Punica granatum(Pomegranate) juice provides an HIV-1 entry inhibitor and candidate topical microbicide
© Neurath et al; licensee BioMed Central Ltd. 2004
Received: 08 July 2004
Accepted: 14 October 2004
Published: 14 October 2004
For ≈ 24 years the AIDS pandemic has claimed ≈ 30 million lives, causing ≈ 14,000 new HIV-1 infections daily worldwide in 2003. About 80% of infections occur by heterosexual transmission. In the absence of vaccines, topical microbicides, expected to block virus transmission, offer hope for controlling the pandemic. Antiretroviral chemotherapeutics have decreased AIDS mortality in industrialized countries, but only minimally in developing countries. To prevent an analogous dichotomy, microbicides should be: acceptable; accessible; affordable; and accelerative in transition from development to marketing. Already marketed pharmaceutical excipients or foods, with established safety records and adequate anti-HIV-1 activity, may provide this option.
Fruit juices were screened for inhibitory activity against HIV-1 IIIB using CD4 and CXCR4 as cell receptors. The best juice was tested for inhibition of: (1) infection by HIV-1 BaL, utilizing CCR5 as the cellular coreceptor; and (2) binding of gp120 IIIB and gp120 BaL, respectively, to CXCR4 and CCR5. To remove most colored juice components, the adsorption of the effective ingredient(s) to dispersible excipients and other foods was investigated. A selected complex was assayed for inhibition of infection by primary HIV-1 isolates.
HIV-1 entry inhibitors from pomegranate juice adsorb onto corn starch. The resulting complex blocks virus binding to CD4 and CXCR4/CCR5 and inhibits infection by primary virus clades A to G and group O.
These results suggest the possibility of producing an anti-HIV-1 microbicide from inexpensive, widely available sources, whose safety has been established throughout centuries, provided that its quality is adequately standardized and monitored.
The global AIDS epidemic has proceeded relentlessly for ≈ 24 years with no promising prophylactic intervention in sight. In 2003 there were 5 million new HIV infections, and 3 million AIDS deaths . To date the number of individuals living with HIV-1 infection/AIDS has reached 40 million, and ≈ 30 million people have already died from AIDS since the beginning of the pandemic [1, 2]. Most new infections have been acquired by the mucosal route, heterosexual transmission playing the major (≈ 80%) role. Although the incidence of transmission per unprotected coital act is estimated to be low (0.0001 – 0.004), but strikingly increased when acutely infected individuals are involved [3, 4], the cumulative effect is overwhelming.
Anti-HIV-1 vaccines applicable to global immunization programs are not expected to become available for many years. Thus, other prevention strategies are urgently needed. This includes educational efforts and application of mechanical and/or chemical barrier methods. The latter correspond to microbicides, i.e. topical formulations designed to block HIV-1 infection (and possibly transmission of other sexually transmitted diseases) when applied vaginally (and possibly rectally) before intercourse [3, 5–7]. Conceptually, it is preferred that the active ingredient(s) of microbicide formulations (1) block virus entry into susceptible cells by preventing HIV-1 binding to the cellular receptor CD4, the coreceptors CXCR4/CCR5 and to receptors on dendritic/migratory cells (capturing and transmitting virus to cells which are directly involved in virus replication), respectively [3, 8–11], and/or (2) are virucidal. The formulations must not adversely affect the target tissues, and should not cause them to become more susceptible to infection after microbicide removal [12, 13].
Treatment with anti-retroviral drugs has decreased mortality from AIDS in industrialized countries but has had a minimal effect so far in developing countries . To avoid a similar dichotomy with respect to microbicides, they should be designed and selected to become affordable and widely accessible, while shortening the time between research and development and their marketing and distribution as much as possible. This would be facilitated if mass manufactured products with established safety records were to be found to have anti-HIV-1 activity. Qualifying candidates to be considered for microbicide development may possibly be discovered by screening pharmaceutical excipients (= "inactive" ingredients of pharmaceutical dosage forms) and foods, respectively, for anti-viral properties. This approach has already led to the discovery of cellulose acetate 1,2-benzenedicarboxylate (used for coating of enteric tablets and capsules) as a promising candidate microbicide [15–19]. Here we report the outcome of screening fruit juices neutralized to pH ≈ 7 to discount nonspecific effects caused by acidity.
Pomegranate juices (PJ) were purchased in local New York City stores; their origin is given in parentheses: PJ1 (Madeira Enterprises Inc., Madeira, CA); PJ2 was prepared from fresh ripe pomegranates in our laboratory; PJ3 (Sadaf®; Sadaf® Foods, Los Angeles, CA; additional ingredients: fructose, citric acid); PJ4 (Cortas Canning & Refrigeration Co. S.A.L., Beirut, Lebanon); PJ5 (Kradjian, Import & Wholesale Distribution, Glendale, CA. Product of Iran); PJ6 (R.W. Knudsen ; Just Pomegranate; Knudsen & Sons, Inc., Chico, CA); PJ7 (Aromaproduct Ltd., Product of Georgia; distributed by Tamani, Inc., New York, NY). Starches used were: PURE-DENT® B815 Corn Starch NF, PURE-DENT® B816 Corn Starch USP, Spress® B825 Pregelatinized corn starch NF, Spress® B820 Pregelatinized corn starch NF, INSTANT PURE-COTE™ B792 Food starch-modified, INSCOSITY™ B656 Food starch-modified (Grain Processing Corporation, Muscatine, IN); PURITY® 21 corn starch NF and PURITY® 826 corn starch NF (National Starch and Chemical Company, Bridgewater, NJ); Remyline AX-DR Waxy rice starch and Remy DR native rice starch, medium grind (A&B Ingredients, Fairfield, NJ); ARGO® corn starch (Best Foods Division, CPC International Inc., Engelwood Cliffs, NJ); STALEY® pure food powder starch (Tate & Lyle, Decatur, IL); STARCH 1500 pregelatinized starch NF (Colorcon, West Point, PA). The following polymers were used: polyethylene glycols (PEG) 1000 NF, 1500 NF and 8000 NF; and hydroxypropyl methylcellulose, 50 cps, USP (Spectrum, New Brunswick, NJ); Carbopol 974P-NF (B. F. Goodrich Co., Cleveland, OH); Carbophil, Noveon AA1 (Noveon, Inc., Cleveland OH); and Pharmaburst B2 (SPI Pharma, New Castle, DE). Fattibase was from Paddock Laboratories, Inc., Minneapolis, MN.
Recombinant proteins employed were: HIV-1 IIIB gp120, biotinyl-HIV-1 IIIB gp120, CD4, and biotinyl-CD4 (ImmunoDiagnostics, Inc., Woburn, MA); HIV-1 IIIB BaL gp120 and FLSC (a full length single chain protein consisting of BaL gp120 linked with the D1D2 domains of CD4 by a 20 amino acid linker) (produced in transfected 293T cells ). Phycoerythrin (PE)-labeled streptavidin was from R & D Systems, Minneapolis, MN. Biotinylated Galanthus nivalis lectin was from EY Laboratories, Inc. San Mateo, CA. Rabbit antibodies to synthetic peptides from gp120 (residue numbering as in reference ) were prepared as described . Monoclonal antibodies (mAb) 588D, specific for the CD4 binding site, and 9284, specific for the gp120 V3 loop, were from Dr. S. Zolla-Pazner and NEN Research Products, Du Pont, Boston, MA, respectively. A "generic" version of the nonnucleoside HIV-1 reverse transcriptase inhibitor TMC-120  was synthesized by Albany Molecular Research, Inc., Albany, NY, and used in control experiments at a final 5 μM concentration. Pelletted, 1000-fold concentrates of HIV-1 IIIB (6.8 × 1010 virus particles/ml) and BaL (2.47 × 1010 virus particles/ml) were from Advanced Biotechnologies, Inc., Columbia, MD. Primary HIV-1 isolates, MT-2 cells, HeLa-CD4-LTR-β-gal and U373-MAGI-CCR5E cells (both contributed by Dr. Michael Emerman) and Cf2Th/synCCR5 cells (contributed by Dr. Tajib Mirzabekov and Dr. Joseph Sodroski) were obtained from the AIDS Research and Reference Reagent Program operated by McKesson BioServices Corporation, Rockville, MD. CEMx174 5.25M7 cells, transduced with an HIV-1 long terminal repeat (LTR)-green fluorescent protein and luciferase reporter construct, expressing CD4 and CXCR4 and CCR5 coreceptors , were obtained from Dr. Cecilia Cheng-Mayer. The cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1 μg/ml puromycin and 200 μg/ml G418. These cells are suitable for titration of both X4 and R5 HIV-1 isolates and for determining the effectiveness of anti-HIV-1 drugs with reliable reproducibility. This is impossible to accomplish by using peripheral blood mononuclear cells (PBMCs) because of their variations in susceptibility to HIV-1 infection among cells derived from distinct individuals [24–26]. PBMCs were isolated from HIV-1 negative donors as described .
In attempts to separate gp120-CD4 binding inhibitory activity from most other ingredients of PJ, 200 mg of distinct starch preparations were added per ml of PJ1. After mixing for 1 h at 20°C, excess juice was decanted, and the pellets resuspended in 1 ml of distilled water. Based on results of enzyme linked immunosorbent assays (ELISA), PURITY® 21 corn starch, NF grade (S21) was selected for further studies, and the corresponding PJ complex was designated as PJ-S21. PJ-S21 was freeze dried and used to prepare the following formulations: PEG suppositories (50% PJ-S21, 45% PEG 1000, 5% PEG 1500); Fattibase suppositories (50% PJ-S21, 50% Fattibase); and mucoadhesive instantly dispersible tablets (50% PJ-S21, 20% HPMC, 20% Pharmaburst, 7.5% Carbopol 974P and 2.5% Carbophil).
Enzyme linked immunosorbent assays (ELISA)
Inhibition of infection by HIV-1 IIIB and BaL, respectively, was determined relying on a β-galactosidase readout system . The enzyme was quantitated with a Galacto-Light Plus System chemiluminescence reporter assay (Applied Biosystems, Foster City, CA) using a Microlight ML 2250 luminometer (Dynatech Laboratories, Inc., Chantilly, VA). To measure virucidal activity, virus was separated from excess inactivating agent by centrifugation and/or precipitation with PEG 8000 [18, 19]. Serial dilutions of the treated virus were assayed for infectivity as described above. Dose response curves (i.e. luminescence vs. dilution) for treated and control viruses were obtained, and the percentages of virus inactivation were calculated . To determine inhibition of infection by primary HIV-1 strains, CEMx174 5.25 M7 cells were incubated with 100 × TCID50 of primary HIV-1 strains in the absence or presence of PJ-S21 at graded concentrations for 3 days at 37°C. The experiments were done in triplicate. Infection was quantitated by measuring luciferase activity  using a kit from Promega (Madison, WI) in an Ultra 384 luminometer (Tecan, Research Triangle Park, NC).
CD4-HIV-1 gp120 binding and its inhibition were measured by ELISA. Wells of 96-well polystyrene plates (Immulon II, Dynatech Laboratories, Inc., Chantilly, VA) were coated with 100 ng/well of either gp120 IIIB or gp120 BaL, and post-coated as described . Dilutions of PJs and of PJ-S21, respectively, in 0.14 M NaCl, 0.01 M Tris, 0.02% sodium merthiolate, pH 7.0 (TS) containing 100 μg/ml bovine serum albumin (BSA) were added to the wells for 1 h at 37°C. The wells were washed 5 × with TS. Biotinyl-CD4 (1 μg/ml) in TS-1% gelatin was added to the wells for 5 h at 37°C. After washing 1 × with TS-0.1% Tween 20 and 5 × with TS, horseradish peroxidase (HRP)-streptavidin (0.625 μg/ml; Amersham, Arlington Heights, IL) in TS-2% gelatin-0.05% Tween 20 was added. After 30 min at 37°C, the wells were washed 4 × with TS-0.1% Tween 20 and 2 × with TS. Bound HRP was detected using a kit from Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD) and the absorbance (A) read at 450 nm. A in the absence of inhibitors was 1.0 to 1.5, and 0 to 0.005 in the absence of biotinyl-CD4. In an alternative assay, CD4 (500 ng/ml) was mixed with biotinyl-gp120 (1 μg/ml) in the presence or absence of inhibitors for 30 min at 20°C. Serial dilutions of the mixtures were added to wells coated with the anti-CD4 mAb OKT 4 (Ortho-Clinical Diagnostics, Rochester, NY) and captured biotinyl-gp120 was detected with HRP-streptavidin as described above. To measure binding to gp120 of antibodies to gp120 peptides, the respective rabbit antisera were diluted 50-fold in a mixture of FBS and goat serum (9:1) containing 0.1% Tween 20 (pH 8.0) and added to gp120 wells. Bound IgG was detected with HRP labeled anti-rabbit IgG (Sigma, St. Louis, MO; 1 μg/ml in TS-10% goat serum-0.1% Tween 20). A cell-based ELISA was used to measure the blocking of CCR5 binding sites on HIV-1 BaL gp120 by PJ and PJ-S21, respectively . Briefly, FLSC (125 ng/ml) in the absence or presence of graded amounts of inhibitors was added to Cf2Th/synCCR5 cells fixed with 5% formaldehyde in wells of 96-well plates. After 1 h at 37°C, bound FLSC was detected with mAb M-T441 (125 ng/ml; Ancell, Bayport, MN) specific for the CD4 D2 domain, followed sequentially by biotinylated anti-mouse IgG and HRP-streptavidin.
Anti-HIV-1 activity of pomegranate juice
Separation of anti-HIV-1 inhibitor(s) from pomegranate juice
To be considered as a topical microbicide, PJ-S21 must be formulated to withstand storage in a tropical environment. Accelerated thermal stability studies revealed that a water suspension of PJ-S21 maintained only 4, 11, and 33%, respectively, of its original activity (measured by inhibition of gp120-CD4 binding) when stored for 30 min at 60°C, and one week at 50°C or 40°C. On the other hand, a dried PJ-S21 powder remained fully active after storage at 50°C for 12 weeks (the longest time used in the evaluation). Consequently, anhydrous formulations should be preferred for further development.
Inhibitory activity of PJ-S21 on infection by primary HIV-1 strains
Subtype, Coreceptor use
0.50 ± 0.05
2.76 ± 0.28
1.42 ± 0.54
3.42 ± 0.98
0.62 ± 0.11
2.86 ± 0.33
3.56 ± 1.10
8.87 ± 2.55
1.02 ± 0.19
3.54 ± 0.90
0.62 ± 0.17
2.94 ± 0.85
0.86 ± 0.01
4.09 ± 0.08
4.25 ± 0.78
8.31 ± 1.04
0.42 ± 0.09
1.54 ± 0.16
Group O, R5
0.59 ± 0.29
3.92 ± 0.27
PJ contains several ingredients [42, 43] which, isolated from natural products other than PJ, were reported to have anti-HIV activity, for example: caffeic acid , ursolic acid , catechin and quercetin [46, 47]. However, these compounds, in purified form, obtained commercially, did not block (at 200 μg/ml) gp120-CD4 binding as measured by the ELISA described above and did not adsorb to corn starch, unlike the entry inhibitor(s) from PJ. In fact, the supernatant after treatment of PJ with starch, and removal of the entry inhibitors, retained anti-HIV-1 activity and also inhibited infection by herpes virus type 1, unlike the HIV-1 entry inhibitors which adsorbed onto starch. Thus, the antiviral activities in the supernatant appeared to be non-specific, and probably similar to those of extracts from pomegranate rind [48, 49], and were not characterized further. Additional information [50–53] has revealed that the findings apply to crude extracts from pomegranate rind prepared at elevated temperatures under conditions which destroy the HIV-1 entry inhibitor described here.
The inhibitor(s) interfering with gp120 binding to CD4 (Fig. 2 and 5) blocked additional sites on gp120 (Fig. 3) involved in interaction with the CXCR4/CCR5 coreceptors (Fig. 4, 6 and 7). This was not completely expected and can be explained either by the presence of multiple inhibitors with distinct or overlapping specificities in PJ-S21 or by induction of gp120 conformational changes  resulting in blockade of both CD4 and CXCR4/CCR5 binding sites on gp120. Similar effects have been noticed for other small molecule inhibitors . Simultaneous blocking of more than a single site on HIV-1 involved in virus entry is expected to increase the effectiveness of candidate microbicides . The target sites for the inhibitor(s) are likely to be located within the protein moiety of gp120 since binding of labeled Galanthus nivalis lectin (specific for terminal mannose residues ; and other lectins to gp120 oligosaccharides was not diminished in the presence of PJ or PJ-S21 (data not shown).
Blocking of CD4 binding sites on HIV-1 gp120 by monoclonal antibodies or a CD4-IgG2 recombinant protein has been shown to be sufficient to inhibit HIV-1 infection of human cervical tissue ex vivo  and in preventing virus transmission to macaque monkeys when applied vaginally . Therefore, it seems likely that PJ-S21 will be similarly effective, an expectation which remains to be confirmed.
The application of PJ-S21 as a topical anti-HIV-1 microbicide requires reasonable uniformity among batches produced at distinct times and locations. Similarities in gp120-CD4 binding inhibitory activity among distinct freshly prepared and commercial juices stored for unknown periods (Fig. 2) suggest that this should be feasible. Pasteurization of juice for 30 seconds at 85°C resulted in complete loss of inhibitory activity. A commercial PJ concentrate exposed to 61°C, and two other concentrates, presumably prepared by evaporation at elevated temperatures, had no or drastically diminished activity. The gp120-CD4 inhibitory activity from PJ3 (juice with fructose and citric acid added), failed to bind to starch. Separate experiments revealed that these compounds interfere with inhibitor binding to corn starch. Therefore, PJs intended for production of the PJ-S21 complex must be sterilized by filtration and be free of additives.
Particular attention must be devoted to the selection of starch, a pharmaceutical excipient generally used in vaginal formulations , for effective binding of the virus entry inhibitors from PJ. Among a dozen starches tested, the best results have been obtained with S21. With other brands, the adsorption of the inhibitors was either incomplete or their binding did not result in a complex having activity in the ELISA measuring gp120-CD4 binding inhibition (ARGO® corn starch), presumably, because of irreversible binding of the PJ inhibitors. Interestingly, there are only a few references available regarding the use of starch as an adsorbent for different compounds: flavors [59, 60], dyes [61–63], low-molecular mass saccharides , lipids [65, 66], proteins  and iodine .
The intended dose of PJ-S21 for vaginal application is 1.0 to 1.5 g, (= 3.17 – 4.76 mg solids from PJ adsorbed onto starch) i.e. ≥ 100-fold higher than the dose needed for blocking HIV-1 infection in vitro (Fig. 10, Table 1), and thus expected to meet requirements for likely in vivo protection against vaginal challenge . This quantity of PJ-S21 is produced from 5 to 7.5 ml of PJ, i.e. ≤ 5% of a single (150 ml) serving of juice, attesting to the safety, feasibility and economy of this proposed candidate topical microbicide.
In an alternative approach to formulation development, PJ-S21 can be incorporated into a water dispersible film (similar to the widely available "breath control" strips) or into water dispersible sponges  which are converted into a gel following topical application . Each of the above formulations would meet the following requirements: (1) minimization of waste disposal problems associated with the use of applicators needed for delivery of microbicidal gels/creams; (2) simplicity; (3) small packaging and discretion related to purchase, portability and storage; (4) low production costs; (5) amenability to industrial mass production at multiple sites globally and (6) potential application as rectal microbicides. Furthermore, it would remain possible to produce for local use PJ-S21 based gel formulations with a limited shelf life, avoiding the costs of producing dry PJ-S21 powders via appropriate low temperature drying processes. Whichever of these formulations is selected, adequate quality control will be needed to assure uniform anti-HIV-1 activity of the final product(s) and to establish reproducible conditions for manufacture.
PJ-S21 can be classified as an AAAA candidate microbicide: Acceptable; Accessible; Affordable; and Accelerative in transition from development to marketing. Thus, PJ-S21 would be expected to circumvent some problems associated with antiretroviral drugs and possibly some of the other candidate microbicides, i.e. uncertainty related to potential side effects, investment and time needed to establish inexpensive large scale production, and monopoly of supply.
acquired immunodeficiency syndrome
bovine serum albumin
- ED50(90) :
effective dose(s) for 50% (90%) inhibition of infection
enzyme linked immunosorbent assays
fetal bovine serum
a full length single chain protein consisting of BaL gp120 linked with the D1D2 domains of CD4 by a 20 amino acid linker
human immunodeficiency virus type 1
long terminal repeat
peripheral blood mononuclear cells
phosphate buffered saline
PURITY®21 corn starch NF grade
We thank Dr. Shibo Jiang and Dr. Hong Lu for carrying out all experiments with primary HIV-1 isolates and for analysis of the resulting data; Dr. Qian Zhao for experiments with the full length single chain protein consisting of HIV-1 BaL gp120 linked with the D1D2 domains of CD4 (FLSC); Veronica L. Kuhlemann for assistance and editorial help in preparing the manuscript and for production of all graphs; and Ruth A. Croson-Lowney for flow cytometry. Financial support for this research was provided by the Marilyn M. Simpson Charitable Trust, the Glickenhaus Foundation, and an NIH grant PO1HD41761.
- UNAIDS: AIDS Epidemic Update (December 2003). 2004, [http://www.unaids.org/html/pub/publications/irc-pub06/jc943-epiupdate2003_en_pdf.htm]Google Scholar
- WHO/SEARO CDS HIV/AIDS: End-2000 global estimates (Children and adults). 2001, [http://w3.whosea.org/hivaids/fact.htm#End-2000%20global%20estimates]
- Shattock RJ, Moore JP: Inhibiting sexual transmission of HIV-1 infection. Nat Rev Microbiol. 2003, 1: 25-34. 10.1038/nrmicro729.View ArticlePubMedGoogle Scholar
- Pilcher CD, Tien H, Eron JJ, Vernazza PL, Leu S-Y, Stewart PW, Goh L-E, Cohen MS: Brief but efficient: Acute HIV infection and the sexual transmission of HIV. J Infect Dis. 2004, 189: 1785-1792. 10.1086/386333.View ArticlePubMedGoogle Scholar
- Stone A: Microbicides: A new approach to preventing HIV and other sexually transmitted infections. Nat Rev Drug Discov. 2002, 1: 977-985. 10.1038/nrd959.View ArticlePubMedGoogle Scholar
- Shattock R, Solomon S: Microbicides - aids to safer sex. Lancet. 2004, 363: 1002-1003. 10.1016/S0140-6736(04)15876-5.View ArticlePubMedGoogle Scholar
- Brown H: Marvellous microbicides. Intravaginal gels could save millions of lives, but first someone has to prove that they work.. Lancet. 2004, 363: 1042-1043. 10.1016/S0140-6736(04)15881-9.View ArticlePubMedGoogle Scholar
- Moore JP, Doms RW: The entry of entry inhibitors: a fusion of science and medicine. Proc Natl Acad Sci U S A. 2003, 100: 10598-10602. 10.1073/pnas.1932511100.View ArticlePubMedPubMed CentralGoogle Scholar
- Pierson TC, Doms RW: HIV-1 entry inhibitors: new targets, novel therapies. Immunol Lett. 2003, 85: 113-118. 10.1016/S0165-2478(02)00235-3.View ArticlePubMedGoogle Scholar
- Davis CW, Doms RW: HIV Transmission: Closing all the Doors. J Exp Med. 2004, 199: 1037-1040. 10.1084/jem.20040426.View ArticlePubMedPubMed CentralGoogle Scholar
- Hu Q, Frank I, Williams V, Santos JJ, Watts P, Griffin GE, Moore JP, Pope M, Shattock RJ: Blockade of attachment and fusion receptors inhibits HIV-1 infection of human cervical tissue. J Exp Med. 2004, 199: 1065-1075. 10.1084/jem.20022212.View ArticlePubMedPubMed CentralGoogle Scholar
- Fichorova RN, Tucker LD, Anderson DJ: The molecular basis of nonoxynol-9-induced vaginal inflammation and its possible relevance to human immunodeficiency virus type 1 transmission. J Infect Dis. 2001, 184: 418-428. 10.1086/322047.View ArticlePubMedGoogle Scholar
- Fichorova RN, Bajpai M, Chandra N, Hsiu JG, Spangler M, Ratnam V, Doncel GF: Interleukin (IL)-1, IL-6 and IL-8 predict mucosal toxicity of vaginal microbicidal contraceptives. Biol Reprod. 2004, 71: 761-769. 10.1095/biolreprod.104.029603.View ArticlePubMedGoogle Scholar
- Weiss R: AIDS: unbeatable 20 years on. Lancet. 2001, 357: 2073-2074. 10.1016/S0140-6736(00)05228-4.View ArticlePubMedGoogle Scholar
- Neurath AR, Strick N, Li Y-Y, Lin K, Jiang S: Design of a "microbicide" for prevention of sexually transmitted diseases using "inactive" pharmaceutical excipients. Biologicals. 1999, 27: 11-21. 10.1006/biol.1998.0169.View ArticlePubMedGoogle Scholar
- Neurath AR, Strick N, Li Y-Y, Debnath AK: Cellulose acetate phthalate, a common pharmaceutical excipient, inactivates HIV-1 and blocks the coreceptor binding site on the virus envelope glycoprotein gp120. BMC Infect Dis. 2001, 1: 17-10.1186/1471-2334-1-17.View ArticlePubMedPubMed CentralGoogle Scholar
- Neurath AR, Strick N, Jiang S, Li YY, Debnath AK: Anti-HIV-1 activity of cellulose acetate phthalate: Synergy with soluble CD4 and induction of "dead-end" gp41 six-helix bundles. BMC Infect Dis. 2002, 2: 6-10.1186/1471-2334-2-6.View ArticlePubMedPubMed CentralGoogle Scholar
- Neurath AR, Strick N, Li Y-Y: Anti-HIV-1 activity of anionic polymers: A comparative study of candidate microbicides. BMC Infect Dis. 2002, 2: 27-10.1186/1471-2334-2-27.View ArticlePubMedPubMed CentralGoogle Scholar
- Neurath AR, Strick N, Li Y-Y: Water dispersible microbicidal cellulose acetate phthalate film. BMC Infect Dis. 2003, 3: 27-10.1186/1471-2334-3-27.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhao Q, Alespeiti G, Debnath AK: A novel assay to identify entry inhibitors that block binding of HIV-1 gp120 to CCR5. Virol. 2004, 326: 299-309. 10.1016/j.virol.2004.06.022.View ArticleGoogle Scholar
- Neurath AR, Strick N, Jiang S: Synthetic peptides and anti-peptide antibodies as probes to study interdomain interactions involved in virus assembly: The envelope of the human immunodeficiency virus (HIV-1). Virol. 1992, 188: 1-13. 10.1016/0042-6822(92)90729-9.View ArticleGoogle Scholar
- Van Herrewege Y, Michiels J, Van Roey J, Fransen K, Kestens L, Balzarini J, Lewi P, Vanham G, Janssen P: In vitro evaluation of nonnucleoside reverse transcriptase inhibitors UC-781 and TMC120-R147681 as human immunodeficiency virus microbicides. Antimicrob Agents Chemother. 2004, 48: 337-339. 10.1128/AAC.48.1.337-339.2004.View ArticlePubMedPubMed CentralGoogle Scholar
- Hsu M, Harouse JM, Gettie A, Buckner C, Blanchard J, Cheng-Mayer C: Increased mucosal transmission but not enhanced pathogenicity of the CCR5-tropic, simian AIDS-inducing simian/human immunodeficiency virus SHIVSF162P3 maps to envelope gp120. J Virol. 2003, 77: 989-998. 10.1128/JVI.77.2.989-998.2003.View ArticlePubMedPubMed CentralGoogle Scholar
- Schwartz DH, Castillo RC, Arango-Jaramillo S, Sharma UK, Song HF, Sridharan G: Chemokine-independent in vitro resistance to human immunodeficiency virus (HIV-1) correlating with low viremia in long-term and recently infected HIV-1-positive persons. J Infect Dis. 1997, 176: 1168-1174.View ArticlePubMedGoogle Scholar
- Wu L, Paxton WA, Kassam N, Ruffing N, Rottman JB, Sullivan N, Choe H, Sodroski J, Newman W, Koup RA, Mackay CR: CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro. J Exp Med. 1997, 185: 1681-1691. 10.1084/jem.185.9.1681.View ArticlePubMedPubMed CentralGoogle Scholar
- Blaak H, Ran LJ, Rientsma R, Schuitemaker H: Susceptibility of in vitro stimulated PBMC to infection with NSI HIV-1 is associated with levels of CCR5 expression and beta-chemokine production. Virol. 2000, 267: 237-246. 10.1006/viro.1999.0111.View ArticleGoogle Scholar
- Gartner S, Popovic M: Virus isolation and production. Techniques in HIV Research. Edited by: Aldovini A and Walker BD. 1990, New York, M. Stockton Press, 53-70.View ArticleGoogle Scholar
- Shattock RJ, Doms RW: AIDS models: Microbicides could learn from vaccines. Nat Med. 2002, 8: 425-10.1038/nm0502-425.View ArticlePubMedGoogle Scholar
- Skinner MA, Ting R, Langlois AJ, Weinhold KJ, Lyerly HK, Javaherian K, Matthews TJ: Characteristics of a neutralizing monoclonal antibody to the HIV envelope glycoprotein. AIDS Res Hum Retroviruses. 1988, 4: 187-197.View ArticlePubMedGoogle Scholar
- Laal S, Zolla-Pazner S: Epitopes of HIV-1 glycoproteins recognized by the human immune system. Immunochemistry of AIDS, Chemical Immunology, Vol. 56. Edited by: NorrbyE. 1993, Basel, Karger, 91-111.View ArticleGoogle Scholar
- Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, Hendrickson WA: Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature. 1998, 393: 648-659. 10.1038/31405.View ArticlePubMedGoogle Scholar
- Xiang SH, Kwong PD, Gupta R, Rizzuto CD, Casper DJ, Wyatt R, Wang L, Hendrickson WA, Doyle ML, Sodroski J: Mutagenic stabilization and/or disruption of a CD4-bound state reveals distinct conformations of the human immunodeficiency virus type 1 gp120 envelope glycoprotein. J Virol. 2002, 76: 9888-9899. 10.1128/JVI.76.19.9888-9899.2002.View ArticlePubMedPubMed CentralGoogle Scholar
- Pantophlet R, Ollmann Saphire E, Poignard P, Parren PWI, Wilson IA, Burton DR: Fine mapping of the interaction of neutralizing and nonneutralizing monoclonal antibodies with the CD4 binding site of human immunodeficiency virus type 1 gp120. J Virol. 2003, 77: 642-658. 10.1128/JVI.77.1.642-658.2003.View ArticlePubMedPubMed CentralGoogle Scholar
- Westervelt P, Gendelman HE, Ratner L: Identification of a determinant within the human immunodeficiency virus 1 surface envelope glycoprotein critical for productive infection of primary monocytes. Proc Natl Acad Sci U S A. 1991, 88: 3097-3101.View ArticlePubMedPubMed CentralGoogle Scholar
- Westervelt P, Trowbridge DB, Epstein LG, Blumberg BM, Li Y, Hahn BH, Shaw GM, Price RW, Ratner L: Macrophage tropism determinants of human immunodeficiency virus type 1 in vivo. J Virol. 1992, 66: 2577-2582.PubMedPubMed CentralGoogle Scholar
- Rizzuto CD, Wyatt R, Hernandez-Ramos N, Sun Y, Kwong PD, Hendrickson WA, Sodroski J: A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding. Science. 1998, 280: 1949-1953. 10.1126/science.280.5371.1949.View ArticlePubMedGoogle Scholar
- Cormier EG, Dragic T: The crown and stem of the V3 loop play distinct roles in human immunodeficiency virus type 1 envelope glycoprotein interactions with the CCR5 coreceptor. J Virol. 2002, 76: 8953-8957. 10.1128/JVI.76.17.8953-8957.2002.View ArticlePubMedPubMed CentralGoogle Scholar
- Suphaphiphat P, Thitithanyanont A, Paca-Uccaralertkun S, Essex M, Lee T-H: Effect of amino acid substitution of the V3 and bridging sheet residues in human immunodeficiency virus type 1 subtype C gp120 on CCR5 utilization. J Virol. 2003, 77: 3832-3837. 10.1128/JVI.77.6.3832-3837.2003.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu S, Fan S, Sun Z: Structural and functional characterization of the human CCR5 receptor in complex with HIV gp120 envelope glycoprotein and CD4 receptor by molecular modeling studies. J Mol Model. 2003, 9: 329-336. 10.1007/s00894-003-0154-9.View ArticlePubMedGoogle Scholar
- Langley P: Why a pomegranate?. BMJ. 2000, 321: 1153-1154. 10.1136/bmj.321.7269.1153.View ArticlePubMedPubMed CentralGoogle Scholar
- Greene WC: The brightening future of HIV therapeutics. Nat Immunol. 2004, 5: 867-871. 10.1038/ni0904-867.View ArticlePubMedGoogle Scholar
- Poyrazoglu E, Goekmen V, Artik N: Organic acids and phenolic compounds in pomegranates (Punica granatum L.) Grown in Turkey. J Food Composition and Analysis. 2002, 15: 567-575. 10.1016/S0889-1575(02)91071-9.View ArticleGoogle Scholar
- Module 2: Phytochemicals (minerals, phytamins, and vitamins). 2003, [http://www.ars-grin.gov/duke/syllabus/module2.htm]
- Mahmood N, Moore PS, De Tommasi N, De Simone F, Colman S, Hay AJ, Pizza C: Inhibition of HIV infection by caffeoylquinic acid derivatives. Antiviral Chem Chemother. 1993, 4: 235-240.View ArticleGoogle Scholar
- Ma C, Nakamura N, Miyashiro H, Hattori M, Shimotohno K: Inhibitory effects of ursolic acid derivatives from cynomorium songaricum, and related triterpenes on human immunodeficiency viral protease. Phytotherapy Research. 1998, 12 : S138-S142. 10.1002/(SICI)1099-1573(1998)12:1+<S138::AID-PTR276>3.0.CO;2-5.View ArticleGoogle Scholar
- Mahmood N, Piacente S, Pizza C, Burke A, Khan AI, Hay AJ: The anti-HIV activity and mechanisms of action of pure compounds isolated from Rosa damascena. Biochem Biophys Res Commun. 1996, 229: 73-79. 10.1006/bbrc.1996.1759.View ArticlePubMedGoogle Scholar
- DeTommasi N, Piacente S, Rastrelli L, Mahmood N, Pizza C: Anti-HIV activity directed fractionation of the extracts of Margyricarpus setosus. Pharmaceutical Biology. 1998, 36: 29-32. 10.1076/phbi.188.8.131.5226.View ArticleGoogle Scholar
- Pomegranates could help in battle against AIDS. Reuters NewMedia, Inc. March 10 1996, [http://www.aegis.com/news/re/1996/RE960310.html]
- Medical breakthrough. British Muslims Monthly Survey. 1996, IV (3): 6-[http://artsweb.bham.ac.uk/bmms/1996/03March96.html#Medical%20breakthrough]
- Jassim SAA, Denyer SP, Stewart GSAB: Antiviral or antifungal composition comprising an extract of pomegranate rind or other plants and method of use. US Patent . 5,840,308-November 24 1998
- Shehadeh AA: Herbal extract composition and method with immune-boosting capability. US Patent . 6,030,622-Febuary 29 2000
- Jassim SAA, Denyer SP, Stewart GSAB: Antiviral or antifungal composition and method. US Patent . 6,187,316-February 2 2001
- Jassim SAA, Denyer SP: Antiviral or antifungal compositon and method. US Patent Application . 20020064567-May 30 2002
- Hsu S-T, Bonvin AMJJ: Atomic insight into the CD4 binding-induced conformational changes in HIV-1 gp120. Proteins. 2004, 55: 582-593. 10.1002/prot.20061.View ArticlePubMedGoogle Scholar
- Neurath AR, Strick N, Lin K, Debnath AK, Jiang S: Tin protoporphyrin IX used in control of heme metabolism in humans effectively inhibits HIV-1 infection. Antiviral Chem Chemother. 1994, 5: 322-330.View ArticleGoogle Scholar
- Hammar L, Hirsch I, Machado AA, de Mareuil J, Baillon JG, Bolmont C, Chermann J-C: Lectin-mediated effects of HIV type 1 infection in vitro. AIDS Res Hum Retroviruses. 1995, 11: 87-95.View ArticlePubMedGoogle Scholar
- Veazey RS, Shattock RJ, Pope M, Kirijan JC, Jones J, Hu Q, Ketas T, Marx PA, Klasse PJ, Burton DR, Moore JP: Prevention of virus transmission to macaque monkeys by a vaginally applied monoclonal antibody to HIV-1 gp120. Nat Med. 2003, 9: 343-346. 10.1038/nm833.View ArticlePubMedGoogle Scholar
- Garg S, Tambweker KR, Vermani K, Garg A, Kaul CL, Zaneveld LJD: Compendium of pharmaceutical excipients for vaginal formulations. Pharmaceutical Technology Drug Delivery. 2001, Sept.: 14-24.Google Scholar
- Yao WR, Yao HY: Adsorbent characteristics of porous starch. Starch/Starke. 2002, 54: 260-263. 10.1002/1521-379X(200206)54:6<260::AID-STAR260>3.0.CO;2-Z.View ArticleGoogle Scholar
- Whistler RL: Microporous granular starch matrix compositions. US Patent . 4,985,082-January 15 1991
- Berset C, Clermont H, Cheval S: Natural red colorant effectiveness as influenced by absorptive supports. J Food Sci. 1995, 60: 858-861, 879.View ArticleGoogle Scholar
- Stute R, Woelk HU: Interaction between starch and reactive dyes. New technique for the investigation of starch. II. Influence on fixation reaction of starch. Starch/Starke. 1974, 26: 1-9.View ArticleGoogle Scholar
- Seguchi M: Dye binding to the surface of wheat starch granules. Cereal Chemistry. 1986, 63: 518-520.Google Scholar
- Tomasik P, Wang Y-J, Jane JL: Complexes of starch with low-molecular saccharides. Starch/Starke. 1995, 47: 185-191.View ArticleGoogle Scholar
- Zhang G, Maladen MD, Hamaker BR: Detection of a novel three component complex consisting of starch, protein, and free fatty acids. J Agric Food Chem. 2003, 51: 2801-2805. 10.1021/jf030035t.View ArticlePubMedGoogle Scholar
- Johnson JM, Davis EA, Gordon J: Lipid binding of modified corn starches studies by electron spin resonance. Cereal Chemistry. 1990, 67: 236-240.Google Scholar
- Tomazic-Jezic VJ, Lucas AD, Sanchez BA: Binding and measuring natural rubber latex proteins on glove powder. J Immunoassay Immunochem. 2004, 25: 109-123.View ArticlePubMedGoogle Scholar
- Conde-Petit B, Nuessli J, Handschin S, Escher F: Comparative charaterization of aqueous starch dispersions by light microscopy, rheometry, and iodine binding behavior. Starch/Starke. 1998, 50: 184-192. 10.1002/(SICI)1521-379X(199805)50:5<184::AID-STAR184>3.3.CO;2-Z.View ArticleGoogle Scholar
- Moore J, Wainberg M, Amman A, Veazey R, Pope M, Shattock RJ, Doms RW: Development of fusion/entry inhibitors as topical microbicides. Microbicides. 2004, March 28-31 2004, London, [http://www.microbicides2004.org.uk/presentations/johnmoore.ppt]Google Scholar
- Neurath AR, Strick N: Biodegradable microbicidal vaginal barrier device. US Patent . 6,572,875-June 3 2003
- Kraulis PJ: MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Cryst. 1991, 24: 946-950. 10.1107/S0021889891004399.View ArticleGoogle Scholar
- Bacon DJ, Anderson WF: A fast algorithm for rendering space-filling molecule pictures. J Mol Graphics. 1988, 6: 219-220. 10.1016/S0263-7855(98)80030-1.View ArticleGoogle Scholar
- Merritt EA, Bacon DJ: Raster3D: Photorealistic molecular graphics. Methods Enzymol. 1997, 277: 505-524.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/4/41/prepub
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