering the use of CCR5 inhibitors as topical microbicides is their inability to block the entry of X4-tropic viruses. Although infection via the CXCR4 co-receptor is less important in sexual transmission, X4-tropic viruses are present in vaginal fluids and semen during acute HIV infection (Grivel et al., 2011). Thus, the development of broad spectrum topical microbicides capable of preventing HIV transmission regardless of viral tropism is crucial. There are several other novel ARV-based and non-ARV-based candidates in the early phases of preclinical development. For instance, the cyclic antimicrobial peptide Retrocyclin RC-101 (Gupta et al., 2013), the NRTI saquinavir (Stefanidou et al., 2012) and rilpivirine (De Clercq, 2012), the fusion inhibitor cyanovirin (Tsai et al., 2003) and zinc-derived compounds (Kenney et al., 2011) are also under development. Interestingly, griffithsin, a carbohydrate-binding agent (CBA) extracted from red algae, is expected to enter in a phase I safety and acceptability trial in the next year (MTN-038) (Emau et al., 2007; Hillier, 2013). A summary of the clinical trials of microbicide candidates is shown in Tables 1– 3. The outcomes of these trials currently underway will significantly impact on the development and search of new candidates.


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Fig. 1. Therapeutic targets of the HIV life cycle. Viral entry requires the binding of gp120 to the cellular receptor CD4 and a co-receptor (CCR5 or CXCR4). Co-receptor binding causes changes in gp120 and the gp41 transmembrane peptide, leading to membrane fusion. CCR5 inhibitors, such as maraviroc (CelsentriÒ), prevent viral binding to this co-receptor. Fusion inhibitors as Enfuvirtide (FuzeonÒ) bind to gp41 and prevent the conformational changes required for viral cell fusion. In the cytoplasmic compartment, the internalized viral core and viral enzyme reverse transcriptase copies the viral RNA into a DNA strand which is transported into the nucleus. Analogue reverse transcriptase inhibitors and non-nucleoside inhibitors (NRTI and NNRTI), such as tenofovir (VireadÒ), abacavir (ZiagenÒ) and efavirenz (SustivaÒ), inhibit this process. In the nucleus, the viral integrase incorporates the viral DNA into the cellular genome, a step blocked by integrase inhibitors, such as raltegravir (IsentressÒ). The integrated proviral DNA is transcribed to generate complete viral RNA sequences, and multiple mRNAs are translated into proteins using cellular machinery. The viral RNA and viral proteins are transported to the cell membrane, where they are assembled with cellular factors within the viral particle. Budding of viral particles occurs simultaneously with the cleavage of precursor Gag and Gag-Pol by the viral protease, resulting in a mature virion. Protease inhibitors, such as atazanavir (ReyatazÒ) and darunavir (PrezistaÒ), block the budding step and prevent the formation of mature virions.

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However, there are some limitations that could explain the modest success of the use of antiretroviral drugs in preventive approaches against HIV, such as their extreme hydrophilic and hydrophobic nature or their low enzymatic and chemical stability.

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3. Nanotechnology: a new field in the design of microbicides

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In recent years, nanotechnology has emerged as a new research area capable of providing solutions to the limitations found in the development of preventive strategies against HIV. This field is focused on designing, engineering and fabricating new materials at the nanosize level. According to the National Nanotechnology Initiative, nanotechnology involves study of materials/architectures of size 1–100 nm in at least one dimension. Nevertheless, materials with size up to several hundred nanometers are also included under nanotechnology. These materials can be classified

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into several groups, such as liposomes, polymers, dendrimers, nanocrystals, nanofibers and metal nanoparticles, according to the material used in their assembly (Table 4). In biomedical applications, nanotechnology has enabled the development of novel nanodrugs and compounds with useful pharmacological advantages in the therapy and diagnosis of cancer and infectious diseases, including HIV (Kim et al., 2010a; Kim and Read, 2010; Siccardi et al., 2013). Various nanosystems have been used in different strategies for the treatment and prevention of HIV-1 infection (Date and Destache, 2013; Mahajan et al., 2012; Mallipeddi and Rohan, 2010; Mamo et al., 2010). Some nanoplatforms, due to their nanometric size, are versatile drug delivery systems because of their capacity to overcome corporal barriers and deliver drugs to specific cells or intracellular compartments. Different methods of cargo delivery include encapsulation in the particle core or absorption onto their functionalized surface, thereby improving the pharma-


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cological properties of several ARV. For instance, nanosuspensions of rilpivirine stabilized by polyethylene–polypropylene glycol and PEGylated tocopheryl succinate ester resulted in extended sustained release of the compound in animal models, suggesting that nanoscale drug delivery may potentially reduce dosing frequency and improve adherence (Baert et al., 2009). Nanoplatforms can also be used to control the release of nucleic acids in gene therapy against HIV, or as new antiviral agents due to their intrinsic antiviral properties (Amiji et al., 2006; Briz et al., 2012; Cordoba et al., 2013b,c; Chonco et al., 2012; das Neves et al., 2010; de Las Cuevas et al., 2012; Farokhzad, 2008; Jimenez et al., 2010; Mallipeddi and Rohan, 2010; Perise-Barrios et al., 2014; Sanchez-Nieves et al., 2014; Weber et al., 2008, 2012; Meng et al., 2011; Steinbach et al., 2012). These nano-architectures have also been used in different approaches to design novel topical microbicides (Date and Destache, 2013; Ensign et al., 2014). Polymeric biodegradable nanoparticles, liposomes, nanofibers and inorganic nanoplatforms are being explored as platforms for vaginal drug delivery. TFV and tenofovir disoproxil fumarate nanoparticles composed of a blend of poly-lactic-co-glycolic acid (PLGA) and pH-responsive polymethylmethacrylate (Eudragit) polymer, dapivirine-loaded poly(epsilon-caprolactone) nanoparticles, raltegravir-efavirenzloaded PLGA nanoparticles or PLGA nanoparticles with encapsulated PSC-RANTES (an antagonist of the CCR5 co-receptor) have already been tested in animal models against HIV (Belletti et al., 2012; Chaowanachan et al., 2013; das Neves et al., 2012a, 2013; Ham et al., 2009; Zhang et al., 2011; Ensign et al., 2014). PSC-RANTES encapsulation within PLGA nanoparticles provided sustained release and increased uptake into ex vivo cervical tissue compared to soluble PSC-RANTES. Other major study has shown that PSCRANTES is effective and provides potent protection against vaginal challenge in rhesus macaques, indicating its potential as microbicide candidate (Lederman et al., 2004). Liposomes composed of phospholipid bilayer structures were investigated as antiviral agents due to discovery that certain lipid compositions can bind to HIV particles and modulate their infectivity. Moreover, lipid-based nanoparticles have been designed for vaginal delivery of drugs or small interfering RNA to silence HIV gene expression and prevent the establishment of primary infection (Malavia et al., 2011; Wang et al., 2012). The ability of nanofibers, which are spaghetti-like compounds made from materials, such as polysaccharides, peptides or polymers with diameters ranging from 1 to 1000 nm, to deliver different microbicides for HIV prevention has been explored. Maravirocloaded nanofibers, nanofibers made of cellulose acetate phthalate (CAP) that acts as an HIV entry inhibitor and CAP fibers loaded with TFV have shown great potential in inhibiting HIV infection (Gunn and Zhang, 2010; Huang et al., 2012). Furthermore, the antiviral properties of inorganic metals, such as silver, gold and zinc, have been exploited in the search for novel topical microbicides. Polyvinyl pyrrolidone-coated silver nanoparticles inhibited viral fusion and demonstrated antiviral activity against various HIV strains, including drug-resistant strains (Lara et al., 2010a,b). Gold nanoparticles contain free thiol groups on their periphery that permit their easy functionalization with various biomolecules. Multivalent oligomannosides-coated gold nanoparticles and anionic sulfate-functionalized gold nanosystems have demonstrated inhibitory activity against HIV in vitro (Di Gianvincenzo et al., 2010; Vijayakumar and Ganesan, 2012). Dendrimers are the most advanced group of nanotech-derived microbicides (Lazniewska et al., 2012; Parboosing et al., 2012; Svenson and Tomalia, 2005). Dendrimers are a class of hyperbranched polymeric nano-structures that are much smaller than traditional nanoparticles, with narrow polydispersity and precise numbers of terminal groups. Dendrimer structure and composi-

tion, including the number of branching units and the composition and number of surface functional groups, can be engineered to obtain compounds with the desired physicochemical and biological properties. The peripheral functional groups can be designed to directly interact with target cells or pathogens, conjugate drugs, or complex with other biomolecules, such as peptides or siRNA. One dendrimer based-compound, VivaGel, is the only nanotechderived microbicide candidate that has reached human clinical trials (Bernstein et al., 2003; Gong et al., 2005; Jiang et al., 2005; Telwatte et al., 2011; Tyssen et al., 2010). The active ingredient in VivaGel is the SPL7013 dendrimer, which is a polylysine anionic dendrimeric agent. It has demonstrated great potency against both HIV-1 and HSV-2 (Price et al., 2011; O’Loughlin et al., 2010), although the phase I clinical trial evaluating its antiviral activity and microbiota tolerance revealed evidence of mild irritation after repeated vaginal use (McGowan et al., 2011). VivaGel is not currently being tested in clinical trials as a topical microbicide against HIV; nevertheless, it is in phase II trials as a treatment for bacterial vaginosis. Other dendrimeric compounds are in development for use as vaginal microbicides (Date and Destache, 2013; Navath et al., 2011). Dendrimers containing a carbosilane core and different anionic ended functional groups have been studied as anti-HIV compounds (Chonco et al., 2012; Galan et al., 2014; Garcia-Gallego et al., 2012; Rasines et al., 2012; Vacas Cordoba et al., 2013). These dendrimers have shown high biosafety in human epithelial cell lines derived from the female genital tract and in primary human blood cells (PBMC), blocked the entry of both X4- and R5-tropic HIV-1 isolates into epithelial cells while protecting the epithelial monolayer from cell disruption, reduced HIV-1 infection of PBMC and were safe in mouse and rabbit models, with no promotion of irritation or vaginal lesions (Chonco et al., 2012; Vacas Cordoba et al., 2013). Anionic dendrimers comprising benzhydryl amide cores and lysine branches and functionalized with naphthalene disulfonic acid or 3,5-disulfobenzoic acid groups have demonstrated activity against HIV-1 and HSV-2 infection, preventing viral entry and HIV envelope-mediated cell-to-cell fusion (Telwatte et al., 2011; Tyssen et al., 2010). Polycationic ‘‘viologen’’ based dendrimers have also shown anti-HIV activity by interaction with the CXCR4 co-receptor (Asaftei et al., 2012). Other dendrimers, such as four generation-PAMAM branched-naphthalene disulfonic surface groups ending with SPL2923 or polysulfonated dendrimer BRI2923, have shown inhibitory effects against HIV-1 not only at the entry step but also at later stages of the viral replicative cycle (Witvrouw et al., 2000). Multivalent dendrimeric compounds coated with carbohydrates expressed on immune cells inhibited HIV-1 infection by targeting viral entry, whereas multivalent-glycodendrimeric compounds prevented HIV transmission via DCSIGN on dendritic cells (Garcia-Vallejo et al., 2013; Rosa Borges et al., 2010). Although nanoparticles are under development as useful and powerful tools in the field of antiviral microbicides, they display a series of limitations that should be addressed because they can affect the transfer of these platforms to clinical trials. These limitations include toxicity, the appearance of non-desirable biological interactions, bioaccumulation, the enzymatic degradation of these nanosystems, their penetrance and absorption features into different tissues and their high manufacturing cost; this last limitation is especially a problem for scale-up production (Mamo et al., 2010).

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4. Moving towards pre-clinical development of nanotechderived microbicides: the main stages

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Rigorous pre-clinical evaluation of vaginal or rectal candidate microbicides is essential for the selection of the best compounds for continued development. This involves a series of basic steps

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Architecture

Features

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Nanocompound

Candidates explored in vivo Anti CCR5 siRNA-loaded liposome (Kim et al., 2010a) Cardiolipin liposome (Malavia et al., 2011)

Liposomes

Biocompatible, completely biodegradable and non-immunogenic Encapsulation of hydrophobic, amphipathic and hydrophilic drugs Ability to protect encapsulated drugs from the external environment and to act as sustained release depots Available technologies for large-scale production

Dendrimers

Controlled synthesis and narrow polydispersity Different molecules/drugs could be conjugated to a single dendrimer Due to their hyperbranched architecture, they can interact in a multivalent way They can be tailored by manipulating their structure or composition and number of surface functional groups to obtain compounds with desired properties

Vivagel (SPL7013 dendrimer) (O’Loughlin et al., 2010) Sulfonated carbosilane dendrimer 2G-S16 (Chonco et al., 2012) Tenofovir-emtricitabine-loaded thiopyridine terminated G4PAMAM dendrimer (Navath et al., 2011)

Polymeric nanoparticles

There are a wide range of biodegradable and non-biodegradable materials for desired application Sustained release of encapsulated agents Improved pharmacokinetic, solubility and stability of encapsulated agents They can be used in targeted-delivery by tailoring their surface

Nanofibers

A wide variety of materials can be assembled into nanofibers Large surface area and many active surface sites Delivery and sustained drug release Easy production process: electrospinning

PSC-RANTES loaded PLGA-nanoparticles (Ham et al., 2009) siRNA-loaded PLGA nanoparticles (Steinbach et al., 2012) Tenofovir-loaded pH-sensitive PLGA nanoparticles (Zhang et al., 2011) Tenofovir-loaded chitosan nanoparticles (Meng et al., 2011) Tenofovir loaded hybrid chitosan/PLGA nanoparticles (Belletti et al., 2012) Dapivirine-loaded polycaprolactone nanoparticles (das Neves et al., 2012a,b) Tenofovir-loaded cellulose acetate phthalate nanofibers (Huang et al., 2012)

Inorganic nanoparticles

High surface-to-volume ratios Manipulable optical and surface characteristics Multiple ligands/drugs could be conjugated to surface using appropriate chemistry Applications in electrooptic devices, biomedical imaging, diagnostic or therapeutic field

Nanocrystal

Drug molecules are micronized in nano-scale crystals using top-down/bottom-up techniques Nanocrystals are composed of 100% drug; there is no carrier material Increase of dissolution velocity by surface area enlargement Increase in saturation solubility and bioavailability There are feasible technologies for large-scale production


Silver nanoparticles coated condoms (Mohammed Fayaz et al., 2012)


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Table 4 Important features and schematic structure of nanosystems used in the development of nano-based HIV topical microbicides. Different nano-architectures (liposomes, dendrimers, polymeric nanoparticles, nanocrystals, nanofibers and inorganic nanoparticles) are being explored as novel microbicide candidates. The most advanced in the microbicide pipeline are liposomes, polymers and dendrimers. Liposomes are spherical vesicles that comprise one or more lipid bilayer structures enclosing an aqueous core. They are high-potency carriers that protect encapsulated drugs from degradation. Polymeric nanoparticles are basically solid polymers with drugs embedded in the polymer matrix or with an inner space loaded with the drug of interest. Dendrimers are highly branched polymers with a controlled three-dimensional structure around a central core that can be easily functionalized. (Adapted from Date and Destache (2013), Biomaterials).

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designed to evaluate and guarantee safety and efficacy (Buckheit and Buckheit, 2012). There are different parameters that need to be studied in several in vitro and in vivo models (Fig. 2).

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4.1. Identification of novel candidates: in vitro screening

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4.1.1. Characteristics of ideal nanotech-derived microbicide candidates The discovery and development of new nanocompounds as microbicides with antiviral properties is difficult because these agents need to exhibit a series of desirable properties. They must be non-toxic, safe for prolonged use and highly effective against HIV-1 and other STDs (Fig. 3). They must also be easy to use, odorless and colorless, inexpensive and easy to manufacture and scaleup. Moreover, they must minimally damage the mucosa, be safe to the microflora found at the target mucosal tissue and not interfere with the efficacy and safety of other therapeutic agents, such as contraceptives. Because microbicides should diminish viral transmission both vaginally and rectally, different formulations need to be designed to prevent HIV transmission by these routes (Morrow and Hendrix, 2010; Turpin, 2011). The microbicide’s effectiveness should not be influenced by factors, such as seminal plasma or pH changes, that occur in the vagina during sexual intercourse. Thus, rational design of these nanoplatforms could be crucial for optimal performance in the vaginal and rectal environments (Fahmy et al., 2008). Properties such as mucus-

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penetration ability and sustained drug release capacity are important for improving vaginal drug delivery in HIV prophylactic approaches (Ensign et al., 2014). For instance, PEG surface density has been shown to be important for efficient mucus penetration (Cu and Saltzman, 2009; Tang et al., 2009). Recent progress in the design of biodegradable compounds, together with the regulation of nanosystem design and preparation, makes it possible to precisely manipulate the nanoparticle’s architecture, molecular weight and chemical composition, resulting in predictable tuning of their biocompatibility as well as their delivery.

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4.1.2. Antiviral assays Initial screening assays provide assessments of the compound’s efficacy (EC50: 50% reduction in HIV production from treated and untreated cells) and allow the identification of inhibitory compounds with direct antiviral activity or with the capacity to inhibit a specific step of the HIV life cycle. Human epithelial cell lines from appropriate target tissues, such as the vaginal mucosa-derived VK2/E6E7, uterine mucosa-derived HEC-1A or colorectal epithelium Caco-2 cell lines are commonly used in the study of transepithelial infection process (Gali et al., 2010a,b). Although infection of epithelial cells has been previously described, it is most probably not productive (Dezzutti et al., 2001). True early target cells of sexually acquired HIV include intra-epithelial cells such as Langerhans cells, subepithelial macrophages or CD4+ T lymphocytes. These

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Fig. 2. Algorithm for selection and evaluation of HIV microbicide candidates. It comprises the main steps for a rationale preclinical development of novel nano-based anti-HIV agents, including the evaluation of compound efficacy and toxicity in relevant cell-based systems, the range and mechanism of action of candidate compounds, the interactions of the compound in combination with other approved antiretrovirals, in vitro and in vivo pharmacokinetic, pharmacodynamics and safety evaluations and efficacy studies in sensitive models such as non-human primate or humanized mice. Adapted from Science Working Group of the Microbicide Initiative, The Science of Microbicides: Accelerating Development, 2002.


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Fig. 3. Potential activity of topical microbicides in vaginal epithelium. Microbicides should have not only anti-HIV activity, but they must be effective against other STDs that can increase the risk of HIV transmission by increasing the risk of epithelial inflammation and ulceration. Polyanionic compounds can disrupt the viral envelop or block cellvirus fusion. An acidic pH is necessary for natural vaginal protection against pathogens. Therefore it is important that microbicides do not alter the normal vaginal microflora. When the virus crosses the epithelial barrier it is taken up dendritic cells prior to infection of T cells and the recruitment of additional susceptible cells (adapted from Shattock et al., Nat Rev Microbiol, 2003).

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PBMCs subets and the CCR5-expressing TZM-bl reporter cell line are the best choice in the primary evaluation of microbicide candidates. Because R5-tropic viral isolates are mainly involved in HIV sexual transmission, the evaluation of the antiviral activity of nanoparticles against these viral strains is relevant because the majority of sexually-transmitted infections are via the CCR5 coreceptor. CD4+ T cells can be found along the lamina propia in the human vagina (endo- and ectocervix), often under the basal membrane (Edwards and Morris, 1985). Most are memory T cells that have increased CCR5 expression compared to T cells circulating in the peripheral blood (Hladik and McElrath, 2008; Hladik et al., 2007; Zhang et al., 1998). Nevertheless, it is important to remember that CXCR4-tropic viral strains are also present in vaginal fluids and semen. Therefore, to assure broad spectrum and potent antiviral activity, microbicide candidates should be tested against CCR5-, CXCR4- and dual-tropic viral strains. The most common R5-tropic laboratory strains are HIV-1JR-CSF, HIV-1BaL and HIV1NL(AD8) (B subtype) and the most common dual-tropic strain is HIV-1CBL23. All of these strains infect human primary cells and cell lines productively. However, to evaluate the efficacy of nanoparticles, testing their antiviral activity against different subtypes of primary clinically isolated viral strains is also necessary. Owing to the difficultly involved in using primary viral strains, working with established laboratory viral strains is an easier and quicker first step of in vitro primary screening of novel candidates.

4.1.3. Safety and biocompatibility of nanoparticles Once the inhibitory potency of new compounds is established, it is important to evaluate their safety and 50% toxic concentration (TC50) in different primary cells and cell lines. Different commercial assays are available for assessing nanoplatform toxicity in cells (Duncan and Izzo, 2005), the most common being the MTT assay. Other closely related colorimetric assays, including XTT and MTS, are also used. Studying cell membrane integrity is another common method to measure cell viability and cytotoxic effects. Vital dyes are frequently employed, such as trypan blue, 7-aminoactinomycin D (7-AAD) or propidium iodide. Alternatively, enzymatic

assays can be used to monitor the passage of molecules though the membrane, such as the LDH assay. Nanoparticle cytotoxicity depends on different features, such as nanoparticle size, surface charge and structure hydrophobicity/ hydrophilicity as reviewed by Date and Destache (2013). For instance, in cationic nano-architectures, the chemical structure of used cationic lipids can dramatically impact their efficacy, cellular uptake and toxicity. Nanosystems composed of cationic lipids with single alkyl chain were more toxic and incapable of transport through cervicovaginal mucus in comparison to nano-architectures composed of cationic lipids with two alkyl chains (das Neves et al., 2012b). Molecular weight is another important property that governs performance and toxicity of nano-architectures. In case of polyethylenimines (PEI), polymer molecular weight has an enormous influence on their transfection capability, antiviral activity and cytotoxicity. Size dependent toxicity has been also observed for silver nanoparticles. 15 nm sized-silver nanoparticles were significantly more toxic to macrophages than 30–55 nm silver nanoparticles (Carlson et al., 2008). On the contrary, PLGA nanoparticles did not show size dependent toxicity in macrophages, showing that, other factors, such as material type, are also crucial in size dependent responses (Xiong et al., 2013). Surface characteristics such as surface charge, hydrophilicity or surface functional groups have significant impact on nanoparticles behavior. Several studies have shown that positively charged nanocarriers have higher cytotoxicity as compared to anionic and neutral nanocarriers. Oral administration of positively charged dendrimers to mice was found to cause adverse effects such as hemobilia and splenomegaly, whereas negatively charged dendrimers did not show adverse effects and were well tolerated even at 10-fold higher dose (Thiagarajan et al., 2013). The decoration of nanosystem surfaces with biocompatible groups and moieties, such as sugars or as PEG, could also improve nanocompound biocompatibility. Therefore, a thorough understanding of the relationships between the physicochemical properties and the behavior of nanomaterials in biological systems is highly necessary for designing safe and efficacious nano-based microbicides. Novel techniques such as quantitative structure–


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activity relationship (QSAR) methods can help to establish such relationships (Burello and Worth, 2011). Another important point to consider when developing a vaginal microbicide is that the compound should not alter the normal vaginal flora or have spermicidal activity. Normal vaginal flora is composed of different Lactobacillus species, mainly Lactobacillus crispatus and Lactobacillus iners but also Lactobacillus jensenii and Lactobacillus , as well as other minority populations such as Atopobium vaginae, Megasphaera or Leptotrichia species (Martin, 2012). Finally, it is important that the compounds remain stable and maintain their antiviral activity in an acidic medium, such as that present in the human female vagina (pH 4–5.8). Factors, such as seminal plasma or pH changes, that occur in the vagina during the sexual act can diminish the effectiveness of microbicides. An acidic pH is necessary for natural protection of the vagina against pathogens. Therefore, it is important to test the effectiveness of compounds in an acidic medium and in the presence of seminal plasma to probe their stability, safety and efficacy. 4.1.4. HIV-transmission inhibition assays Sexual transmission is one of the main routes of HIV transmission, mediated by exposure to infected cells or seminal fluid secretions in the mucosa. Currently, whether primary HIV infection is due to transmission of cell-free virus or cell-associated viruses is not well understood. The vaginal or rectal epitheliums are the first sites of deposition for cell-free or cell-associated viruses contained in the semen during sexual intercourse. The use of transwell permeable supports has been proposed as a model of these physiological conditions that can be used to study the potential of microbicide candidates to block the transmission of cell free or cell-associated HIV particles through the genital epithelium (Chonco et al., 2012; Gali et al., 2010a). This dual-chamber system consists of a tight epithelial cell layer or explants from vaginal, uterine cervical, endometrial or rectal mucosa grown on a transwell insert, and a basal chamber with PBMC. This model can be used to monitor whether treatment of the epithelial cells with the microbicide candidate prevents transmission of cell-free or cell-associated virus through the epithelial cell monolayer, simulating natural viral transmission in the vaginal/rectal epithelium. The infection of indicator cells, such as PBMC, in the lower chamber of the transwell is used to evaluate HIV transepithelial transmission. Moreover, transepithelial electrical resistance (TEER, Xxcm2) across the monolayer of epithelial cells can be monitored using a voltmeter and an electrode to investigate whether alterations of tight junction dynamics in the cell cultures are induced by treatment with the compounds. Several studies have shown the importance of using human vaginal or rectal explants to test the antiviral characteristics of the compounds in a physiologically relevant manner (Gali et al., 2010b; McElrath et al., 2010; Rohan et al., 2010). Interestingly, some nanoparticles have been already tested using these models. Polyanionic carbosilane dendrimers G3-S16 and G2-NF16 were able to prevent the transmission of cell-associated R5-HIV particles through transepithelial monolayers in transwell assays and they could block HIV internalization inside epithelial cells showing ability to protect the integrity of the monolayers (Vacas Cordoba et al., 2013). 4.1.5. Investigation of nanoparticle inhibitory mechanisms Following determination of the potency and biosafety of microbicide candidates, the next relevant step is to investigate the mechanisms by which these compounds inhibit infection. Ideal agents are those that block HIV-1 infection at early stages of the HIV-1 life cycle prior to viral integration, primarily by impeding HIV attachment, entry, fusion or reverse transcription. To date, the majority of nanotech-based microbicides act as HIV entry inhibitors. Due to their mechanism, they prevent the integration of the viral gen-

ome into the DNA of target cells, avoiding the primary infection and the establishment of a viral latent reservoir. Nevertheless, other nanocompounds with a late mode of action such as protease inhibitors (PI) could be also very efficacious blocking HIV sexual infection. PI are successfully used in HAART and present some advantages for their development as microbicide candidates. They exhibit a low adsorption in epithelium that may be advantageous in ensuring high local drug concentrations with minimal systemic exposure. Moreover, PI are the agents with the highest genetic barrier to resistance among ARV and due to their delayed antiviral activity, inhibiting maturation but not viral integration, an increased window of protection could be offered by these microbicide candidates (Herrera and Shattock, 2012), even exhibiting efficacy after post-coital application. Molecular modeling is an easy and potent tool to evaluate the targets on which the compounds exert their effects (Xue et al., 2014). For instance, molecular modeling was useful to establish the interaction between carbosilane dendrimer 2G-S16 and Gp120 and CD4 proteins (Chonco et al., 2012). Atomistic molecular dynamics simulations of complexes 2G-S16/CD4 and 2G-S16/Gp120 showed that dendrimer bound to cationic areas in those proteins such as Gp120-V3 loop region, ultimately blocking Gp120–CD4 interaction and thus the attachment step. Furthermore, molecular modeling can also be used to tailor and improve candidates in the search for more effective compounds. Other mechanistic studies, including cell-based and biochemical and enzymatic assays, can define the mode of action of the tested nanocompound. These include time-of-addition assays, in which a variety of antiretrovirals targeting different stages of the viral life cycle are compared to the activity of the tested compound at different time points, or gp120 capture ELISAs, which study the inhibitory activity of compounds on gp120–CD4 binding (Lara et al., 2010a).

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4.1.6. Combination assays The combination of at least two families of ARV is more effective than the use of monotherapy for HIV treatment. Therefore, the combination of different compounds should be taken into consideration when designing new microbicide strategies (Anton, 2012; Balzarini and Schols, 2012). However, combination therapy has various significant disadvantages such as multiple drug–drug interactions, the potential development of multidrug resistances or the additive toxicity from the combination therapy leading to poor adherence. Nanotechnology can help to overcome these limitations. Nanomaterials have emerged as promising tools to increase the delivery of antiretroviral drugs or improve their pharmacokinetic and pharmacodynamic properties, certainly generating positive interactions. Polyanionic carbosilane dendrimers combined with different ARV, such as maraviroc or TFV, show synergistic activity against several laboratory and primary HIV isolates (Cordoba et al., 2013a; Sepulveda-Crespo et al., 2014; VacasCordoba et al., 2014). Efavirenz and saquinavir were loaded into Q3 PLGA nanoparticles in another combinatorial microbicide candidate study. The drug released from the nanoparticles was effective at inhibiting R5-tropic HIV in the human TZM-bl cell line (Chaowanachan et al., 2013). These combinatorial strategies have the potential to block HIV-1 infection at different stages of the HIV-1 life cycle, and may explain the positive interaction outcomes that are commonly observed. Moreover, targeting multiple stages within the HIV-1 replication cycle may decrease the risk of transmission of resistant viral isolates as well as reduce the development of resistance. Decreasing the amounts of antiviral compounds used in vivo might result in the reduction of adverse effects, limiting local toxicity and inflammation caused by topical application of the microbicide before sexual intercourse. Several software packages are available to analyze and statistically evalu-

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ate the outcome of combination assays (Buckheit and Lunsford, 2009; Chou and Talalay, 1984). 4.1.7. Broad spectrum activity of topical microbicides A desirable consideration in the development of new microbicides is not only effectiveness against diverse primary isolates, including clinical, drug resistant and multidrug resistant HIV viral strains, but also against other sexually transmitted pathogens, such as herpes simplex virus, hepatitis viruses, human papillomavirus and causative agents of bacterial and fungal vaginosis including Candida albicans, Neisseria gonorrhoeae or Gardnerella vaginalis. Therefore, it is crucial to evaluate the range of action of topical microbicide agents. It is especially important to develop effective microbicides against herpes simplex type 2 (HSV-2), the virus that causes genital herpes, HSV-2 infection is the most prevalent STD worldwide, with more than 500 million people infected and approximately 20 million new infections occurring each year. Moreover, genital herpes is an important co-factor for HIV-1 transmission; individuals infected with HSV-2 have a threefold higher risk of acquiring HIV-1 infection (Thurman and Doncel, 2012). Several mechanisms may account for this HSV-2-mediated facilitation of HIV-1 infection. HSV-2 infection causes ulceration of the genital mucosa, increasing the risk of HIV-1 acquisition. Furthermore, latent HSV2 infection increases the levels of CD4 and CCR5 expression on lymphocytes and macrophages and the levels of DC-SIGN on dendritic cells in the genital mucosa 5 6, increasing the probability of HIV infection (Rebbapragada et al., 2007; Sartori et al., 2011). As opposed to HIV, which primarily infects cells of the immune system, HSV-2 infects epithelial cells, neurons and other cell types. Therefore, distinct attachment and entry pathways are involved in HSV-2 entry into target cells. This attachment includes interactions between several glycoproteins in the viral envelope (gB, gD, gH, and gL) and a variety of cell surface molecules, including heparan sulfate moieties (Spear and Longnecker, 2003). Compounds capable of inhibiting this initial association and binding, as well as subsequent membrane fusion, could be potential microbicide candidates. The prediction algorithm and follow-up steps used to evaluate the antiviral activity of nanostructures against HSV-2 are the same as those used for HIV infection. However, several distinct in vitro and in vivo models have been successfully developed to evaluate inhibitory potency, such as the use of epithelial Vero cells for infection or plaque reduction assays for viral titration (Blaho et al., 2005). Some nanotech-derived architectures, such as membrane interacting peptide-functionalized dendrimers or zinc acetate gel, have been tested as potential anti-HSV-2 microbicides (Kenney et al., 2013; Luganini et al., 2011). Several nanoparticles have shown a broad spectrum of activity. For instance, phosphatidylcholine liposomes containing the microbicide octylglycerol were effective in vitro against HSV-1, HSV-2 and HIV-1 (Wang et al., 2012). SPL7013 dendrimer-based VivaGel demonstrated antiviral activity against HIV and HSV in animal models and antibacterial activity preventing bacterial vaginosis (Price et al., 2011), and silver nanoparticle-coated condoms were also able to inhibit HIV and HSV infection and demonstrated antibacterial and antifungal activities against several microorganism such as Escherichia coli, Staphylococcus aureus, Micrococcus luteus, Klebsiella pneumoniae, Candida tropicalis, Candida krusei, Candida glabrata, and Candida albicans (Mohammed Fayaz et al., 2012). 4.2. Ex vivo and in vivo evaluation: a proof-of-concept of compound efficacy prior to human clinical trials Because HIV replication is restricted to humans, and the level of biological complexity of vaginal and rectal transmission of HIV

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cannot be mimicked in cell or tissue culture models, the research community has been searching for alternative in vivo study strategies for a long time. Sensitive animal models, that express the specific receptor and coreceptors that HIV-1 uses for attachment and entry in target cells (CD4, CCR5, or CXCR4), such as non-human primates and humanized mice (Brehm et al., 2010; Denton and Garcia, 2011; Veazey et al., 2012) play an important role in preclinical testing of microbicides, specially on safety, pharmacokinetic and efficacy testing. However, these animal models are not yet validated for US FDA (U.S. Food and Drug Administration) for predicting efficacy in humans (Administration, 2012). To date, the in vivo test based on the rabbit remains the only model recommended by the US FDA for the safety evaluation of vaginal products. The most widely used non-human primates models are macaques, including the rhesus (Macaca mulatta), pigtail (Macaca nemestrina), and cynomolgus (Macaca fascicularis) sub-species. These species have anatomical, immunological, and reproductive physiological properties broadly comparable to those of humans and they can be efficiently infected by different HIV strains. In case of rodents, their cells are non-permissive for the replication of HIV-1. Although rats, mice and rabbits, are certainly useful for preclinical safety studies of microbicide candidates, their reproductive cycles and tissues are quite different from those of humans or primates. Nevertheless, several humanized mouse models have been engineered recently to study HIV infection in vivo: (i) mice with single or multiple human transgenes; (ii) immunodeficient mice transplanted with human cells or tissues (healthy or diseased); (iii) immunodeficient mice transplanted with human CD34+ cells; or (iv) a combination of these three types. Currently, the most common model is the recently developed NOD/ SCIDcc / , Rag2 / cc / and blood, liver and thymus human (h-BLT) mouse model, which is reconstituted with human CD34 cells (NSG/BLT). In this model, immunodeficient mice are transplanted with human bone marrow (containing T cell progenitors), liver (for fetal immune development), and thymus (for T cell maturation) cells. Following engraftment, the mice are allowed to recover for 12 weeks prior to assessment for engraftment efficiency. These mice can completely reconstitute and self-renew all major human hematopoietic lineages (T and B cells, monocyte/ macrophage, dendritic and NK cells), essentially reproducing a functional human immune system that responds to X4- and R5tropic HIV-1 infection. The female reproductive tract of humanized BLT mice is reconstituted with functional human immune cells, rendering them susceptible to vaginal HIV-1 infection and allowing for testing of microbicide candidates. Recently, several laboratories have used this mouse model to evaluate the effectiveness of novel HIV microbicide candidates (Chateau et al., 2013; Denton et al., 2008, 2010, 2011; Di Fabio et al., 2003; Neff et al., 2011; Veselinovic et al., 2012; Wheeler et al., 2011). To test the ability of microbicides to prevent HIV infection, these studies focused on the evaluation of the presence of HIV particles in treated mice through different molecular techniques. Viral infection and viral loads are commonly assessed by Q-RT-PCR and western blot and flow cytometry is used to measure the levels of CD4 T cells in the peripheral blood of monitored mice. Although humanized mice are certainly useful, they present some obstacles, like the high cost and complexity of the surgical techniques required to generate them. Non-human primate models, due to their similarities to humans, remain the best option for safety and efficacy testing of new microbicide candidates. These in vivo models, together with ex vivo explant cultures, are also highly useful in evaluating the biosafety profiles of new nanobased microbicides in the final steps of their preclinical development, prior to toxicological analysis in humans. To study the impact of the compounds in the mucosal tissues, compounds must


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be tested vaginally and/or rectally in order to evaluate potential toxic effects though direct observation of lesions in the vaginal or rectal area and the quantification of pro-inflammatory cytokines. Histopathological analysis of vaginal tissues can also be performed using semi-quantitative clinical scores to determine a vaginal irritation index (Eckstein et al., 1969). Epithelial lesions are evaluated based on the existence of hyperplasia or hyperkeratosis and ulceration, and/or inflammatory infiltrate in the epithelium. The inflammation process is characterized by an increased infiltration of lymphocytes compared to healthy mucosal tissues. Therefore, analysis of the lymphocyte score could help determine microbicide efficacy. Pro-inflammatory chemokines and cytokines plays an important role in HIV infection, owing to the recruitment and activation of CD4+ T cells to the mucosa (Fichorova et al., 2004). As mentioned previously, the concentration of inflammatory chemokines and cytokines in culture supernatants, serum samples or vaginal secretions is important in assessing the biosafety of microbicides. Furthermore, immunohistochemical studies on vaginal explants could be performed to study the levels of several biomarkers of cervicovaginal inflammation, such as IL-1, IL-6, TNFa, IL-8, macrophage inflammatory protein (MIP) or RANTES. Some nanoplatforms have been tested as topical microbicides in animal models. RNAi-mediated CCR5 silencing by LFA-1-targeted nanoparticles prevented HIV infection in BLT mice (Kim et al., 2010b). SPL7013 gel was protective against vaginal challenge with X4- and R5-tropic simian humanized immunodeficiency virus (SHIV) in a macaque model (Jiang et al., 2005). Moreover, activity against HSV-2 infection was demonstrated in mice and guinea pigs (Bernstein et al., 2003).

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5. Future perspectives, obstacles and conclusions

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Nanomedicine has provided new tools and rationales in the development and search for new and more powerful PrEP strategies, particularly in the design and engineering of more potent topical microbicides. The rational design and manipulation of diverse nanosystems, including dendrimers, liposomes and polymeric nanoparticles, has provided new microbicide candidates against HIV and other STDs with improved solubility, stability and pharmacokinetic and adherence properties. Furthermore, the combination of these nanoplatforms with other inhibitory drugs targeting different stages in the virus life cycle is essential for the development of more powerful and effective microbicides and for the improvement of existing ones. Since one of the most important problems in microbicide development is the lack of adherence in patients, a critical point to consider is the optimization of the nano-based microbicide formulation (Wong et al., 2014). Topical gel, intravaginal rings, films, tablets, ovules and other novel formulations such as female condoms coated with these nanostructures could be used. In each case, the formulation should be designed to maximize the activity, pharmacokinetics, safety, acceptability and adherence to the regimen of the different candidates. However, other limitations such as the adherence measurement or the comprehension of microbicide instructions for use in participating patients should be also improved to overcome adherence problems as reviewed in depth by Woodsong et al. (2013). In addition, novel routes of administration must be explored in order to discover novel nanotech-based prophylactic strategies against HIV. Although there are benefits and advantages of using nanotechnology in the development of novel microbicides to stop the spread of HIV worldwide, there are features and challenges that must be overcome in the future for successful translation of nano-based microbicides from in vitro and in vivo assays to clinical settings. These include the biocompatibility, safety, stability and cost of nanocompounds, as well as their scale-up for large-scale

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production. More efficient and ecologically sustainable production methods or searching for active natural nanostructures like vegetable polymers, that can be large-scale produced in biofactories, could be essential points to reduce the production cost of nano-based microbicides. Plant systems offer unique advantages in comparison with conventional techniques (bacteria or yeast) or chemical synthesis such as lower production costs or an easy scale-up and purification process. These economic aspects are especially important because the main target of these products are the most vulnerable populations that live mainly in poor and developing countries, particularly in Africa. Because the majority of microbicides have failed in the last phases of human clinical trials, it is critical to begin the evaluation of topical microbicide candidates in conditions as similar as possible to physiological conditions that occur during sexual intercourse at the moment of HIV transmission. Pivotal points are the study of the impact of microbicides on vaginal and rectal fluid, on mucosal immune-cross-talk and the consequences of physical trauma during sex on the use of the microbicides. Currently, several nanosystems are under investigation as potential microbicides, with several on the threshold of clinical trials in humans. The encouraging results obtained in the CAPRISA 004 clinical trial and with VivaGel are proof-of-concepts that topical microbicides, and specifically nanotechnology-derived systems, can be effective approaches in the context of blocking HIV1 sexual transmission. These results support further clinical trials and in vivo research studies to develop a powerful strategy in the fight against the spread of the HIV/AIDS epidemic worldwide.

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Acknowledgments

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The authors do not have a commercial or other association that might pose a conflict of interest. This work was supported by grants provided through Fondo de Investigación Sanitaria (FIS) [grant number PI13/02016], Red Q4 Española de Investigación en SIDA (RIS) [grant numbers RD120017-0037, PT13/0010/0028 and RD12/0017/0029], CYTED 214RT0482, ‘‘Fundación para la Investigación y la Prevención del Sida en España’’ (FIPSE), Comunidad de Madrid [grant numbers, S-2010/BMD-2351 and S-2010/BMD-2332], ME&C (Ref. CTQ201123245). CIBER-BBN is an initiative funded by the VI National Q5 R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.

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11 November 2014 14 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181


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Topical microbicides and HIV prevention in the female genital tract.

The development of rectal microbicides for HIV prevention.

Non-Antiretroviral Microbicides for HIV Prevention.

Beyond HIV microbicides: multipurpose prevention technology products.

Precise engineering of dapivirine-loaded nanoparticles for the development of anti-HIV vaginal microbicides.

Development of a protocol for predicting bacterial resistance to microbicides.

ONC201: a bend in the road to clinical development.

Microbicides for the Treatment of Sexually Transmitted HIV Infections.

Application of Design of Experiment and Simulation Methods to Liquid Chromatography Analysis of Topical HIV Microbicides Stampidine and HI443.

ADA clinical recommendations on topical fluoride for caries prevention.

HIV PrEP Trials: The Road to Success.

Historical development of vaginal microbicides to prevent sexual transmission of HIV in women: from past failures to future hopes.

Silencing sexually transmitted infections: topical siRNA-based interventions for the prevention of HIV and HSV.

Development of HIV-1 rectal-specific microbicides and colonic tissue evaluation.

AIDS.

HIV treatment-as-prevention research: taking the right road at the crossroads.

Heart disease prevention in children: the road to 2020.

2014 Federal Recommendations for Human Immunodeficiency Virus (HIV) Prevention With Persons With HIV: A New Road Map for High Impact Prevention.

Combining biomedical preventions for HIV: Vaccines with pre-exposure prophylaxis, microbicides or other HIV preventions.

Dyslipidaemia: cardiovascular prevention--end of the road for niacin?

Topical fluoride for caries prevention: executive summary of the updated clinical recommendations and supporting systematic review.

Developments in the prevention of road injuries.

Baby steps on the road to HIV eradication.

Prevention of portal hypertension: from variceal development to clinical decompensation.