Nanogel-Conjugated Reverse Transcriptase Inhibitors and Their Combinations as Novel Antiviral Agents with Increased Efficacy against HIV-1 Infection.

Nucleoside reverse transcriptase inhibitors (NRTIs) are an integral part of the current antiretroviral therapy (ART), which dramatically reduced the mortality from AIDS and turned the disease from lethal to chronic. The further steps in curing the HIV-1 infection must include more effective targeting of infected cells and virus sanctuaries inside the body and modification of drugs and treatment schedules to reduce common complications of the long-term treatment and increase patient compliancy. Here, we describe novel NRTI prodrugs synthesized from cholesteryl-ε-polylysine (CEPL) nanogels by conjugation with NRTI 5'-succinate derivatives (sNRTI). Biodegradability, small particle size, and high NRTI loading (30% by weight) of these conjugates; extended drug release, which would allow a weekly administration schedule; high therapeutic index (>1000) with a lower toxicity compared to NRTIs; and efficient accumulation in macrophages known as carriers for HIV-1 infection are among the most attractive properties of new nanodrugs. Nanogel conjugates of zidovudine (AZT), lamivudine (3TC), and abacavir (ABC) have been investigated individually and in formulations similar to clinical NRTI cocktails. Nanodrug formulations demonstrated 10-fold suppression of reverse transcriptase activity (EC90) in HIV-infected macrophages at 2-10, 2-4, and 1-2 µM drug levels, respectively, for single nanodrugs and dual and triple nanodrug cocktails. Nanogel conjugate of lamivudine was the most effective single nanodrug (EC90 2 µM). Nanodrugs showed a more favorable pharmacokinetics compared to free NRTIs. Infrequent iv injections of PEGylated CEPL-sAZT alone could efficiently suppress HIV-1 RT activity to background level in humanized mouse (hu-PBL) HIV model.

New sustained release of zidovudine matrix tablets - cytotoxicity toward Caco-2 cells.

OBJECTIVE: The aim of this study was to adjust the zidovudine (AZT) release from solid tablets to an ideal profile, by developing matrices comprising swellable polymers with nonswellable ones. METHODS: Directly compressed matrices comprised different ratios of hydroxypropylmethylcellulose K15M and K100M, ethylcellulose, and methacrylic acid (Eudragit(®) RS PO and Eudragit(®) RL PO) were prepared. Technological characterization and evaluation of the in vitro release behavior were carried out. Cell density and viability following drug exposure were evaluated by the SRB method, for the Caco-2 line, while cell morphology was assessed upon Trypan blue staining. RESULTS: A specific formulation containing 5% of each excipient - HPMC K15M, HPMC K100M, Eudragit(®) RS PO, and Eudragit(®) RL PO - was found to yield the best release profile. Application of the Korsmeyer-Peppas model to the dissolution profile evidenced that a non-Fickian (anomalous) transport is involved in the drug release. Regarding the influence of the tablets' composition on the drug's cytotoxic effect toward the Caco-2 cell line, a reduction of cell biomass (0-15%) was verified for the distinct AZT formulations tested, F19 having displayed the highest cytotoxicity, after 24 and 48 h of incubation. Additionally, a high reversibility of the AZT effect was observed. CONCLUSIONS: The results showed that the simultaneous application of both hydrophilic and hydrophobic polymers can modulate the drug release process, leading to an improved efficacy and patient compliance. All AZT formulations studied were found to be cytotoxic against Caco-2 cells, F19 being the most effective one.

Effect of the HIV nucleoside reverse transcriptase inhibitor zidovudine on the growth and differentiation of primary gingival epithelium.

OBJECTIVES: Oral complications associated with HIV infection and with the antiretroviral drugs used to treat it are of increasing concern in HIV-infected patients. Protease inhibitors have been shown to change the proliferation and differentiation state of oral tissues but the effect of nucleoside reverse transcriptase inhibitors is currently unknown. This study examined the effect of zidovudine on the growth and differentiation of the gingival epithelium. METHODS: Gingival keratinocyte organotypic (raft) cultures were established. The raft cultures were treated with a range of zidovudine concentrations. Haematoxylin and eosin staining was performed to examine the effect of zidovudine on gingival epithelium growth and stratification. Raft cultures were immunohistochemically analysed to determine the effect of this drug on the expression of key differentiation and proliferation markers, including cytokeratins and proliferating cell nuclear antigen (PCNA). RESULTS: Zidovudine dramatically changed the proliferation and differentiation state of gingival tissues both when it was present throughout the growth period of the tissue and when it was added to established tissue at day 8. Zidovudine treatment increased the expression of cytokeratin 10, PCNA and cyclin A. Conversely, cytokeratin 5, involucrin and cytokeratin 6 expression was decreased. The tissue exhibited characteristics of increased proliferation in the suprabasal layers as well as an increased fragility and an inability to heal itself. CONCLUSIONS: Zidovudine treatment, even when applied at low concentrations for short periods of time, deregulated the cell cycle/proliferation and differentiation pathways, resulting in abnormal epithelial repair and proliferation. Our system could potentially be developed as a model for studying the effects of HIV and highly active antiretroviral therapy in vitro.

Does maternal treatment with zidovudine affect changes in mandibles of newborns? Laser induced fluorescence study.

The influence of antiretroviral drug zidovudine treatment during pregnancy on mandible development in newborn rats was studied. The fluorescence of mandibles from 7-, 14- and 28-days old individuals was measured by means of fiber-optical fluorescence analyzer with 407 nm laser excitation. Obtained results revealed disturbing effect of maternal zidovudine administration on mandible fluorescence intensity which should decrease with bone development. Small changes in fluorescence of porphyrin forms are maintaining in the first month of newborns life while the changes observed in 440-585 nm range disappear.

Shuttle-mediated drug delivery to the brain.

Advances in the field of shuttle-mediated drug delivery have been made in the last decade; however, the treatment of brain disorders still remains a great challenge because of the presence of the blood-brain barrier (BBB), a structure that limits the access of drugs to their site of action in the central nervous system. Several strategies have been proposed to enhance the transport of drugs across the BBB. In this Review, we focus on the vector-mediated approach, in which a drug is coupled to a molecule (shuttle) that has the ability to cross the BBB and deliver the drug to the brain.

Synthesis, characterization, and in vitro assay of folic acid conjugates of 3'-azido-3'-deoxythymidine (AZT): toward targeted AZT based anticancer therapeutics.

Conjugates of three components namely folic acid, poly(ethyleneglycol) and 3 '-azido-3 '-deoxythymidine (AZT) are presented. Folate-PEG units were coupled to AZT to facilitate delivery of the nucleoside into the cell. A convenient separation of the polydisperse PEGylated-folic acid regioisomers produced upon conjugation is described. This is to select for the active gamma-regioisomer over the inactive alpha-regioisomer. In vitro cytotoxicity assays were conducted against an ovarian cell line (A2780/AD) that overexpresses the folate receptor (FR) and compared to a FR free control cell line. Compared to AZT a approximately 20-fold greater potency against the resistant ovarian line was observed for the conjugates.

Raman spectroscopy of pH-induced release of zidovudine from lactobionic acid-conjugated PEGylated gold colloids.

Zidovudine (AZT) adsorbed on colloidal gold nanoparticles (AuNPs) undergoes pH-induced conformational changes according to spectral changes in surface-enhanced Raman scattering (SERS). In acidic pH values conditions, AZT assumes the C(2')-endo conformer, which binds more weakly to AuNPs than under neutral and alkaline conditions. In this study, density functional theory (DFT) calculations were performed; these calculations also supported the conformation-dependent binding energies. A lactobionic acid-conjugated PEGylated (LA-PEG-SH; molecular weight: 3400) unit was attached to AuNPs to target the asialoglycoprotein receptors overexpressed in hepatocarcinoma cells of Huh7 and SNU-354. The loading efficiency values were measured to be ∼44-49% and ∼66-68% at pH values of 7 and 10, respectively. At an acidic pH of 4.5, they were estimated to be only ∼35-38%. pH-dependent spectral changes were observed for the asymmetric stretching modes of the azide (NNN) bands at 2183 cm-1 (in acidic pH) and at 2129 cm-1 (in basic pH). Cell viability analysis indicated that the LA-PEG-capped, AZT-coated AuNPs specifically inhibited the growth of the targeted hepatocarcinoma cells with better cancer cell killing efficiency than was observed with the LA-PEG-capped AuNPs without AZT.

Versatile iron-catechol-based nanoscale coordination polymers with antiretroviral ligand functionalization and their use as efficient carriers in HIV/AIDS therapy.

A novel chemical approach integrating the benefits of nanoparticles with versatility of coordination chemistry is reported herein to increase the effectiveness of well-known HIV antiretroviral drugs. The novelty of our approach is illustrated using a catechol ligand tethered to the known antiretroviral azidothymidine (AZT) as a constitutive building block of the nanoparticles. The resulting nanoscale coordination polymers (NCPs) ensure good encapsulation yields and equivalent antiretroviral activity while significantly diminishing its cytotoxicity. Moreover, this novel family of nanoparticles also offers (i) long-lasting drug release that is dissimilar inside and outside the cells depending on pH, (ii) triggered release in the presence of esterases, activating the antiviral activity in an on-off manner due to a proper chemical design of the ligand and (iii) improved colloidal stabilities and cellular uptakes (up to 50-fold increase). The presence of iron nodes also adds multifunctionality as possible contrast agents. The present study demonstrates the suitability of NCPs bearing pharmacologically active ligands as an alternative to conventional antiretroviral treatments.

Poly (vinyl pyrrolidone)-lipid based hybrid nanoparticles for anti viral drug delivery.

Zidovudine (AZT) is an antiviral drug with moderate solubility in water. It has limited application due to its short half life in vivo and consequent requirement for frequent administrations. To solve this problem, zidovudine loaded polyvinylpyrrolidone (PVP)/stearic acid (SA)-polyethylene glycol (PEG) nanoparticles (PSNPs) were developed.The hybrid nanoparticles were prepared by emulsification-solvent evaporation method. The physico chemical characterizations of the PSNPs was done by dynamic light scattering (DLS), transmission electron microscopy (TEM), and fourier transform infra-red spectroscopy (FT-IR). The in vitro release behavior and haemocompatibility studies were also performed. The in vitro cytotoxicity and cell uptake studies of the PSNPs were assessed in murine neuro-2a and HeLa cells. Our results revealed that the core shell PSNPs prepared from lipid and polymer led to significant improvement in cellular internalization. Therefore, it is envisaged that nanoparticles composed of lipid and polymer moieties may constitute a preferred embodiment for anti-viral drug delivery for use in HIV/AIDS therapy.

Polymers fight HIV: potent (pro)drugs identified through parallel automated synthesis.

Macromolecular (pro)drugs interfere with the proliferation of HIV through both inhibition of viral cell entry and via intracellular delivery of antiviral drugs. Lead polymer conjugates exhibit longevity of action exceeding that of parent nucleoside analogue drug and are active in primary T cell over at least 72 h.

The changes in bone organic and inorganic matrix in newborn rats after maternal application of antiretroviral agents: Indinavir and zidovudine.

This work presents results concerning influence of indinavir (protease inhibitor, PI(1)) and zidovudine (nucleoside and nucleotide inhibitor of reverse transcriptase, NRTI) administered to pregnant Wistar rat females on organic and mineral constituents of bones and teeth (mandibles, skulls, tibiae, femurs, and incisors) of their offspring at the age of: 7, 14, and 28 days studied by means of induced laser and X-ray fluorescence spectroscopy supported by digital radiography. Influence of indinavir administered to pregnant female rats on bone of their offspring revealed mainly in changes of mineral concentration: lowered Ca concentration and disturbances of trace elements. Zidovudine influenced organic matter more than inorganic matrix which was seen in enhancement of LIF fluorescence. However, there was also an unexpected increase of bone density for rats from zidovudine group, unlike indinavir group, observed. Our studies suggest that studied antiretroviral agents given to pregnant women, may have different destructive impact on bone state of their offspring in the first period of life. Maternal administration of zidovudine may delay development of organic matrix, while indinavir may have adverse effects on inorganic structure.

The Alpha trial: European/Australian randomized double-blind trial of two doses of didanosine in zidovudine-intolerant patients with symptomatic HIV disease. Alpha International Coordinating Committee.

OBJECTIVES: To compare the efficacy and toxicity of two doses of didanosine (ddI) in patients with symptomatic HIV disease who are intolerant of zidovudine (ZDV). DESIGN: The Alpha trial is a randomized double-blind multicentre trial of two doses of ddI. ddI was given as one buffered sachet twice daily in doses adjusted for weight: 750 mg per day for patients > or = 60 kg in the higher-dose group, and 200 per day for the lower-dose group. RESULTS: Patients (n = 1775; 907 higher-dose, 868 lower-dose) from nine European countries and Australia were randomized and started trial treatment. Sixty per cent had AIDS, 65% had CD4 cell counts < 50 x 10(6)/l and 55% had received ZDV for more than 12 months. Follow-up was to death or 30 September 1992, and only 33 patients (20 higher-dose, 13 lower-dose) had been lost to follow-up for at least 3 months at that time. The longest follow-up was 28.5 months and the mean (SD) was 12.4 (6.9) months. There was no significant difference in survival between the groups: 67% of patients in each group died, the median survival being 13.0 months in the higher-dose and 12.5 in the lower-dose groups, a difference of about 0.5 months (95% confidence interval, -0.9 to 2.0; log-rank P = 0.7). There was also no significant difference in progression to AIDS or death, development of HIV encephalopathy or death, or development of new AIDS events or death. There were small (but statistically significant) differences in the changes in CD4 cell count and in p24 antigen levels between the groups, with greater increases in CD4 and greater decreases in p24 in the higher-dose group. There were also clear differences in adverse events: pancreatitis developed more frequently in the higher-dose group, 66 patients compared to nine patients in the lower-dose group, of whom 37 and six, respectively, were classified as definite cases. Nine cases (seven higher-dose, two lower-dose) were reported to have died because of or with pancreatitis. Peripheral neuropathy, abnormal liver function and dry mouth were also reported more often in the higher-dose group. CONCLUSIONS: The Alpha trial is not able to provide direct evidence for the clinical efficacy of ddI. There was no significant difference between the two doses in mortality or disease progression. However, the higher dose was more toxic.

Synthesis and anti-HIV activity of [AZT]-[TSAO-T] and [AZT]-[HEPT] dimers as potential multifunctional inhibitors of HIV-1 reverse transcriptase.

In an attempt to combine the HIV-inhibitory capacity of 2',3'-dideoxynucleoside (ddN) analogues and non-nucleoside reverse transcriptase (RT) inhibitors (NNRTI), we have designed, synthesized, and evaluated for their anti-HIV activity several dimers of the general formula [ddN]-(CH2)n-[NNRTI]. These dimers combine in their structure a ddN such as AZT and a NNRTI such as TSAO-T and HEPT linked through an appropriate spacer between the N-3 of the thymine base of both compounds. The [TSAO-T]-(CH2)n-[AZT] dimers proved markedly inhibitory to HIV-1. Also, if AZT was replaced by thymidine in the dimer molecules, potent anti-HIV-1 activity was observed. However, although the compounds proved inhibitory to HIV-1, they were less potent inhibitors than the parent compounds from which they were derived. None of the dimers were endowed with anti-HIV-2 activity. In contrast with the TSAO-T monomers, none of the TSAO-T-containing dimers proved markedly cytotoxic to the cells. There was a clear trend toward decreased antiviral potency with lengthening the methylene spacer in the [TSAO-T]-(CH2)n-[AZT] dimers.

Inhibition of proliferation of human immunodeficiency virus type 1 by novel heteropolyoxotungstates in vitro.

Fifteen heteropolyoxotungstates were tested for their effects on the proliferation of human immunodeficiency virus type 1 (HIV-1) using an in vitro system consisting of MT-4 cells and HTLV-IIIb. Eight heteropolyoxotungstates (HPOTs) with the Keggin structure or dimerized deficient Keggin structure proved to be potent inhibitors of HIV-1. In contrast, seven non-Keggin HPOTs including HPA 23 did not have significant effects on HIV-1 proliferation at non-toxic doses. [PTi2W10O40]7- (PM-19) was the most potent inhibitor of HIV-1 among the 15 HPOTs tested. The inhibition of HIV-1 replication by PM-19 presumably results from impaired virus adsorption and/or penetration into target cells. Viral spread of HIV-1 and HIV-2 on cell-to-cell basis was also susceptible to PM-19. In combination, PM-19 and 3'-azido-3'-deoxythymidine were synergistic in inhibiting HIV-1 proliferation.

Evaluation of anti-AIDS drugs in conventional mice implanted with a permeable membrane device containing human T cells infected with HIV.

We now report the confirmation of the work of Hollingshead et al. (1995) on development of a cell based hollow fiber (HF) system for evaluating potential anti-AIDS drugs in vivo using conventional mice rather than SCID mice. CD4 +, CEM-SS cells infected with HIV/1, strain RF, at a multiplicity of infection of 0.1 were placed into HFs. The fibers were implanted into the peritoneal cavity of outbred Swiss mice. Using this model, the antiviral activity of azidothymidine (AZT) at doses of approximately 150, 75 and 37.5 mg/kg/day was evaluated by administering AZT to the mice in drinking water. Upon fiber removal on day 6, AZT treatment was shown to significantly increase CEM cell viability over the untreated, virus control group and significantly reduced the levels of HIV p24 and HIV RT activity.

New lipophilic alkyl/acyl dinucleoside phosphates as derivatives of 3'-azido-3'-deoxythymidine: inhibition of HIV-1 replication in vitro and antiviral activity against Rauscher leukemia virus infected mice with delayed treatment regimens.

The antiretroviral activity of two new lipophilic derivatives of azidothymidine (AZT), N4-hexadecyl-2'-deoxyribocytidylyl-(3',5')-3'-azido-2',3'-deoxythy midine (N4-hexadecyldC-AZT) and N4-palmitoyl-2'-deoxyribocytidylyl-(3',5')-3'-azido-2',3'-deoxythy midine (N4-palmitoyldC-AZT) was evaluated in comparison to AZT. In vitro the drugs were tested in human immunodeficiency virus 1 (HIV-1) infected CD4+ HeLa and H9 cells. The in vivo antiviral effect of these derivatives was analysed in Rauscher leukemia virus (RLV) infected mice. The derivatives were incorporated into small liposomes. In vitro both derivatives inhibited virus proliferation in both HIV-1 infected cell lines in a similar dose-responsive manner as AZT. In a plaque reduction assay, using HeLa cells, the IC50 values were 0.035 microM for AZT, 0.5 microM for N4-hexadecyldC-AZT and 4.5 microM for N4-palmitoyldC-AZT, whereas p24 antigen analysis on H9 cells gave IC50 values of 0.005 microM, 0.05 microM and 0.05 microM, respectively. RLV infected mice were treated with intermittent schedules i.p. or i.v. on days 1, 6, 11, and days 16 or 0, 3, 7, and 11 after infection. Regimens with further delayed drug application were on days 3, 7, and 11 and 7 and 11 only. While i.p. treatment with total doses of 380-1140 mg/kg free AZT resulted in 10-30% inhibition of RLV induced splenomegaly, the derivatives gave inhibitions of 37-94%. Late onset of treatment with the derivatives was significantly more effective as compared to free AZT. Intravenous treatment with N4-hexadecyldC-AZT was effective, but with AZT was inactive. The discrepancy in antiviral activity of the AZT derivatives found between the in vitro and in vivo test systems emphasizes the importance of investigating the activity of drug derivatives in vivo.

Contraceptive activity of a spermicidal aryl phosphate derivative of bromo-methoxy-zidovudine (compound WHI-07) in rabbits.

OBJECTIVE: To determine the vaginal contraceptive activity of WHI-07 in the rabbit model. DESIGN: Prospective, controlled study. SETTING: Center for advanced preclinical sciences. ANIMAL(S): Subgroups of 15, 16, or 24 New Zealand White does and 24 bucks per experiment. INTERVENTION(S): Ex vivo (Experiment 1) and in vivo (Experiments 2 and 3) treatment of semen with WHI-07 or Nonoxynol-9 (N-9). In Experiment I, ovulated does in subgroups of 15 were artificially inseminated with semen mixed with WHI-07 or vehicle. In Experiment 2, ovulated does in subgroups of 24 were artificially inseminated within 2 min after intravaginal administration of 2% WHI-07 gel-microemulsion or 2% N-9 gel and allowed to complete term pregnancy. In Experiment 3, ovulated does in subgroups of 16 were artificially inseminated at 15, 30, or 60 minutes. MAIN OUTCOME MEASURE(S): The numbers of implanted embryos on postinsemination day 8 or the proportion of does that became pregnant and delivered newborn rabbits; the litter size, weight, growth, and viability of pups until lactation day 5. RESULT(S): Exposure of semen to WHI-07 at the time of artificial insemination completely inhibited pregnancy rates (WHI-07-pretreated, 0%, vs. control, 60%) and embryo implantation (WHI-07-pretreated, 0/175 vs. control, 68/170). Intravaginal administration of a 2% WHI-07 gel-microemulsion or 2% N-9 gel before artificial insemination significantly inhibited pregnancy rates (81% and 85% inhibition, respectively) when compared with control. Furthermore, the 2% WHI-07 gel-microemulsion provided >90% inhibition of fertility even when insemination was delayed until 60 minutes after intravaginal application. Rabbits that delivered litters despite intravaginal application of 2% WHI-07 gel-microemulsion had healthy offsprings with no perinatal or postnatal repercussions. CONCLUSION(S): WHI-07 is a potent contraceptive agent in vivo. Intravaginal use of WHI-07 gel-microemulsion has clinical potential as a safe prophylactic contraceptive, in addition to its microbicide activity to curb the sexual transmission of HIV.

In vitro anti-HIV-1 activity of sn-2-substituted 1-O-octadecyl-sn-glycero-3-phosphonoformate analogues and synergy with zidovudine.

Monoalkyl ether lipid analogues of foscarnet (phosphonoformate, PFA) exhibit substantially greater in vitro antiviral activity than unmodified PFA against human immunodeficiency virus type 1 (HIV-1). Our previous studies indicate that the length of the alkyl chain must be 14-22 carbons for optimal antiviral activity. To further evaluate the structure-activity relationship, we prepared 1-O-octadecyl-sn-glycerol analogues of PFA with various substitutions at the sn-2 position of glycerol and determined the effect of structure on in vitro antiviral activity and selectivity against HIV-1 in MT-2 and CD4-expressing HeLa cells (HT4-6C). We also studied combinations of zidovudine with PFA, 1-O-octadecyl-2-O-methyl-sn-glycero-3-PFA, or 1-O-octadecyl-sn-glycero-3-PFA and calculated their combination index values against HIV-1 in HT4-6C cells. Alkyl substitutions of one to four carbons at the sn-2 position of glycerol showed optimal antiviral activity. Both alkyl ether lipid analogues were strongly synergistic with zidovudine over a wide range of drug ratios and concentrations. 1-O-octadecyl-sn-glycerol analogues of PFA have selective antiviral properties and warrant further evaluation as potential antiretroviral drugs.

Synthesis of dual function (5R,6R)- and (5S,6S)-5-bromo-6-methoxy-5,6-dihydro-AZT-5'-(para-bromophenyl methoxyalaninyl phosphate) as novel spermicidal and anti-HIV agents.

We synthesized a novel compound, 5-bromo-6-methoxy-5,6-dihydro-AZT-5'- (p-bromophenyl methoxyalaninyl phosphate), which had an EC50 value of 5 microM in sperm motility assays. This is > 1 log10 better than that of the detergent spermicide nonoxynol-9 (EC50 81 microM). The compound also displayed a potent anti-human immunodeficiency virus (HIV) activity with an IC50 value of 0.005 microM in HIV replication assays, which was virtually identical to that of AZT (IC50 0.006 microM) and > 2 log10 more potent than that of nonoxynol-9 (IC50 2.2 microM). The promising results reported herein recommend the further development of the dual function 5-halo-6-alkoxyl-5,6-dihydro-AZT derivatives as a new class of vaginal contraceptives capable of preventing the sexual transmission of HIV while providing fertility control for women who are at high risk of acquiring HIV by heterosexual transmission. These dual function 5-halo-6-alkoxyl-5,6-dihydro-AZT derivatives may also have utility in curbing domestic and wildlife animal retroviral transmissions.

Synergistic antiviral effect of PEG-asparaginase (ONCASPAR), with protease inhibitor alone and in combination with RT inhibitors against HIV-1 infected T-cells: a model of HIV-1-induced T-cell lymphoma.

We evaluated the anti-HIV-1 activity of the T-cell-specific protein inhibitor PEG-asparaginase (PEG-ASNase) in human HIV-1-infected T-cells. We further examined the drug synergism between PEG-ASNase and the protease inhibitor Saquinavir (SAQ), both alone and in combination with nucleoside analog reverse transcriptase inhibitors (NRTI). Our drug synergism studies served as a model for an HIV-induced T-cell lymphoma. Phytohemagglutinin [PHA(+)] stimulated T-cells were infected with HIV-1 and then treated with one or more drugs 90 minutes from the viral exposure. To measure inhibition of viral replication, we examined HIV-1 RT and HIV-1 RNA in the supernatant and intracellularly on day 7 post-infection and drug treatment. Last, we examined the effect of administering drugs immediately after HIV-1 infection of T-cells to simulate treatment after an accidental exposure to the virus. PEG-ASNase, even when used alone, has anti-HIV-1 activity in PHA(+)-stimulated T-cells due to inhibition of protein synthesis. When the drug was used with SAQ, the combination was synergistic in inhibiting HIV-1 RT and RNA in the supernatant and intracellularly by 2.5 log10 in comparison with controls. PEG-ASNase and SAQ were even more effective in inhibiting HIV-1 replication when combined with the NRTI inhibitors azidothymidine (AZT) and (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine). The addition of ribonucleotide reductase inhibitor, 2-methyl-1H-isoindole-1,3-dione (MISID), further potentiated the antiviral effect of the regimen. HIV-1 RT and RNA analyses showed that the administration of the PEG-ASNase + SAQ drug combination immediately following exposure to HIV-1 completely inhibited the infection of T-cells in our in vitro T-cell model. From these results we conclude that PEG-ASNase is a valuable T-cell-specific protein inhibitor against HIV-1 infection, when used singly or in combination with a protease inhibitor, an RT inhibitor and an RR inhibitor. Since PEG-ASNase is a drug of choice for the treatment of T-cell lymphomas, a combination regimen containing PEG-ASNase could be very effective in the treatment of HIV-1-induced T-cell lymphoma and possibly AIDS. Future studies are needed in HIV-infected and/or HIV-induced T-cell lymphoma patients to investigate these findings.