PubMed Health⌕ Search

Biomedical subjects

Arnold B Rabson

Publications and source records attributed to Arnold B Rabson.

At least 19 recordsLinked to original sources
PubMed2006

Alternative pathways of NF-kappaB activation: a double-edged sword in health and disease.

While the classical pathway of NF-kappaB activation plays critical roles in a wide range of biological processes, the more recently described "non-canonical" NF-kappaB pathway has important but more restricted roles in both normal and pathological processes. The non-canonical NF-kappaB pathway, based on processing of the nf-kappab2 gene product p100 to generate p52, appears to be involved in B-cell maturation and lymphoid development. Deregulated activation of this pathway has been observed in a variety of malignant and autoimmune diseases, thus inhibitors that specifically target p100 processing might be predicted to have potential roles as immunomodulators and in the therapy of malignant diseases. We review current understandings of NF-kappaB activation, particularly the mechanisms of p100 processing under both physiological and pathological conditions.

Autoimmune Diseases↗
PubMed2006

Effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) in combination with paclitaxel (Taxol) on prostate Cancer LNCaP cells cultured in vitro or grown as xenograft tumors in immunodeficient mice.

PURPOSE: To investigate the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) in combination with paclitaxel (Taxol) on prostate cancer cells cultured in vitro or grown as tumors in immunodeficient mice. EXPERIMENTAL DESIGN: Human prostate cancer LNCaP cells in culture were treated with TPA alone or in combination with paclitaxel. NCr immunodeficient mice with well-established LNCaP tumors received i.p. injections with vehicle or with TPA, paclitaxel, or TPA in combination with paclitaxel. The animals either received daily treatment for 5 consecutive days followed by a 2-day intermission, which was repeated for a total of 28 days (experiment 1), or continuous daily treatment for 28 days (experiment 2). RESULTS: Treatment of LNCaP cells with a combination of TPA and paclitaxel synergistically inhibited the growth and induced apoptosis in cultured LNCaP cells, and this treatment also induced a marked increase in phosphorylated c-Jun-NH2-kinase (JNK). In animal experiments, tumor growth occurred in all mice treated with vehicle. When treated with TPA alone, the percentage of animals with some tumor regression was 33% in experiment 1 and 100% in experiment 2. Treatment of animals with paclitaxel alone caused some tumor regression in 17% and 57% of the animals in experiments 1 and 2, respectively. All animals treated with TPA + paclitaxel in both experiments had some tumor regression. CONCLUSIONS: TPA and paclitaxel in combination had a stronger inhibitory effect on the growth of LNCaP cells in culture or as xenograft tumors in immunodeficient mice than either agent alone. Clinical trials with TPA alone or in combination with paclitaxel in patients with prostate cancer may be warranted.

Animals↗
PubMed2006

Novel multi-component nanopharmaceuticals derived from poly(ethylene) glycol, retro-inverso-Tat nonapeptide and saquinavir demonstrate combined anti-HIV effects.

BACKGROUND: Current anti-AIDS therapeutic agents and treatment regimens can provide a dramatically improved quality of life for HIV-positive people, many of whom have no detectable viral load for prolonged periods of time. Despite this, curing AIDS remains an elusive goal, partially due to the occurrence of drug resistance. Since the development of resistance is linked to, among other things, fluctuating drug levels, our long-term goal has been to develop nanotechnology-based drug delivery systems that can improve therapy by more precisely controlling drug concentrations in target cells. The theme of the current study is to investigate the value of combining AIDS drugs and modifiers of cellular uptake into macromolecular conjugates having novel pharmacological properties. RESULTS: Bioconjugates were prepared from different combinations of the approved drug, saquinavir, the antiviral agent, R.I.CK-Tat9, the polymeric carrier, poly(ethylene) glycol and the cell uptake enhancer, biotin. Anti-HIV activities were measured in MT-2 cells, an HTLV-1-transformed human lymphoid cell line, infected with HIV-1 strain Vbu 3, while parallel studies were performed in uninfected cells to determine cellular toxicity. For example, R.I.CK-Tat9 was 60 times more potent than L-Tat9 while the addition of biotin resulted in a prodrug that was 2850 times more potent than L-Tat9. Flow cytometry and confocal microscopy studies suggest that variations in intracellular uptake and intracellular localization, as well as synergistic inhibitory effects of SQV and Tat peptides, contributed to the unexpected and substantial differences in antiviral activity. CONCLUSION: Our results demonstrate that highly potent nanoscale multi-drug conjugates with low non-specific toxicity can be produced by combining moieties with anti-HIV agents for different targets onto macromolecules having improved delivery properties.

Journal Article↗
PubMed2006

Role of NF-kappaB in regulation of PXR-mediated gene expression: a mechanism for the suppression of cytochrome P-450 3A4 by proinflammatory agents.

It is a long-standing observation that inflammatory responses and infections decrease drug metabolism capacity in human and experimental animals. Cytochrome P-450 3A4 cyp304 is responsible for the metabolism of over 50% of current prescription drugs, and cyp3a4 expression is transcriptionally regulated by pregnane X receptor (PXR), which is a ligand-dependent transcription factor. In this study, we report that NF-kappaB activation by lipopolysaccharide and tumor necrosis factor-alpha plays a pivotal role in the suppression of cyp3a4 through interactions of NF-kappaB with the PXR.retinoid X receptor (RXR) complex. Inhibition of NF-kappaB by NF-kappaB-specific suppressor SRIkappaBalpha reversed the suppressive effects of lipopolysaccharide and tumor necrosis factor-alpha. Furthermore, we showed that NF-kappaB p65 disrupted the association of the PXR.RXRalpha complex with DNA sequences as determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. NF-kappaB p65 directly interacted with the DNA-binding domain of RXRalpha and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by the PXR.RXRalpha complex. This mechanism of suppression by NF-kappaB activation may be extended to other nuclear receptor-regulated systems where RXRalpha is a dimerization partner.

Cytochrome P-450 CYP3A↗
PubMed2005

Regulation of Bcl-3 through interaction with the Lck tyrosine kinase.

bcl-3 is a protooncogene which undergoes chromosomal translocation in a subset of chronic B-cell lymphocytic leukemia cells. Bcl-3 is a unique IkappaB family protein that regulates transcription of a number of NF-kappaB target genes through interactions with NF-kappaB dimers. Based on previous studies, suggesting that Bcl-3 interacts with the Fyn tyrosine kinase in platelets, we investigated possible interactions of Bcl-3 with Lck, a related tyrosine kinase important in lymphoid cells. Protein-protein interactions between Bcl-3 and the Lck tyrosine kinase were identified both in vitro and in vivo. Lck enhanced Bcl-3-mediated activation of a p52/Bcl-3-responsive promoter in reporter gene assays independent of its tyrosine kinase activity, but requiring the Lck SH3 protein interaction domain. These studies suggest that Bcl-3 might participate in oncogenic pathways involving Lck.

B-Cell Lymphoma 3 Protein↗
PubMed2005

Activation of human T cell leukemia virus type 1 LTR promoter and cellular promoter elements by T cell receptor signaling and HTLV-1 Tax expression.

Human T cell leukemia virus 1 (HTLV-1) gene expression is regulated by both the viral Tax protein and by cellular transcriptional factors. We have previously shown that immune activation stimuli such as phorbol esters (PMA) and phytohemagglutinin (PHA) cooperate with HTLV-1 Tax expression to enhance HTLV-1 gene expression in infected T cells through increased activity of the HTLV-1 LTR. We now extend these studies to demonstrate roles for the T cell receptor complex, Lck, and Ras molecules in the coactivation of the HTLV-1 LTR by Tax and T cell activation stimuli. We also observe coactivation of Tax-responsive cellular promoter elements containing NF-kappaB and serum response factor (SRF) binding sites by Tax and T cell activation stimuli. These results suggest a model whereby T cell receptor stimulation and Tax expression coactivate HTLV-1 gene expression and cellular gene expression, enhancing activation of latent HTLV-1 and expression of cellular genes involved in disease pathogenesis.

Cell Line↗
PubMed2005

Inhibitory effects of 12-O-tetradecanoylphorbol-13-acetate alone or in combination with all-trans retinoic acid on the growth of cultured human pancreas cancer cells and pancreas tumor xenografts in immunodeficient mice.

Treatment of cultured PANC-1, MIA PaCa-2, and BxPC-3 human pancreatic adenocarcinoma cells with 0.1 to 1.6 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 96 h inhibited the proliferation of these cells in a dose-dependent manner, and PANC-1 and MIA PaCa-2 cells were more sensitive to TPA than BxPC-3 cells. Inhibition of proliferation by TPA in PANC-1 cells was associated with an increase in the level of p21, but this was not observed in MIA PaCa-2 or BxPC-3 cells. The TPA-induced increase of p21 in PANC-1 cells was blocked by bisindolylmaleimide or rottlerin (inhibitors of protein kinase C). Studies in NCr-immunodeficient mice with well established PANC-1 tumor xenografts indicated that daily i.p. injections of TPA strongly inhibited tumor growth, increased the percentage of caspase-3-positive cells, and decreased the ratio of mitotic cells to caspase-3-positive cells in the tumors. Studies with BxPC-3 tumors in NCr mice receiving daily i.p. injections of vehicle, TPA, all-trans retinoic acid (ATRA), or a TPA/ATRA combination showed that TPA had an inhibitory effect on tumor growth, but treatment of the animals with the TPA/ATRA combination had a greater inhibitory effect on tumor growth than TPA alone. Treatment with the TPA/ATRA combination resulted in a substantially decreased ratio of the percentage of mitotic cells to the percentage of caspase-3-positive cells in the tumors compared with tumors from the vehicle-treated control animals. The inhibitory effects of TPA on tumor growth occurred at clinically achievable blood levels.

Animals↗
PubMed2005

Adenovirus infection and cytotoxicity of primary mantle cell lymphoma cells.

Mantle cell lymphoma (MCL) is a distinct form of non-Hodgkin's lymphoma (NHL) derived from CD5+ B cells. MCL cells overexpress cyclin D1 as a consequence of translocation of the gene into the immunoglobulin heavy-chain gene locus. MCL is an aggressive form of NHL with frequent relapses after standard-dose chemotherapy. In this context, a variety of novel therapies for patients with MCL have been investigated. In this study, we use an expanded panel of attenuated adenoviruses to study adenovirus-mediated cytotoxicity of MCL cells. Our results demonstrate: 1) adenovirus infection of MCL cells despite the absence of receptor/coreceptor molecules known to be important for adenovirus infection of other cells types; 2) cytotoxicity of MCL cells after infection with specific adenovirus mutants; 3) a high degree of cytotoxicity after infection of some patient samples with viruses lacking the E1B 19k "antiapoptotic" gene; and 4) cytotoxicity after infection with viruses containing mutations in E1A pRb or p300 binding. The extent of cytotoxicity with the panel of viruses demonstrated interpatient variability, but 100% cytotoxicity, as determined by molecular analysis, was detected in some samples. These studies provide the foundation for: 1) the development of adenoviruses as cytotoxic agents for MCL and 2) analyses of key regulatory pathways operative in MCL cells.

Adenoviridae↗
PubMed2005

Enhancement of TPA-induced growth inhibition and apoptosis in myeloid leukemia cells by BAY 11-7082, an NF-kappaB inhibitor.

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent stimulator of differentiation and apoptosis in myeloid leukemia cells. In the present study, we investigated the role of the transcription factor NF-kappaB in TPA-induced growth inhibition and apoptosis in the myeloid leukemia HL-60 cell line and its TPA-resistant cell variant HL-525. Unlike the parental cell line, HL-525 cells are protein kinase C (PKC)-beta deficient and resistant to TPA-induced differentiation and apoptosis. We found that treatment of HL-60 cells with TPA resulted in a concentration-dependent growth inhibition and an increase in apoptotic cells. TPA only had a small effect on growth and apoptosis in HL-525 cells. Treatment of HL-60 cells with TPA (0.64-3.2 nM) caused a rapid activation of NF-kappaB as determined by electrophoresis mobility shift assay (EMSA) and immunocytochemistry. Although the basal level of NF-kappaB activity was low in HL-60 cells, TPA-resistant HL-525 cells had a high basal level of NF-kappaB activity. Treatment of HL-525 cells with higher concentrations of TPA (16-80 nM) resulted in a further increase in NF-kappaB activity. (E)3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile (BAY 11-7082; BAY), which inhibits IkappaB alpha phosphorylation and thus decreases NF-kappaB activation, was found to decrease TPA-induced nuclear translocation of NF-kappaB. Furthermore, BAY enhanced TPA-induced growth inhibition and apoptosis in both HL-60 and HL-525 cells. Results from the present study indicate that inhibition of NF-kappaB by BAY was associated with enhanced TPA-induced growth inhibition and apoptosis in human myeloid leukemia cells. TPA in combination with pharmacological inhibitors of NF-kappaB may improve the therapeutic efficacy of TPA and overcome the resistance to TPA in some myeloid leukemia patients.

Active Transport, Cell Nucleus↗
PubMed2004

Hypoxia and defective apoptosis drive genomic instability and tumorigenesis.

Genomic instability is a hallmark of cancer development and progression, and characterizing the stresses that create and the mechanisms by which cells respond to genomic perturbations is essential. Here we demonstrate that antiapoptotic BCL-2 family proteins promoted tumor formation of transformed baby mouse kidney (BMK) epithelial cells by antagonizing BAX- and BAK-dependent apoptosis. Cell death in vivo correlated with hypoxia and induction of PUMA (p53 up-regulated modulator of apoptosis). Strikingly, carcinomas formed by transformed BMK cells in which apoptosis was blocked by aberrant BCL-2 family protein function displayed prevalent, highly polyploid, tumor giant cells. Examination of the transformed BMK cells in vivo revealed aberrant metaphases and ploidy changes in tumors as early as 9 d after implantation, which progressed in magnitude during the tumorigenic process. An in vitro ischemia system mimicked the tumor microenvironment, and gain of BCL-2 or loss of BAX and BAK was sufficient to confer resistance to apoptosis and to allow for accumulation of polyploid cells in vitro. These data suggest that in vivo, even in cells in which p53 function is compromised, apoptosis is an essential response to hypoxia and ischemia in the tumor microenvironment and that abrogation of this response allows the survival of cells with abnormal genomes and promotes tumorigenesis.

Animals↗
PubMed2004

Drug targeting of HIV-1 RNA.DNA hybrid structures: thermodynamics of recognition and impact on reverse transcriptase-mediated ribonuclease H activity and viral replication.

RNA degradation via the ribonuclease H (RNase H) activity of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) is a critical component of the reverse transcription process. In this connection, mutations of RT that inactivate RNase H activity result in noninfectious virus particles. Thus, interfering with the RNase H activity of RT represents a potential vehicle for the inhibition of HIV-1 replication. Here, we demonstrate an approach for inhibiting the RNase H activity of HIV-1 RT by targeting its RNA.DNA hybrid substrates. Specifically, we show that the binding of the 4,5-disubstituted 2-deoxystreptamine aminoglycosides, neomycin, paromomycin, and ribostamycin, to two different chimeric RNA-DNA duplexes, which mimic two distinct intermediates in the reverse transcription process, inhibits specific RT-mediated RNase H cleavage, with this inhibition being competitive in nature. UV melting and isothermal titration calorimetry studies reveal a correlation between the relative binding affinities of the three drugs for each of the chimeric RNA-DNA host duplexes and the relative extents to which the drugs inhibit RT-mediated RNase H cleavage of the duplexes. Significantly, this correlation also extends to the relative efficacies with which the drugs inhibit HIV-1 replication. In the aggregate, our results highlight a potential strategy for AIDS chemotherapy that should not be compromised by the unusual genetic diversity of HIV-1.

Aminoglycosides↗
PubMed2004

NF-kappaB activation in human prostate cancer: important mediator or epiphenomenon?

The NF-kappaB family of transcription factors has been shown to be constitutively activated in various human malignancies, including leukemias, lymphomas, and a number of solid tumors. NF-kappaB is hypothesized to contribute to development and/or progression of malignancy by regulating the expression of genes involved in cell growth and proliferation, anti-apoptosis, angiogenesis, and metastasis. Prostate cancer cells have been reported to have constitutive NF-kappaB activity due to increased activity of the IkappaB kinase complex. Furthermore, an inverse correlation between androgen receptor (AR) status and NF-kappaB activity was observed in prostate cancer cell lines. NF-kappaB may promote cell growth and proliferation in prostate cancer cells by regulating expression of genes such as c-myc, cyclin D1, and IL-6. NF-kappaB may also inhibit apoptosis in prostate cancer cells through activation of expression of anti-apoptotic genes, such as Bcl-2, although pro-apoptotic activity of NF-kappaB has also been reported. NF-kappaB-mediated expression of genes involved in angiogenesis (IL-8, VEGF), and invasion and metastasis (MMP9, uPA, uPA receptor) may further contribute to the progression of prostate cancer. Constitutive NF-kappaB activity has also been demonstrated in primary prostate cancer tissue samples and suggested to have prognostic importance for a subset of primary tumors. The limited number of samples analyzed in those studies and the relative lack of NF-kappaB target genes identified in RNA expression microarray analyses of prostate cancer cells suggest that further studies will be required in order to determine if NF-kappaB actually plays a role in human prostate cancer development, and/or progression, and to characterize its potential as a therapeutic target.

Cell Division↗
PubMed2002

Ah receptor and NF-kappaB interactions: mechanisms and physiological implications.

The aryl hydrocarbon (Ah) receptor mediates most of the toxic effects induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds, which are ubiquitous environmental contaminants causing toxic responses in human and wildlife. Nuclear factor kappa B (NF-kappaB) is a pleiotropic transcription factor that plays a pivotal role in a wide array of physiological and pathological responses including immune modulation, inflammatory responses and apoptosis. Many physiological functions adversely affected by TCDD are also known to be regulated by NF-kappaB, such as immune activation, maintenance of skin differentiation, control of cell proliferation and survival, as well as induction of xenobiotic metabolizing enzymes. In the past few years, evidence has emerged to show that the Ah receptor and NF-kappaB interact and transcriptionally modulate each other. This review discusses Ah receptor-NF-kappaB interactions and examines potential mechanistic explanations for toxic responses as a result of TCDD exposure and the suppression of cytochrome P450 1A1/1A2 by stress stimuli such as inflammation and infection.

Animals↗
PubMed2002

Mechanisms of constitutive NF-kappaB activation in human prostate cancer cells.

BACKGROUND: Activation of the NF-kappaB transcription factor has been previously demonstrated in two androgen receptor negative prostate cancer cell lines. We wished to extend this work to additional prostate cancer cells and to characterize the mechanisms responsible for constitutive NF-kappaB activation. METHODS: Electrophoretic mobility shift assays were performed to measure NF-kappaB DNA-binding activity in prostate cancer cell lines, and immunohistochemistry was performed to detect nuclear localization of NF-kappaB in prostate cancer tissues. Western blot analysis was used to study the status of IkappaBalpha. Transient transfection assays were employed to characterize the contributions of IkappaB kinase (IKK), MAPK kinase kinases (MAPKKKs), androgen receptor (AR), and tyrosine phosphorylation to the constitutive activation of NF-kappaB in the prostate cancer cell lines. RESULTS: Constitutive NF-kappaB activity was observed in AR-negative cell lines as well as in the prostate cancer patient samples, but was not present in AR positive cells. A "super-repressor" IkappaBalpha, as well as dominant negative forms of IKKbeta and NF-kappaB-inducing kinase (NIK), and tyrosine kinase inhibition were able to suppress NF-kappaB activity in the cells with constitutive activation. CONCLUSIONS: The constitutive activation of NF-kappaB observed in prostate cancer cells is likely due to a signal transduction pathway involving tyrosine kinases, NIK, and IKK activation.

DNA-Binding Proteins↗
PubMed2002

Adenovirus infection of primary malignant lymphoid cells.

Adenovirus infection represents a cellular stress that induces host cell pro-apoptotic responses. To overcome this barrier to productive infection, viral polypeptides modulate a variety of host cell pathways. The interface of these early viral gene products with key cellular regulatory proteins has provided considerable information concerning basic cellular mechanisms operative in cell cycle regulation, transcriptional control and apoptosis. The overlap of these mechanisms with those impacted during oncogenesis provides the opportunity to use adenoviruses and adenovirus mutants to characterize the state of key regulatory pathways in specific malignant cells. For example, adenoviruses mediate cytotoxicity after infection of chronic lymphocytic leukemia (CLL) cells, mantle cell lymphoma (MCL) cells and multiple myeloma cell lines. Specific adenovirus mutants demonstrate enhanced cytotoxicity and, in many cases, apoptosis is not the primary mechanism of cell death. Analysis of these infections with respect to both the features of the primary malignant cell and the mechanisms of adenovirus-mediated cytotoxicity holds the prospect of providing novel insights into the status of key regulatory pathways in individual patient malignant cells. These studies also hold the prospect of supporting the development of specific attenuated adenoviruses as therapeutic agents with selective cytotoxicity for specific primary lymphoid malignancies.

Adenoviridae↗
PubMed2002

Administration of a phorbol ester to patients with hematological malignancies: preliminary results from a phase I clinical trial of 12-O-tetradecanoylphorbol-13-acetate.

PURPOSE: Phorbol esters are capable of inducing a broad range of cellular effects,including the maturation/differentiation of hematopoietic cell lines (E. Huberman and M. F. Callaham, Proc. Natl. Acad. Sci. USA, 76: 1293-1297, 1979; J. Lotem and L. Sachs, Proc. Natl. Acad. Sci. USA, 76: 5158-5162, 1979; G. Rovera et al., Proc. Natl. Acad. Sci. USA, 76: 2779-2783, 1979; H. P. Koeffler, J. Clin. Investig., 66: 1101-1108, 1980). The ability to induce this differentiation at very low concentrations stimulated investigators to administer a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), to patients with myeloid leukemias in the People's Republic of China (Z. T. Han et al., Proc. Natl. Acad. Sci. USA, 95: 5357-5361, 1998). The tolerability of this therapy in China prompted Phase I studies of TPA in the United States. The purpose of this report is to demonstrate the tolerance of TPA at doses that result in detectable biological activity in blood and malignant cells. EXPERIMENTAL DESIGN: TPA was administered to patients with relapsed/refractory hematological malignancies. RESULTS: Phenotypic effects were detected in malignant cells and TPA-associated biological activity was present in blood for up to several hours after the infusion. CONCLUSIONS: These studies confirm the feasibility of TPA administration to humans and establish the foundation for the development of phorbol esters as therapy for patients with a variety of malignant and nonmalignant disorders.

Adult↗
PubMedDate not supplied

Synthesis of poly(ethylene glycol)-based saquinavir prodrug conjugates and assessment of release and anti-HIV-1 bioactivity using a novel protease inhibition assay.

Various poly(ethylene glycol)(PEG)-based prodrug conjugates of the HIV-1 protease inhibitor (PI) saquinavir (SQV) were prepared using several types of chemical groups potentially capable of modifying its pharmacokinetic properties. These prodrug conjugates included SQV-cysteine-PEG3400, SQV-cysteine-PEG3400-biotin, SQV-cysteine(R.I.CK-Tat9) [a cationic retro-inverso-cysteine-lysine-Tat nonapeptide]-PEG3400, and SQV-cysteine(R.I.CK(stearate)-Tat9)-PEG3400. SQV was linked to cysteine to form a releasable SQV-cysteine ester bond in all of the conjugates. The amino group of the cysteine moiety provided an attachment site for a slower-degrading amide bond with N-hydroxysuccinimide-activated forms of PEG- and PEG-biotin. Disulfide bonds were used to attach the cationic peptides, R.I.CK-Tat9 and R.I.CK(stearate)-Tat9 to the cysteine moiety in order to provide cell-specific release. An assay was established and validated for measuring the activity of SQV and other protease inhibitors in biological samples. In this assay, cleavage of an internally quenched fluorescent substrate, Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gly-Lys(DABCYL)-Arg by HIV-1 protease was inhibited by SQV in a dose-dependent manner at concentrations of 0.05-0.5 microM. All prodrug conjugates were shown to be inactive in this assay until the ester bond was cleaved and active SQV was released. The prodrug reconversion half-lives in 0.1 N HCl, phosphate-buffered saline (PBS) at pH 7.4 and in spiked plasma at 37 degrees C were 9, 14, and 0.9 h, respectively. The anti-HIV-1 activity (ED(50)) of the PEG-based SQV prodrug conjugates was evaluated in MT-2 cells using an MTT assay. The activity of conjugated SQV was reduced (ED(50) = 900 nM) for the PEG only conjugate, but restored with the addition of biotin (ED(50) = 125 nM), R.I.CK-Tat9 (ED(50) = 15 nM), and R.I.CK(stearate)-Tat9 (ED(50) = 62 nM) as compared to maximum achievable anti-HIV-1 activity (unconjugated SQV, control, ED(50) = 15 nM), suggesting enhanced cellular uptake of conjugates. Cytotoxicity (LD(50)) was assessed for all prodrug conjugates using non-HIV-1 infected cells and was found to be in the micromolar range. The difference between the LD(50) and ED(50) suggests a favorable therapeutic index for the prodrug conjugates. In conclusion, these promising initial results demonstrate that the reconversion of the conjugate prodrugs was complete and that active SQV was released. Since the major delivery advantages of PEG prodrug conjugates can only be observed in vivo, issues of reconversion and elimination half-lives in plasma will have to be further studied in an in vivo model. The current results also demonstrate that the protease inhibition assay is a simple yet effective bioanalytical tool that can be used to assess the release and anti-HIV-1 activity of HIV-1 PIs from their prodrug forms.

Anti-HIV Agents↗