HK1253011B - Combination hiv therapeutic - Google Patents

Description

Field of the Invention

The present invention relates to the field of latent viral diseases and compositions for the treatment of such diseases.

Background of the Invention

Antiretrovirus therapy (ART) is an indispensable life-saving therapy for millions of HIV+ individuals. However, the persistence of latent HIV-infected cellular reservoirs remains the last major hurdle to virus eradication. Latently infected cells represent a permanent source of potential viral reactivation. For this reason, the eradication of viral reservoirs is now the major goal for HIV-1 therapeutics (Richman et al., 2009).

MICHAEL KOVOCHICH ET AL: "Activation of Latent HIV Using Drug-Loaded Nanoparticles (PLOS ONE, PUBLIC LIBRARY OF SCIENCE, US, vol. 6, no. 4, 5 April 2011, pages 1-8) disclose a proof-of-concept study demonstrating that HIV latency activators can be packed into nanoparticles, either alone or in conjunction with an antiretroviral drug. Bryostatin-2 loaded liquid nanoparticles (LNP-Bry) produced in this way have an enhanced activity compared with drug alone, and can be further modified by adding targeting antibodies to make them more selective for CD4+ T cells.

Summary of the Invention

The present invention is directed to a medicament as defined by claim 1 for treating latent viral disease. As used herein, the term "medicament" broadly means any agent used in the treatment of a disease, such as, for example, without limitation, tablets, capsules, gelcaps, powders, patches, emulsion, suspensions and solutions which are administered orally, rectally, buccally, sublingually, subcutaneously, intramuscularly, intravenously and intraperitoneal.

One embodiment features a Bryoid selected from the group of Bryostatins consisting of Bryostatin 1-20. One embodiment features a HDAC inhibitor is selected from the group consisting of valproic acid, Vorinostat, Romidepsin and Panobinostat.

Embodiments of the present invention feature the administration of the Bryoid in an effective dose range of 10 to 100 microgram/ Kg subject every other day for up to 180 days.

Embodiments of the present invention feature the administration of the HDAC inhibitor in an effective dose range of 10 to 100 mg/Kg subject every other day for up to 180 days.

According to the invention, the HDAC inhibitor and Bryoid are carried by one or more particles. As used herein, the term "carried by" refers to any configuration in which the HDAC inhibitor and Bryoid are associated with the particle.

According to the invention, the core has a mixture of a hydrophilic material and an HDAC inhibitor, and the surrounding material has a mixture of a hydrophobic material and a Bryoid. As used herein, the term "mixture" denotes a distribution whether in solution, in suspension or as an emulsion.

One embodiment of the invention features that the one or more particles have a virus associated with the latent viral disease having one or more viral components. The one or more particles comprise one or more ligands specific for the viral component and the one or more ligands associated with the outer surface of the one or more particles. For example, without limitation, the viral components comprise protein markers specific for Human Immunodeficiency Virus (HIV) and the particle surface comprises ligand such as antibodies, aptomers and similar constructs.

One embodiment of the invention features the one or more particles having one or more upregulating ligands to upregulate CD-4 cells. The one or more upregulating ligands are associated with the surface.

One embodiment of the present invention is directed to a medicament according to any of claims 1 to 7 for use in a method of treating a latent viral disease .

A further embodiment of the present invention is directed to an article of manufacture comprising a medicament according to any of claims 1 to 7.

These and other features and advantages will be apparent to those skilled in the art upon viewing the drawings which are described in brief below and studying the Detailed Description of the Invention which follows.

Brief Description of the Drawings

• Figure 1 depicts a cross sectional view of a particle embodying features of the present invention; and
• Figure 2 depicts a schematic view of an apparatus for making the particle of the present invention.

Detailed Description of the Invention

Different investigators have suggested that reactivation of the latent reservoirs with immunoactivation therapy would allow effective targeting and possible eradication of the virus. It is thought that viral reactivation by this therapy would result in lytic cell death of CD4+ T cells because of the cytopathic effect of the virus or through recognition of infected cells by the immune system. In addition, viral reactivation in the presence of ART would prevent new infections. In this sense an Histone Deacetylase (HDAC) inhibitor, Vorinostat, induced a significant and sustained increase in HIV transcription from latency in some HIV-infected patients but failed to clear HIV-1 reservoirs. These results indicate that additional strategies will be needed to eliminate latently infected cells.

Embodiments of the present invention feature Protein Kinase C (PKC) agonists such as the non-tumorigenic Bryoids combined with HDAC inhibitors to purge latent HIV-1 from cellular reservoirs. Currently, over 22 million people have died from AIDS and there are over 42 million people living with HIV/AIDS worldwide. In the United States, an estimated 1 million people are currently living with HIV and approximately 40,000 infections occur each year. There is no vaccine against HIV and AIDS, if untreated, will lead to the death of over 95% of infected individuals 10 years post-infection. HIV infects several cell types during the course of infection and progression to acquired immune deficiency syndrome (AIDS).

The persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication with anti-retroviral therapy (ART), since latently infected cells remain a permanent source of viral reactivation. It has been hypothesized that intensification of ART could reduce the residual viremia but recent studies strongly suggest that this is not the likely scenario.

Moreover, ART is problematic because of long-term toxicity, inhibitor resistance, and the inability to target persistent reservoirs. Therefore, other pharmacological approaches targeting the HIV-1 reservoir have been suggested by several investigators as a promising strategy to develop new drugs able to activate latent HIV-1 without inducing a global T cell-activation.

HIV-1 infects several cell types during the course of infection and progression to AIDS. In the absence of ART, HIV-1 replication is active in most of the infected cells and in the majority of patients. However, HIV-1 establishes long-term infection in a small pool of memory CD4+ T cells and in other cell types, which contain integrated but transcriptionally silent HIV provirus. These latently infected cells constitute a viral reservoir in which a replication-competent form of the virus persists with more stable kinetics than the main pool of actively replicating virus.

Although ART is undoubtedly a life-saving therapy for millions of AIDS patients, the persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication, since latently infected cells remain a permanent source of viral reactivation. As a result, a sudden rebound of the viral load after interruption of HAART is generally observed. For this reason, eradication of viral reservoirs is at present the major goal for HIV-1 therapeutics.

Early introduction and intensification of ART have been suggested to diminish the frequency of latently infected memory CD4+ T cells. However, a recent report has shown that ART intensification does not reduce residual viremia in a small cohort of patients. Moreover, it is believed that even a few, or a single, residually infected cell would be sufficient to produce systemic viremia upon ART interruption. Therefore, it has been hypothesized that reactivation of the latent reservoirs could allow effective targeting and possible eradication of the virus.

It is thought that viral reactivation would result in lytic cell death of CD4+ T cells because of the cytophatic effect of the virus or through recognition of infected cells by the immune system. In addition, viral reactivation in the presence of ART would also prevent new infection events. Developing drugs directed against different targets of the HIV cycle is urgently needed, especially the development of drugs able to diminish or eradicate latent reservoirs. This therapy should not induce polyclonal T cell activation.

The present invention features Protein Kinase C (PKC) agonists such as the non-tumorigenic Bryoids combined with Histone Deacetylase (HDAC) inhibitors to purge latent HIV-1 from cellular reservoirs. HDAC is an enzyme that removes acetyl groups from DNA bound histone proteins, affecting gene expression and contributing to HIV latency. Inhibitors of HDAC have been shown to reverse latency in vitro, ex vivo, and recently in a human clinical trial. Vorinostat, a HDAC inhibitor, failed to eliminate HIV-1 reservoirs in patients. Bryoids such as Bryostatin-1, as well as many PKC agonists, activates cellular transcription factors such as NF-κB that binds the HIV-1 promoter and regulates its transcriptional activity. In HIV-1 latency the viral promoter is less accessible to cellular transcription factors because nuclear histones surrounding the viral promoter are deacetylated (compacted chromatin). Thus HDAC inhibitors may increase the acetylation of histones (relaxed chromatin) and then transcription factors may have an easier access to the HIV promoter.

As used herein, the term "administer" or "administration" refers to the taking or receiving of a medicament in an effective manner, such as taking orally a tablet, capsule, powder, gelcap, liquid, suspension, emulsion or the like orally; or a liquid, emulsion or suspension for injection. The Bryoid is administered in an effective dose of 10 to 100 microgram/ Kg subject every other day for up to 180 days.

The HDAC inhibitor is administered in an effective dose of 10 to 100 mg/Kg subject every other day for up to 180 days.

Turning now to Figure 1, a particle having features of the present invention, generally designated by the numeral 11, is depicted. The particle has a core 15, at least one surrounding material 17 and an outer surface 21. The core 15 has a mixture of a hydrophilic material and an HDAC inhibitor. The surrounding material 17 has a mixture of a hydrophobic material and a Bryoid. The surrounding material envelopes 17 the core 15 and the outer surface 21 surrounding thesurrounding material 17.

The core 15 is an aqueous solution that forms a mixture with the HDAC inhibitor. The aqueous solution may comprise other constituents such as salts and buffering agents.

The surrounding material 17 is selected from hydrophobic compositions including phospholipids and like materials which form substantially uniform mixtures with a selected Bryoid. For example, without limitation, the phospholipid is selected from one or more of the group consisting of phosphotidylcholine (PC), phosphotidylglycerol (PG), phosphatidylserine (PS), dimyristoylphophotidylcholine (DMPC), dimyristoylphophotidylglycerol (DMPG), phosphatidylethanolamine (PE), and polyethylene conjugated distearylphosphatidylethanolamine (either DSPE-PEG₂₀₀₀ or DSPE-PEG₃₅₀₀). Hydrophobic compositions include by way of example, without limitation α-tocopherol (vitamin E) and cholesterol. The phospholipids forming the hydrophobic material are depicted as a hydrophilic head 31 and a hydrophobic tail 33.

The virus associated with the latent viral disease has one or more viral components.. For example, without limitation, the viral components comprise protein markers specific for Human Immunodeficiency Virus (HIV). As depicted, the outer surface 21 of the particle 11 comprises one or ligands 23 such as antibodies, nanobody, dual-variable domain ligands and similar constructs which bind to such protein markers. The antibody depicted is a broadly neutralizing antibody (bNAb).

As depicted, the particle has one or more upregulating ligands to upregulate CD-4 cells, an anti-PD -L1 antibody designated by the numeral 25. The one or more upregulating ligands are associated with the surface, similar to the ligand to the protein markers. That is, the head groups 31 of the phospholipids are modified to covalently carry a ligand.

As depicted, one or more head groups of one or more phospholipid compositions carry a polyethylene glycol modification 35. Polyethylene glycol modification of the phospholipid conveys decreased recognition by phagocytes.

Embodiments of the present invention feature targeting a combination of a Bryoid and an HDAC inhibitor co-encapsulated in a long-circulation pegylated immunonanosomes with coatings of broadly neutralizing antibodies and anti-PD-L1 nanobodies, as shown in Figure 1, will provide efficient HIV latency activation and immunological depletion of latent reservoirs while significantly reducing systemic toxicities of both Bryostatin-1 and the HDAC inhibitor.

Using an in vitro model of HIV-1 latency, Jurkat-LAT-GFP, Bryostatin-1 re-activates HIV-1 latency in T cells via classical PKCs pathways. Bryostatin-1, at concentrations higher than 10 nM, induced translocation of cPKCs to the plasma membrane, and activated the canonical NF-κB and MAPKs (JNK and ERK) pathways.

In contrast, lower concentrations of Bryostatin-1 (10 nM) translocated cPKCs and Ras-GRP1 to the endoplasmic reticulum, activated ERK and the nuclear phosphorylation of p65 that fully reactivates HIV-1 latency. Low concentrations of Bryostatin-1 also down-regulated the expression of the human HIV-1 receptors CD4 and CXCR4 and prevent de novo HIV-1 infection (Perez, et al., 2010). Low concentrations of Bryostatin-1 activate the cPKC-Ras-Raf-ERK pathway and synergize with an HDAC inhibitor, valproic acid (VPA), to activate the transcription factor SP1.

Transcriptome studies found that low vs. high concentrations of Bryostatin-1 at 10 and 100 nM differentially regulate gene expression in T cells. Therefore, therapeutic activity can be achieved at concentrations that do not activate signal transduction pathways that may result in negative side effects.

Bryostatin-1 antagonized HIV-1 latency ex vivo in PBMC isolated from HIV-1 patients, and Bryostatin-1 at the doses of 10 and 20 µg/m2 did not induced significant adverse events in HIV-1 patients in a Phase I clinical study, Madrid, Spain (ClinicalTrials.gov NCT02269605).

In vitro studies suggest that very low concentrations of Bryostatin-1 (1-10 nM) synergizes with HDAC inhibitors such as valproic acid to antagonise HIV-1 latency (Perez et al., 2010). Thus, the therapeutic activity of Bryostatin-1 can be drastically improved in humans by utilizing a HDAC inhibitor. Our research indicates that combination therapy will be most effective, and reduce the therapeutic concentration of a Bryoid from 10 nM to 1 nM reducing systemic toxicities. Toxicities will be further reduced by encapsulating the combination therapeutic in liposomes which have been clinically shown to significantly reduce the in vivo toxicity of therapeutic drugs, e.g. the anti-fungal, amphotericin B.

The particle 11, as described, nanoencapsulates a non-tumorogenic Bryoid such as Bryostatin-1, which is quite hydrophobic in the lipid bilayer of a phospholipid nanosomes that are small, uniform liposomes, and co-encapsulate an HDAC inhibitor such as Romidepsin or Panobinostat in the aqueous core. Particles, of the type described in Figure 1, are made in a process for the formation of small, uniform liposomes as described in U.S. Patent 8,637,074 to Castor (2014 ).

Bryostatin-1 is encapsulated at concentrations of 1 to 100 nm with a preference of 1 to 10 nM and an HDAC inhibitor at concentrations of 30 to 1,000 nM with a preference of 30 to 100 nM. The utility of the co-encapsulation is that both drugs will reach their intended target at the same time, will be guided to the target with broadly neutralizing antibodies and the anti-PD-L1 nanobodies will keep CD4+ T-cells activated for clearing the activated HIV-1 virus. The immunonanosomes will further reduce systemic toxicities while pegylation will increase residence time of the circulating nanoparticle increasing therapeutic efficacy and overall therapeutic index.

Targeting a combination of a Bryoid and an HDAC inhibitor co-encapsulated in a long-circulation pegylated immunonanosomes with coatings of broadly neutralizing antibodies and anti-PD-L1 nanobodies, as shown in Figure 1, will provide efficient HIV latency activation and immunological depletion of latent reservoirs while significantly reducing systemic toxicities of both Bryostatin-1 and the HDAC inhibitor.

Using an in vitro model of HIV-1 latency, Jurkat-LAT-GFP, we have shown that Bryostatin-1 re-activates HIV-1 latency in T cells via classical PKCs pathways. Bryostatin-1, at concentrations higher than 10 nM, induced translocation of cPKCs to the plasma membrane, and activated the canonical NF-κB and MAPKs (JNK and ERK) pathways.

In contrast, lower concentrations of Bryostatin-1 (10 nM) translocated cPKCs and Ras-GRP1 to the endoplasmic reticulum, activated ERK and the nuclear phosphorylation of p65 that fully reactivates HIV-1 latency. Low concentrations of Bryostatin-1 also down-regulated the expression of the human HIV-1 receptors CD4 and CXCR4 and prevent de novo HIV-1 infection (Perez, et al., 2010). We also found that low concentrations of Bryostatin-1 activate the cPKC-Ras-Raf-ERK pathway and synergize with an HDAC inhibitor, valproic acid (VPA), to activate the transcription factor SP1.

Transcriptome studies found that low vs. high concentrations of Bryostatin-1 at 10 and 100 nM differentially regulate gene expression in T cells. Therefore, therapeutic activity can be achieved at concentrations that do not activate signal transduction pathways that may result in negative side effects.

Bryostatin-1 antagonized HIV-1 latency ex vivo in PBMC isolated from HIV-1 patients, and Bryostatin-1 at the doses of 10 and 20 ng/m2 did not induced significant adverse events in HIV-1 patients in a Phase I clinical study, Madrid, Spain (ClinicalTrials.gov NCT02269605).

In vitro studies that very low concentrations of Bryostatin-1 (1-10 nM) synergizes with HDAC inhibitors such as valproic acid to antagonise HIV-1 latency (Perez et al., 2010). Thus, the therapeutic activity of Bryostatin-1 can be drastically improved in humans by utilizing a HDAC inhibitor. Our research indicates that combination therapy will be most effective, and reduce the therapeutic concentration of a Bryoid from 10 nM to 1 nM reducing systemic toxicities. Toxicities will be further reduced by encapsulating the combination therapeutic in liposomes which have been clinically shown to significantly reduce the in vivo toxicity of therapeutic drugs, e.g. the anti-fungal, amphotericin B.

To summarize the process of making the particle 11, of Figure 1, in accordance with the teaching of Castor US Patent 8,637,074 , reference is made to Figure 2. Supercritical, critical or near-critical fluids with or without polar co-solvents at appropriate conditions of pressure and temperature are utilized to solvate phospholipids, cholesterol and other nanosomal raw materials. After a specific residence time, the resulting mixture is decompressed via a backpressure regulator (valve) though a dip tube with a nozzle into a decompression chamber that contains phosphate-buffered saline or other biocompatible solution. Bubbles will form at the injection nozzle tip because of SFS depressurization and phase-conversion into a gas, and the solvated phospholipids will deposit out at the phase boundary of the aqueous bubble. As the bubbles detach from the nozzle into the aqueous solution, they rupture, causing bilayers of phospholipids to peel off, thereby encapsulating solute molecules and spontaneously sealing themselves to form phospholipid nanosomes. Product volatilization and oxidation as well as processing time and organic solvent usage can be significantly reduced with the use of supercritical, critical or near critical fluids.

A Bryoid and HDAC inhibitor will be co-encapsulated in phospholipid immunonanosomes in the immunonanosomes apparatus shown in Figure 2 with an supercritical, critical or near critical fluid such as carbon dioxide, nitrous oxide, fluorocarbon or alkane such as propane with or without a cosolvent such ethanol. A preferred supercritical, critical or near critical fluid is 80% propane and 20% ethanol at 3,000 psig and 40°C. We plan to use near-critical propane which, with a dipole moment of 0.084 Debyes, exhibits a much higher solvation power for phospholipids and hydrophobic drugs. Propane is considered GRAS (generally regarded as safe) by the FDA when used under GMP conditions in the food and pharmaceutical industries. Lipid materials will be selected on the basis of previous studies and the solubility of these lipids in the supercritical, critical or near critical fluid under appropriate operational conditions.

The presence of cholesterol in nanosomes transforms the bilayer into an ordered fluid phase over a wide temperature range, and therefore, improves the stability of nanosomes in plasma. Nanosomal compositions are listed in Table 1. Table 1: Lipid Compositions and Molar Ratios

Lipid Compositions	Molar Ratio
PC:CH	1:1 and 2:1
PC:PG:CH	1:0.1:0.4
PC:PS:CH	1:0.1:0.4
DMPC:DMPG:CH	1:0.1:0.4
PC:DMPG:CH:DSPE-PEG2000	1:0.1:0.35:0.05

The supercritical, critical or near critical fluid is utilized to first solvate phospholipids and liposomal raw materials, then mixed with a solution of the Bryoid prior to decompression and injection into a biocompatible solution containing the HDAC inhibitor. After decompression through a nozzle, the supercritical, critical or near critical fluid evaporates off, leaving an aqueous solution of liposomes entrapping hydrophobic Bryoid within the lipid bilayer and HDAC inhibitor in the aqueous core of the phospholipid nanosomes.

Phospholipids spliced with specific antibodies are utilized to target the co-encapsulated drugs to the latent HIV virus. The phospholipid nanosomes are coated with antibodies or nanobodies and are referred to as immunonanosomes by using phospholipids functionalized with the ligand.

One of the problems with nanosomes is phagocytosis by leukocytes and the reticuloendothelial system, which causes their rapid removal from circulation and makes them unavailable for uptake by tumor cells. This problem is overcome by coating the particles with polyethylene glycol (PEG) which prevents them from being recognized by phagocytic cells.

PEG coating is used to produce 'stealth' liposomes which make them non-recognizable by phagocytes and hence resistant to their uptake. Commercially available phospholipids with head groups linked to PEG of various molecular weights will be utilized. Pegylated phospholipids will be utilized to provide steric hindrance, increasing residence time and therapeutic index.

We also hypothesize that targeting a combination of a Bryoid and an HDAC inhibitor co-encapsulated in a long-circulation pegylated immunonanosomes with coatings of broadly neutralizing antibodies and anti-PD-L1 nanobodies, as shown in Figure 1 will provide efficient HIV latency activation and immunological depletion of latent reservoirs while significantly reducing systemic toxicities of both Bryostatin-1 and the HDAC inhibitor.

Immunonanosomes are produced by various lipid materials in the size range of 100 to 200 (± 50) nm. Immunonanosomal suspensions of this size range can be filtered by a 0.22 µm filter as a final sterilization step.

Particles, such as a plurality of particle 11, are used as a suspension in solution for administration by way of intravenous injection.

Claims (9)

1. A medicament for treating a latent viral disease, comprising: a Histone Deacetylase (HDAC) inhibitor and a Bryoid, said HDAC inhibitor and said Bryoid are carried by one or more particles (11), wherein said HDAC inhibitor and said Bryoid are co-encapsulated in said one or more particles (11), and wherein said one or more particles (11) has a core (15), at least one surrounding material (17) and an outer surface (21), said core (17) having a mixture of a hydrophilic material and said HDAC inhibitor, and said surrounding material having a mixture of a hydrophobic material and said Bryoid, said surrounding material (17) enveloping said core (15) and said outer surface (21) surrounding said surrounding material (17).
2. The medicament of claim 1, wherein said Bryoid is selected from the group of Bryostatins consisting of Bryostatin 1-20.
3. The medicament of claim 1, wherein said HDAC inhibitor is selected from the group consisting of valproic acid, Vorinostat, Romidepsin and Panobinostat.
4. The medicament of claim 1, wherein said Bryoid is administered in an effective dose range of 10 to 100 microgram/Kg subject.
5. The medicament of claim 1, wherein said HDAC inhibitor is administered in an effective dose range of 10 to 100 mg/Kg subject.
6. The medicament of claim 1, wherein said one or more particles (11) has a virus associated with said latent viral disease having one or more viral components, and wherein said one or more particles (11) comprise one or more ligands specific for said viral component, and said one or more ligands associated with the outer surface (21) of said one or more particles.
7. The medicament of claim 1, wherein said one or more particles (11) comprise one or more upregulating ligands to upregulate CD-4 cells, said one or more upregulating ligands associated with the outer surface (21).
8. A medicament according to any of claims 1 to 7 for use in a method of treating a latent viral disease.
9. An article of manufacture comprising a medicament according to any of claims 1 to 7.