In situ forming implant for controlled delivery of an anti-HIV fusion inhibitor.

An injectable, phase sensitive, in situ forming, implantable delivery system was developed for enfuvirtide, a therapeutic peptide used in the treatment of HIV infection. The development studies were carried out using poly (D,L-lactide-co-glycolide), a smart, biodegradable polymer. Different formulations were designed, prepared and evaluated by employing response surface, optimal design of experiment technique. The optimized formulation was identified and validated for its performance by using numerical optimization technique. The in vitro evaluation parameters included rheology, compatibility studies, drug release as well as conformational and physicochemical stability studies. In vivo pharmacokinetic parameters and biocompatibility studies were determined in rat models and were statistically analyzed. It was found that the optimized formulation extended the enfuvirtide release and maintained the drug plasma concentration within therapeutically effective range up to 48 h. The optimized formulation maintained physicochemical and conformational stability for at least 6 months and was biocompatible with the animal tissue.

Safety, tolerability, and plasma pharmacokinetics of high-strength formulations of enfuvirtide (T-20) in treatment-experienced HIV-1-infected patients.

BACKGROUND: Enfuvirtide, a HIV-1 membrane fusion inhibitor, is the first viral entry inhibitor approved for the treatment of HIV-1 infected patients in the USA. Parenteral administration of enfuvirtide in clinical trials has been safe and has resulted in significant decreases in plasma viral load, even in the setting of extensive previous treatment and multi-drug resistance to conventional antiretroviral (ARV) therapy. Previous formulations have required two injections administered twice-daily (BID). OBJECTIVES: The primary objectives of this study were to evaluate the safety, tolerability, and pharmacokinetics of two high-strength 100 mg/ml formulations of enfuvirtide (carbonate [CO(3)] and tromethamine [TRIS] buffer) and of the current formulation (50 mg/ml CO(3) formulation) at doses of 90 mg (deliverable) BID and 67.5 mg (deliverable) BID in treatment-experienced patients. STUDY DESIGN: This was a phase II, multi-center, open-label, sequential cross-over pharmacokinetic, efficacy, and safety study. Study design included two treatment variables; dose (90 mg or 67.5 mg BID) and formulation (A: 50 mg/ml CO(3), B: 100 mg/ml CO(3) or C; 100 mg/ml TRIS). RESULTS: Forty-six treatment-experienced participants were sequentially enrolled into three treatment cohorts. All cohorts had similar safety profiles and only one patient discontinued due to an adverse event. Pharmacokinetic data indicated that the high-strength 100 mg/ml CO(3) formulation was bioequivalent to the 50 mg/ml CO(3) formulation whereas the TRIS formulation was not. At 48 weeks, 59.1%, 66.7% and 16.7% had <400 copies per milliliter HIV-1 RNA in the 90 MgCO(3), 67.5 MgCO(3) and 90 mg TRIS cohorts with median suppression of HIV-1 RNA of 2.97, 3.48, and 0.87 log(10)copies per milliliter, respectively. CONCLUSIONS: Based upon bioequivalence data and the convenience and similarity in safety and virological effect with the 50 mg/ml formulation, the 100 mg/ml CO(3) formulation was selected for use in clinical efficacy studies of enfuvirtide.

Selective toxicity of engineered lentivirus lytic peptides in a CF airway cell model.

Lentivirus lytic peptides (LLPs) are derived from HIV-1 and have antibacterial properties. LLP derivatives (eLLPs) were engineered for greater potency against Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA). Minimum bactericidal concentration (MBC) was determined in low and physiologic salt concentrations. MBC was decreased against SA and equivalent against PA in physiologic salt when compared to the parent compound LLP1. In a novel cystic fibrosis (CF) airway cell model, one derivative, WLSA5, reduced the number of adherent PA and only moderately affected CF cell viability. Overall, eLLPs are selectively toxic to bacteria and may be useful against CF airway infections.

Protective effect of glutathione in HIV-1 lytic peptide 1-induced cell death in human neuronal cells.

To elucidate the pathogenic mechanisms involved in neurodegeneration in AIDS patients with cognitive deficits, we have examined the toxic effect of the lentivirus lytic peptide 1 (LLP-1) corresponding to the carboxyl terminus of HIV-1 transmembrane glycoprotein gp41 on human neuronal and glial cell lines. LLP-1 induced a significant lactate dehydrogenase (LDH, a marker of cell death) release from these cells in a concentration- and time-dependent manner, while the noncytolytic LLP-1 analog 2 had little effect. Application of LLP-1 to SH-SY5Y, a well-characterized human neuronal cell line, caused the decline of intracellular glutathione (GSH) content that appeared to occur before a significant LDH release. Furthermore, LLP-1 elicited a significant loss of mitochondrial function as measured by mitochondrial transmembrane potential (MTP). Among the reducing agents and antioxidants tested, GSH and a GSH prodrug N-acetylcysteine (NAC) provided protection against LLP-1-induced neuronal cell death, evidently by restoring the intracellular GSH levels and blocking the disruption of mitochondrial integrity. Thus, gp41-derived LLP-1 may be a potential neurotoxic agent capable of causing the intracellular GSH depletion and disturbing the mitochondrial function, possibly contributing to the neurodegenerative cascade as seen in HIV-1-associated dementia. Our data indicate that restoring both GSH concentration and mitochondrial function may hold promise as possible therapeutic strategies for slowing disease progression of dementia in AIDS patients.

Direct role of plasma membrane-expressed gp120/41 in toxicity to human astrocytes induced by HIV-1-infected macrophages.

OBJECTIVE: To compare astrocyte toxicity induced by plasma membrane-expressed gp120/41 and soluble gp120. DESIGN: Analysis of morphological alterations and lactate dehydrogenase (LDH) release from astrocytes in culture with monocytes infected with HIV-1, microglia expressing Env of a macrophage-tropic HIV-1 isolate or soluble Env. METHODS: Primary human embryonic astrocytes were cultured with: monocytes infected with two M-tropic HIV-1 isolates (HIV-1(9533), HIV-1(BX08)); human microglia infected with the defective Semliki Forest virus (SFV) vector coding for the env gene of HIV-1(BX08) isolate (SFVenvBX08); and soluble gp140 purified from baby hamster kidney cells transfected with the env gene of HIV-1(BX08) lacking the intracytoplasmic region of gp41 (SFVdelta envBX08). Gp120 mRNA levels were assessed by quantitative reverse transcriptase-polymerase chain reaction and the protein was detected by immunofluorescence in infected monocytes or microglia. RESULTS: Contact of HIV-infected monocytes induced morphological changes in astrocytes and a 137% increase in LDH release at day 2 of co-culture compared with controls (uninfected monocytes). Gp120/41(BX08)-expressing microglia induced a 170% increase in LDH release (relative to SFVLacZ-infected microglia). Pretreatment of co-cultures with an anti-gp120 monoclonal antibody (mAb; NEA-9305) directed against the V3 loop inhibited LDH release. Soluble purified gp140 from BX08 isolate induced only a weak LDH release (104%). Finally, cytotoxicity was not blocked by treatment of the co-culture with Bordetella pertussis toxin, an inhibitor of Gi alpha protein-dependent receptors. CONCLUSION: HIV envelope glycoprotein expressed at the plasma membrane induced astrocyte damage more efficiently than its soluble counterpart. The V3 loop was involved in toxicity induction through a pathway independent of the Gi alpha protein-coupled receptor.

Mechanisms and structural determinants of HIV-1 coat protein, gp41-induced neurotoxicity.

Of the individuals with human immunodeficiency virus type 1 (HIV-1) infection, 20-30% will develop the neurological complication of HIV-associated dementia (HAD). The mechanisms underlying HAD are unknown; however, indirect immunologically mediated mechanisms are theorized to play a role. Recently, the HIV-1 coat protein gp41 has been implicated as a major mediator of HAD through induction of neurocytokines and subsequent neuronal cell death. Using primary mixed cortical cultures from neuronal nitric oxide synthase (NOS) null (nNOS-/-) mice and immunological NOS null (iNOS-/-) mice, we establish iNOS-derived NO as a major mediator of gp41 neurotoxicity. Neurotoxicity elicited by gp41 is markedly attenuated in iNOS-/- cultures compared with wild-type and nNOS-/- cultures. The NOS inhibitor L-nitroarginine methyl ester is neuroprotective in wild-type and nNOS-/- cultures, confirming the role of iNOS-derived NO in gp41 neurotoxicity. Confirming that iNOS-/- cultures lack iNOS, gp41 did not induce iNOS in iNOS-/- cultures, but it markedly induced iNOS in wild-type and nNOS-/- cultures. We elucidate the region of gp41 that is critical for iNOS induction and neuronal cell death by monitoring iNOS induction with overlapping peptides spanning gp41. We show that the N-terminal region of gp41, which we designate as the neurotoxic domain, induces iNOS protein activity and iNOS-dependent neurotoxicity at picomolar concentrations in a manner similar to recombinant gp41 protein. Our experiments suggest that gp41 is eliciting the induction of iNOS through potential cell surface receptors or binding sites because the induction of iNOS is dose dependent and saturable and occurs at physiologically relevant concentrations. These data confirm that the induction of iNOS by gp41 and the production of NO are primary mediators of neuronal damage and identify a neurotoxic domain of gp41 that may play an important role in HAD.

Alterations in cell membrane permeability by the lentivirus lytic peptide (LLP-1) of HIV-1 transmembrane protein.

Previously we reported that synthetic peptide homologs of an amphipathic region (designated the lentivirus lytic peptide, or LLP-1) near the carboxy terminus of HIV-1 transmembrane protein (TM) were toxic for both prokaryotic and eukaryotic cells when added exogenously to cell cultures. We postulated that these peptides may exert their toxic effects in much the same manner as natural cytolytic peptides such as magainins, cecropins, and melittin by forming pores through cellular membranes. Here we show the results of 51Cr-release assays and membrane flux measurements of peptide treated cells that support our hypothesis. We have also tested a limited panel of LLP-1 peptide analogs in these assays and found that relatively minor alterations in peptide charge or amphipathicity in the parent HIV LLP-1 sequence resulted in total loss of membrane perturbative properties. These results demonstrate that the peptide homolog of HIV-1 LLP-1 can indeed perturb membranes by forming pores of defined size in cytoplasmic membranes. Furthermore, the analog studies described here reveal that the amphipathy and high positive charge of this protein segment are required for the membrane perturbative properties.