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VIR-576 (Active substance against HIV infection), New HIV Entry Inhibitor Against gp41 Fusion Peptide - VIRO Pharmaceuticals GmbH & Co. KG
 
 
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A new kind of experimental HIV medicine can halt one of the earliest stages of HIV infection and may eventually lead to a novel class of drugs to fight other dangerous viruses, German scientists said on Wednesday. "What the virus does is a bit like throwing an anchor to get hooked up to the cell," Frank Kirchhoff, of the University Hospital of Ulm in Germany, said in a telephone interview. "This drug occupies the anchor - which is called the fusion peptide - and prevents its insertion into the cell membrane. So then the virus cannot get into the cell."
 
Kirchhoff said VIR-576 is similar to other fusion inhibitors such as Fuzeon, sold by Trimeris and Roche, but is designed block the infection process at an earlier stage.VIR-576 is the main product of VIRO Pharmaceuticals GmbH & Co. KG. The company was founded in 2005 and develops antiviral peptides. VIR-576 prevents the HIV virus from entering into the immune cells. The results showed that at the highest dose of 5 grams per day there was a 95 percent reduction in the patients' viral load - a measurement of the amount of HIV in the blood. Side effects were minimal and mainly involved soreness around the injection sites. After completion of the preclinical work, a clinical Phase I/II study is currently being performed with the aim of proving the effects of VIR-576 (reducing the viral load) and its safety and tolerability.
 
The researchers said the results were encouraging but stressed that the experimental drug has some drawbacks which would need further work. Because VIR-576 is a peptide and must be given through injections, it will be costly and inconvenient to use, they said, and the high dose of 5 grams a day would also make it relatively expensive. The team's focus now is to hunt for a small molecule that works just like just like VIR-576 but that could be made cheaply and given orally in the form of a pill. the discovery that VIR-576 can fight early HIV infection suggests it may be possible in future to develop similar blockers for these other viruses.
 
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Short-Term Monotherapy in HIV-Infected Patients with a Virus Entry Inhibitor Against the gp41 Fusion Peptide - pdf attached
 
Sci Transl Med 22 December 2010:
Vol. 2, Issue 63, p. 63re3
 
Wolf-Georg Forssmann,1,2,3 Yu-Han The,1 Matthias Stoll,1 Knut Adermann,1,2,3 Uwe Albrecht,4 Kleomenis Barlos,5,6 Annette Busmann,1,2 Angeles Canales-Mayordomo,7 Guillermo Gimenez-Gallego,7 Jochen Hirsch,1,2,3 Jesus Jimenez-Barbero,7 Dirk Meyer-Olson,1 Jan Munch,8 Javier Perez-Castells,9 Ludger Standker,1 Frank Kirchhoff,8* Reinhold E. Schmidt1 1Department of Immunology and Rheumatology, Hannover Medical University, Carl- Neuberg-Strasse 1, D-30625 Hannover, Germany. 2VIRO Pharmaceuticals GmbH & Co. KG, Feodor-Lynen-Strasse4 31, D-30625 Hannover, Germany. 3Pharis Biotec GmbH, 30625 Hannover,5Germany. Mediconomics GmbH, Misburger-Strasse 81b, D-30625 Hannover, Germany. DepartmentofChemistry,UniversityofPatras,GR-26500Rion-Patras,Greece. 6CBL-Patras, 26333 Patras, Greece. 7Departamento de Biologia Fisico-Quimica, Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas, E-28040 Madrid, Spain. 8Institute of Molecular Virology, University Hospital of Ulm, Meyerhofstrasse 1, D-89081 Ulm, Germany. 9Departamento de Quimica, Facultad de Farmacia, Universidad San Pablo CEU, E-28664 Madrid, Spain. *To whom correspondence should be addressed. E-mail: frank.kirchhoff@uni-ulm.de
 
Abstract
 
To infect host cells, most enveloped viruses must insert a hydrophobic fusion peptide into the host cell membrane. Thus, fusion peptides may be valuable targets for developing drugs that block virus entry. We have shown previously that a natural 20-residue fragment of α1-antitrypsin, designated VIRus-Inhibitory Peptide (VIRIP), that binds to the gp41 fusion peptide of HIV-1 prevents the virus from entering target cells in vitro. Here, we examine the efficacy of 10-day monotherapy with the optimized VIR-576 derivative of VIRIP in treatment-naive, HIV-1-infected individuals with viral RNA loads of ≥10,000 copies per ml. We report that at the highest dose (5.0 grams per day), intravenous infusion of VIR-576 reduced the mean plasma viral load by 1.23 log10 copies per ml without causing severe adverse effects. Our results are proof of concept that fusion peptide inhibitors suppress viral replication in human patients, and offer prospects for the development of a new class of drugs that prevent virus particles from anchoring to and infecting host cells.
 
"All patients in the high-dose group responded to VIR-576 treatment, albeit to various extents ranging from 0.30 to 1.90 log10 reduction in the plasma viral load.....we observed a significant correlation between the reduction in the viral load and the plasma concentrations of VIR-576.....VIR-576 was well tolerated and did not cause major adverse effects......we are currently working on the development of small-molecule inhibitors with an analogous mode of action that can be administered orally......VIRIP-related compounds do not show cross-resistance with other antiretroviral drugs including fusion inhibitors such as T-20, and the gp41 fusion peptide is highly conserved and essential for HIV-1 entry. Therefore, further clinical development of fusion peptide inhibitors seems warranted and may provide a useful addition to the current armamentarium of antiretroviral drugs, particularly for salvage therapy of individuals infected with multiresistant HIV-1 strains and for the risk reduction of transmission between an HIV-infected mother and her fetus during birth."
 
INTRODUCTION
 
Blocking the ability of HIV-1 to enter host cells has significant advantages for therapeutic intervention compared to targeting later steps in the virus life cycle, such as reverse transcription, integration, or maturation of virions (1, 2). First, the virus can be blocked without the inhibitor having to enter the host cells, thus reducing the risk of undesired side effects. Second, unlike protease inhibitors, HIV-1 entry inhibitors block the virus before the integration of the provirus into the host cell genome and hence prevent the establishment and maintenance of latent viral reservoirs. Finally, HIV-1 entry involves several distinct steps that can be blocked by different classes of antiretroviral agents that do not show cross-resistance (1, 2). Despite these advantages, only two HIV-1 entry inhibitors have currently been approved for clinical use. One of them, maraviroc (Celsentri), binds to the HIV-1 co-receptor CCR5 (R5) and blocks infection by R5-tropic HIV-1 strains (3, 4) (Fig. 1A). The second, T-20 enfuvirtide (Fuzeon), is derived from the HIV-1 glycoprotein transmembrane (gp41) C-terminal heptad repeat region. It prevents the formation of the six-helix bundle between the gp41 N-terminal and C-terminal heptad repeat regions required for fusion of viral and target cell membranes (5) (Fig. 1A).
 
Although maraviroc and T-20 are successfully used in the clinic, both have significant drawbacks. For example, maraviroc is not active against HIV-1 strains that use the second major co-receptor (CXCR4) for host cell entry, and T-20 needs to be administered by subcutaneous injection and is frequently associated with the rapid emergence of drug-resistant virus variants (6, 7). T-20-related HIV-1 fusion inhibitors, such as T-1249, with improved resistance profiles have been developed (8), but their further clinical development has been halted because of problems with formulation, production costs, and lack of oral bioavailability.
 
By screening a complex blood-derived peptide/protein library, we have previously identified a 20-amino acid residue peptide [VIRus-Inhibitory Peptide (VIRIP)] corresponding to the C-proximal region of α1-antitrypsin (the most abundant circulating serine protease inhibitor) that naturally blocks HIV-1 infection (9). Structural and functional analyses showed that VIRIP specifically targets the fusion peptide of HIV-1 gp41 and blocks viral entry by preventing insertion of the fusion peptide into the target cell membrane (9). Thus, VIRIP can be considered a natural "anchoring" inhibitor. Recently, it has been shown that VIRIP is generated from α1-antitrypsin by matrix metalloproteinases and can be detected before activation of most other immune responses in some individuals with acute HIV-1 infection (10). Thus, this natural inhibitor of the gp41 fusion peptide of HIV-1 may constitute part of the earliest systemic antiviral host response induced after virus infection.
 
The HIV-1 gp41 fusion peptide is required for HIV-1 infection, is well conserved, and can hardly tolerate alterations without losing function (9). Thus, the gp41 fusion peptide is a promising target for antiretroviral therapy, and several properties make VIRIP interesting for clinical development: (i) Its mode of action and viral target are different from that of all other HIV-1 inhibitors. (ii) It is active against a broad range of HIV-1 strains, including those resistant to current antiretroviral drugs. (iii) Compared to other inhibitors, it is difficult to select resistant HIV-1 variants in cell culture (9). (iv) It is nontoxic even at exceedingly high concentrations in vitro and in vivo in various animal species, and (v) its antiviral potency in vitro could be increased by more than two orders of magnitude by introduction of a few specific amino acid changes (9). Here, we evaluated the safety, pharmacokinetics, and antiviral efficacy of an optimized derivative of VIRIP (VIR-576) in a Phase I/II clinical trial in 18 patients. We show that short-term monotherapy with VIR-576 is well tolerated by treatment-naive HIV-1 patients and reduces the plasma viral load by more than one order of magnitude.
 
DISCUSSION
 
We show that the optimized VIR-576 derivative of a naturally occurring HIV-1 entry inhibitor reduces the viral load during short-term monotherapy by 1.2 orders of magnitude. This reduction is similar to those previously observed in short-term monotherapy with the CCR5 antagonist maraviroc (-1.6 log10), T-20 (-1.96 log10), the second-generation fusion inhibitor T-1249 (-1.26 log10), reverse transcriptase inhibitors [for example, cidovudine (-0.52 log10), lamivudine (-1.19 log10), and efavirenz (-1.68 log10)], and the protease inhibitor ritonavir (-1.38 log10) (8, 12-16). The high antiviral efficiency of VIR-576 in vivo is surprising because the most plausible "sticky finger" model of HIV-1 entry suggests that its viral gp41 fusion peptide shoots like a "harpoon" into the lipid membrane of the host cell and is only transiently exposed and accessible for inhibition (17). Our data demonstrate that inhibition of the viral fusion peptide in vivo in HIV-infected humans is feasible.
 
A number of natural host factors have been implicated in the inhibition of HIV-1 replication in HIV-1-infected individuals (18), such as various chemokines, cytokines, and defensins. The discovery that chemokines bind to cofactors of HIV-1 entry and block viral replication has facilitated the development of new antiretroviral agents that target various steps in the viral entry process (1, 2). Modified forms of the chemokine RANTES are highly potent inhibitors of host cell entry in vitro by HIV strains that require the CCR5 co-receptor (19). Despite their high in vitro potency, however, they were never successfully tested in clinical trials. Here, we show that VIR-576, a derivative of a factor produced naturally in the human body, has antiretroviral activity in a small Phase I/II clinical trial. The fact that VIR-576 is less effective in vivo than in vitro is not surprising, because many factors, such as proteolytic degradation, tissue distribution, and absorption to the cell surface, extracellular matrix, or serum components, may all affect its antiviral potency in HIV-infected individuals. VIR-576 has some advantages over other virus entry inhibitors such as maraviroc and T-20. For example, VIR-576 is substantially smaller than T-20 (20 instead of 36 amino acids) and thus easier and cheaper to produce. Furthermore, it seems to be more difficult for HIV-1 to become resistant to VIR-576 than to T-20. It is well established that single-amino acid changes in the heptad repeat region of gp41 targeted by T-20 can easily be selected in cell culture and may rapidly emerge in T-20-treated patients, rendering the virus resistant to this fusion inhibitor (6, 7). In comparison, the selection of VIR-576-resistant HIV-1 variants (at least in cell culture) has proven difficult because most mutations in the gp41 fusion peptide inactivate the virus and a combination of changes is required to reduce viral susceptibility to inhibition by VIR-576 (9). Finally, although further long-term safety studies are required, our preliminary data suggest that VIR-576 is well tolerated in HIV-infected patients, possibly because it is derived from a natural peptide and does not need to enter cells to block HIV-1 infection.
 
Despite its efficacy in vivo, however, treatment with VIR-576 has drawbacks; in particular, it requires high treatment doses and intravenous injection or infusion. Furthermore, the large-scale production of peptides under GMP conditions is still relatively expensive (T-20 therapy costs an estimated $25,000 per year compared to about $14,000 for maraviroc, a small-molecule inhibitor). Although the smaller size of VIR-576 will allow some reduction in the cost of generating highly pure peptide preparations for clinical use, this advantage is counterbalanced by the higher treatment dose of VIR-576 (5.0 g/day) compared to T-20 (usually 0.18 g/day). To overcome these drawbacks in costs and administration, we are currently working on the development of small-molecule inhibitors with an analogous mode of action that can be administered orally. VIRIP-related compounds do not show cross-resistance with other antiretroviral drugs including fusion inhibitors such as T-20, and the gp41 fusion peptide is highly conserved and essential for HIV-1 entry. Therefore, further clinical development of fusion peptide inhibitors seems warranted and may provide a useful addition to the current armamentarium of antiretroviral drugs, particularly for salvage therapy of individuals infected with multiresistant HIV-1 strains and for the risk reduction of transmission between an HIV-infected mother and her fetus during birth.
 
We have shown previously that it is difficult to generate VIRIP-resistant HIV-1 variants in vitro and that changes in the gp41 fusion peptide region either are not tolerated or do not confer resistance to VIR-576 (9). In agreement with these results, we found that patient-specific variations in the gp41 fusion peptide sequences did not significantly reduce the susceptibility of HIV-1 to inhibition by VIR-576. Furthermore, all patients in the high-dose group responded to treatment with VIR-576, albeit to different extents. The plasma concentrations of VIR-576 in the two poor responders were only slightly lower compared to those in the remaining four patients of the high treatment group, arguing against a causal role of reduced bioavailability of the inhibitor in the limited reduction of plasma viral load. Because none of the variations in the gp41 fusion peptide region detected in the patients conferred resistance to VIR-576, it is tempting to speculate that different fusion kinetics of the viral Env proteins (20) may affect HIV-1 sensitivity to VIR-576 and play a role in the antiviral efficacy of this inhibitor in vivo. Further analyses are necessary to elucidate the mechanisms governing the susceptibility of HIV-1 to VIR-576 and to clarify whether VIRIP derivatives with further increased affinity for the gp41 viral fusion peptide may be more potent in blocking HIV-1 entry and replication in infected humans.
 
Our results provide clinical evidence that viral fusion peptides are suitable targets for developing antiviral treatments. Fusion peptides are essential for host cell entry by most enveloped viruses, including many human pathogens, such as the influenza, mumps, measles, hepatitis B and C, and SARS (severe acute respiratory syndrome) viruses (21). For many of these viral pathogens, there is no specific therapy available. Recent data show that antibodies against viral fusion peptides block infection by influenza (orthomyxovirus) and Junin (arenavirus) viruses in cultured cells and in mice, respectively (22, 23). It may be possible to develop fusion peptide inhibitors against other enveloped viruses that are dangerous human pathogens.
 
RESULTS
 
Development of an optimized VIRIP derivative for clinical evaluation

 
In our previous study (9), we have shown that VIRIP blocks a step in HIV-1 entry that is distinct from those targeted by other virus entry inhibitors (Fig. 1A). To identify variants of the natural VIRIP peptide with improved therapeutic potential, we performed systematic structure-activity relationship studies and analyzed ∼600 chemically synthesized derivatives. One dimeric variant (VIR-576) that differs from the native form of VIRIP by four-amino acid changes (Fig. 1B) blocked HIV-1 infection with enhanced potency of about two orders of magnitude (9), and showed suitable properties in pharmacological and toxicological studies (table S1 and Supplementary Material), and thus was selected for clinical development. To gain insight into the interaction between VIR-576 and its viral target, we characterized the structural features of VIR-576 in its free form and also in a complex with the gp41 fusion peptide (FP1-23) by nuclear magnetic resonance (NMR) spectroscopy. The NMR spectra of VIR-576 corresponded to that of a monomer, both in the presence and in the absence of FP1-23 (see Supplementary Material). However, matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy carried out after NMR data acquisition confirmed that VIR-576 remained a dimer. These results show that both subunits of the VIR-576 dimer behave independently. Our NMR-based structural analyses demonstrate that the average backbone structures of bound and free VIR-576 are remarkably similar (figs. S1 to S3 and table S2). Thus, the entropy component of its binding to FP1-23 should be negligible. A model of the three-dimensional structure of VIR-576 in a complex with FP1-23 computed on the basis of the NMR data (described in the Supplementary Material) is illustrated in Fig. 1C. This model is compatible with a dimeric structure of VIR-576 (Fig. 1C). The three-dimensional structure of the VIR-576-FP1-23 complex suggests that a low entropy cost of the binding process, along with a large complementary interaction surface between the two polypeptides (Fig. 1C), and its dimerization contribute to the increased antiviral potency of VIR-576 compared to the previously examined derivative VIR-165 and the parent peptide VIRIP (9). For clinical studies, VIR-576 was produced under Good Manufacturing Practice (GMP) conditions. The GMP conformation of VIR-576 inhibited both X4- and R5-tropic HIV-1 strains in vitro with an IC50 (median inhibitory concentration) of ∼10 to 50 nM (examples shown in Fig. 1D).
 
VIR-576 inhibits HIV-1 replication in infected individuals
 
To examine the antiviral efficacy of VIR-576 in humans, we performed a short-term dose-escalation Phase I/II study in 18 treatment-naive, clinically asymptomatic HIV-1-infected individuals who had plasma HIV-1 RNA levels of ≥10,000 copies per ml and a CD4+ T cell count of ≥350 cells/µl. Patients were randomly assigned to three treatment groups, receiving continuous intravenous infusion of escalating quantities of VIR-576 (0.5, 1.5, and 5 g/day) over a 10-day period. Baseline virological, immunological, and disease characteristics were similar in all three treatment groups, with mean viral loads of 4.82 ± 0.49 (range, 4.00 to 5.62) log10 copies/ml (Table 1). VIR-576 was readily detectable in the patient plasma (Fig. 2A), and the average viral load declined by 0.061 log10 copies/ml under the low-dose treatment, 0.303 log10 copies/ml under the medium-dose treatment, and 1.233 log10 copies/ml under the high-dose treatment (Fig. 2B). Notably, we observed a significant correlation between the reduction in the viral load and the plasma concentrations of VIR-576 (Fig. 2C). After the termination of treatment, the viral load rebounded to baseline levels within 1 to 2 weeks. As expected for such a short treatment period, no major changes in the levels of CD4+ T cells were observed (fig. S4).
 
Patient-specific sequence variations in the gp41 fusion peptide do not confer resistance to VIR-576
 
All patients in the high-dose group responded to VIR-576 treatment, albeit to various extents ranging from 0.30 to 1.90 log10 reduction in the plasma viral load. Sequence analyses of Env gene reverse transcription polymerase chain reaction (RT-PCR) fragments amplified from patient plasma samples showed that the gp41 fusion peptide domains of the HIV-1 strains present in the two individuals who responded most poorly to VIR-576 contained either an unusual insertion (patient 23) or five-amino acid changes (patient 18) compared to the gp41 fusion peptide sequence of the well-characterized HIV-1 NL4-3 clone (Fig. 3A). To examine whether these sequence variations affected susceptibility of the virus to VIR-576, we introduced them into the gp41 fusion peptide of the X4-tropic HIV-1 NL4-3 molecular clone and into a CCR5-tropic strain containing the V3 loop region of the HIV-1 92TH014 strain (11). Virus stocks were generated by transfection of 293T cells and quantified by p24 enzyme-linked immunosorbent assay (ELISA) and infectivity assays. All HIV-1 fusion peptide variants showed comparable infectivity in TZM-bl indicator cells (fig. S5). We found that none of the patient-specific sequence variations in the gp41 fusion peptide region significantly reduced the sensitivity of HIV-1 to inhibition by VIR-576 (Fig. 3B). This result is not surprising because most amino acid substitutions in the gp41 fusion peptide are conservative or located at positions not involved in the interaction with the VIR-576 peptide, suggesting that differences in the viral fusion kinetics rather than changes in the primary drug interaction site are responsible for the differential responses of HIV-1-infected patients to treatment.
 
VIR-576 is well tolerated by HIV-1-infected patients
 
All patients were carefully monitored for adverse events, and comprehensive laboratory testing was performed to ensure the safety of the treatment throughout the investigation period. These investigations showed that two study participants from the low- and middle-dose treatment groups suffered from moderate allergic reactions, such as exanthemas (acute rashes), which appeared at days 9 and 11, respectively. When both patients discontinued treatment, the allergic reactions rapidly disappeared. Other adverse effects were generally transient and mild to moderate and included constipation, headache, and fever (Table 2). With the exception of hyper bilirubinemia found in two patients (Table 2), the side effects were less severe in the high-dose treatment group and thus were not related to the dosage of VIR-576. Together, VIR-576 was well tolerated and did not cause major adverse effects.
 
 
 
 
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