Reproductive and developmental toxicology studies of iminosugar UV-4.

UV-4 (N-(9-methoxynonyl)-1-deoxynojirimycin) is a host-targeted antiviral agent, which targets mammalian proteins (endoplasmic reticulum glucosidases) rather than virally encoded proteins. This mechanism confers both broad-spectrum activity and low potential for generation of viral drug resistance mutations. Reproductive and developmental studies of UV-4 evaluated effects on fertility and early embryonic development in rats, embryo-fetal development in rats and rabbits, and pre- and postnatal development including maternal function in rats.

Randomized single oral dose phase 1 study of safety, tolerability, and pharmacokinetics of Iminosugar UV-4 Hydrochloride (UV-4B) in healthy subjects.

Background: UV-4 (N-(9'-methoxynonyl)-1-deoxynojirimycin, also called MON-DNJ) is an iminosugar small-molecule oral drug candidate with in vitro antiviral activity against diverse viruses including dengue, influenza, and filoviruses and demonstrated in vivo efficacy against both dengue and influenza viruses. The antiviral mechanism of action of UV-4 is through inhibition of the host endoplasmic reticulum-resident α-glucosidase 1 and α-glucosidase 2 enzymes. This inhibition prevents proper glycan processing and folding of virus glycoproteins, thereby impacting virus assembly, secretion, and the fitness of nascent virions.

Investigational New Drug Enabling Nonclinical Safety Assessment of the Iminosugar UV-4, a Broad-Spectrum Host-Targeted Antiviral Agent.

The iminosugar UV-4 is a broad-spectrum antiviral drug candidate with activity and against multiple, diverse viruses. The toxicological profile of UV-4, dosed as the hydrochloride salt, was evaluated in single-dose and repeat-dose oral toxicity studies in mice, rats, dogs, and non-human primates (NHP). No moribundity or deaths were associated with the drug up to the maximum tolerated dose.

Investigational New Drug Enabling Nonclinical Safety Pharmacology Assessment of the Iminosugar UV-4, a Broad-Spectrum Host-Targeted Antiviral Agent.

UV-4 (N-(9-methoxynonyl)-1-deoxynojirimycin) is a broad-spectrum antiviral drug candidate with demonstrated activity in vitro and in vivo against multiple, diverse viruses. Nonclinical safety pharmacology studies were conducted to support the filing of an Investigational New Drug (IND) application. Preliminary in vitro pharmacology testing evaluating potential for binding to "off-target" receptors and enzymes indicated no significant liability for advanced development of UV-4.

Lack of selective resistance of influenza A virus in presence of host-targeted antiviral, UV-4B.

Development of antiviral drug resistance is a continuous concern for viruses with high mutation rates such as influenza. The use of antiviral drugs targeting host proteins required for viral replication is less likely to result in the selection of resistant viruses than treating with direct-acting antivirals. The iminosugar UV-4B is a host-targeted glucomimetic that inhibits endoplasmic reticulum α-glucosidase I and II enzymes resulting in improper glycosylation and misfolding of viral glycoproteins.

Assessment of the potential for host-targeted iminosugars UV-4 and UV-5 activity against filovirus infections in vitro and in vivo.

Iminosugars are host-directed antivirals with broad-spectrum activity. The iminosugar, N-butyl-deoxynojirimycin (NB-DNJ or Miglustat), is used in humans for treatment of Gaucher's disease and has mild antiviral properties. More potent analogs of NB-DNJ have been generated and have demonstrated activity against a variety of viruses including flaviviruses, influenza, herpesviruses and filoviruses.

The Iminosugar UV-4 is a Broad Inhibitor of Influenza A and B Viruses ex Vivo and in Mice.

Iminosugars that are competitive inhibitors of endoplasmic reticulum (ER) α-glucosidases have been demonstrated to have antiviral activity against a diverse set of viruses. A novel iminosugar, UV-4B, has recently been shown to provide protection against lethal infections with dengue and influenza A (H1N1) viruses in mice. In the current study, the breadth of activity of UV-4B against influenza was examined ex vivo and in vivo.

Inhibition of endoplasmic reticulum glucosidases is required for in vitro and in vivo dengue antiviral activity by the iminosugar UV-4.

The antiviral activity of UV-4 was previously demonstrated against dengue virus serotype 2 (DENV2) in multiple mouse models. Herein, step-wise minimal effective dose and therapeutic window of efficacy studies of UV-4B (UV-4 hydrochloride salt) were conducted in an antibody-dependent enhancement (ADE) mouse model of severe DENV2 infection in AG129 mice lacking types I and II interferon receptors. Significant survival benefit was demonstrated with 10-20 mg/kg of UV-4B administered thrice daily (TID) for seven days with initiation of treatment up to 48 h after infection.

In vivo therapeutic protection against influenza A (H1N1) oseltamivir-sensitive and resistant viruses by the iminosugar UV-4.

Our lead iminosugar analog called UV-4 or N-(9-methoxynonyl)-1-deoxynojirimycin inhibits activity of endoplasmic reticulum (ER) α-glucosidases I and II and is a potent, host-targeted antiviral candidate. The mechanism of action for the antiviral activity of iminosugars is proposed to be inhibition of ER α-glucosidases leading to misfolding of critical viral glycoproteins. These misfolded glycoproteins would then be incorporated into defective virus particles or targeted for degradation resulting in a reduction of infectious progeny virions.

Dengue Virus Evolution under a Host-Targeted Antiviral.

Unlabelled: The host-targeted antiviral drug UV-4B reduces viral replication and promotes survival in a mouse model of experimental dengue virus (DENV) infection. UV-4B is an iminosugar that inhibits the α-glucosidase family of enzymes and subsequently the folding of glycosylated proteins, both viral and host. Here, we utilized next-generation sequencing to investigate evolution of a flavivirus under selective pressure by a host-targeted antiviral in vivo.

In vivo electroporation enhances the potency of poly-lactide co-glycolide (PLG) plasmid DNA immunization.

Immunization with plasmid DNA that has been encapsulated in poly lactide-co-glycolide (PLG) microparticles targets the plasmid DNA to antigen presenting cells and elicits immune responses to the encoded antigen(s). Application of a series of electrical pulses (EPT) immediately following unformulated DNA injection enhances expression of the encoded antigen and increases immune responses. The combination of using EPT before or after PLG-encapsulated plasmid DNA immunization was tested to determine if enhanced immune responses would be generated.

Repeated immunization with plasmid DNA formulated in poly(lactide-co-glycolide) microparticles is well tolerated and stimulates durable T cell responses to the tumor-associated antigen cytochrome P450 1B1.

Injection of microparticle-encapsulated DNA elicits immune responses to plasmid-encoded antigens in mice and humans. Cytochrome P450 CYP1B1 (CYP1B1) is a member of the CYP1 P450 enzyme family that is overexpressed in a variety of solid tumors. The work described herein was performed to study the kinetics of stimulating T cell responsiveness with an encapsulated DNA encoding CYP1B1 and provides support for the clinical development of this formulation.

The shared tumor-associated antigen cytochrome P450 1B1 is recognized by specific cytotoxic T cells.

Cytochrome P450 1B1 (CYP1B1), a drug-metabolizing extrahepatic enzyme, was recently shown to be overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for anticancer therapy, particularly cancer immunotherapeutics. We identified HLA-A*0201-binding peptides and a naturally processed and presented T-cell epitope capable of inducing CYP1B1-specific cytotoxic T lymphocytes (CTLs) in HLA-A2 transgenic mice.

Generation of expression constructs that secrete bioactive alphaMSH and their use in the treatment of experimental autoimmune encephalomyelitis.

alpha Melanocyte-stimulating hormone (alphaMSH) is a 13 amino acid peptide with potent anti-inflammatory effects. We created two DNA expression constructs (miniPOMC and pACTH1-17) that encode bioactive versions of the alphaMSH peptide, and tested these constructs for therapeutic effects in experimental autoimmune encephalomyelitis (EAE). Each construct contained the sequences for alphaMSH, as well as the sequences that are involved in the secretion and processing of the POMC gene with the assumption that these sequences would promote processing and release of the encoded alphaMSH peptide.

Inhibition of human immunodeficiency virus replication and growth advantage of CD4+ T cells and monocytes derived from CD34+ cells transduced with an intracellular antibody directed against human immunodeficiency virus type 1 Tat.

Current clinical gene therapy protocols for the treatment of human immunodeficiency virus type 1 (HIV-1) infection involve the ex vivo transduction and expansion of CD4+ T cells derived from HIV-positive patients at a late stage in their disease (CD4+ cell count <400 cells/mm3). We examined the efficiency of transduction and transgene expression in adult bone marrow (BM)- and umbilical cord blood (UCB)-derived CD34+ cells induced to differentiate into T cells and monocytes in vitro with an MuLV-based vector encoding the neomycin resistance gene and an intracellular antibody directed against the Tat protein of HIV-1 (sFvtat1-Ckappa). The expression of the marker gene and the effects of antiviral construct on subsequent challenge with monocytotropic and T cell-tropic HIV-1 isolates were monitored in vitro in purified T cells and monocytes generated in culture from the transduced CD34+ cells.

Inhibition of human immunodeficiency virus type 1 replication in vitro in acutely and persistently infected human CD4+ mononuclear cells expressing murine and humanized anti-human immunodeficiency virus type 1 Tat single-chain variable fragment intrabodies.

We have previously reported that a murine anti-Tat sFv intrabody, termed sFvtat1Ck, directed against the proline-rich N-terminal activation domain of HIV-1, is a potent inhibitor of HIV-1 replication [Mhashilkar, A. M., et al.

Inhibition of human immunodeficiency virus replication and growth advantage of CD4+ T cells from HIV-infected individuals that express intracellular antibodies against HIV-1 gp120 or Tat.

Current clinical gene therapy protocols for the treatment of human immunodeficiency virus type 1 (HIV-1) infection often involve the ex vivo transduction and expansion of CD4+ T cells derived from HIV-positive patients at a late stage in their disease (CD4 count <400). These protocols involve the transduction of T cells by murine leukemia virus (MLV)-based vectors encoding antiviral constructs such as the rev m10 dominant negative mutant or a ribozyme directed against the CAP site of HIV-1 RNA. We examined the efficiency and stability of transduction of CD4+ T cells derived from HIV-infected patients at different stages in the progression of their disease, from seroconversion to AIDS.

Cyclic AMP-mediated growth arrest is associated with increased expression of human T cell leukemia virus type I structural and transforming genes.

The effect of increased intracellular cyclic AMP levels on gene expression of the human T cell leukemia virus type I (HTLV-I) provirus was examined. Induction of infected cells to produce elevated levels of cyclic AMP was associated with specific increases in viral surface antigen expression, protein synthesis, p24 release into the supernatant, and RNA levels. The patterns of HTLV-I proviral gene expression observed support results from transfection experiments regarding the function of Tax, Rex, and cyclic AMP in HTLV-I gene regulation.

Role of human T-cell leukemia virus type 1 X region proteins in immortalization of primary human lymphocytes in culture.

Human T-cell leukemia virus type 1 (HTLV-1) immortalizes human CD4+ T lymphocytes in culture. Previous studies show that in the context of a herpesvirus saimiri vector, the sequence of the X region at the 3' end of the HTLV-1 genome is also capable of immortalizing CD4+ lymphocytes in the absence of HTLV-1 structural proteins. The X region of HTLV-1 encodes two trans-acting viral proteins, the 42-kDa Tax protein and the 27-kDa Rex protein.

Impact of 90Sr on mouse natural killer cells and their regulation by alpha interferon and interleukin 2.

Male CBA/SU mice were exposed to ionizing radiation by intraperitoneal injection of the bone-seeking beta-emitter 90Sr. NK-cell lytic activities in spleen, peripheral blood, and lymph nodes were severely depressed or completely abolished. In contrast, production of the NK regulatory proteins alpha interferon (IFN-alpha) and interleukin 2 (IL-2) was normal 5-8 weeks after 90Sr injection.

Mutant monoclonal antibodies with select alteration in complement activation ability. Impact on immune complex functions in vivo.

Mutagenesis of mAb is a useful means for studying the biologic and pathologic functions of immune complexes. Treatment of the Hy-1.2 hybridoma-producing IgG2a-anti-TNP antibodies with ethylmethanesulfonate provided us with a mutant clone, producing antibodies with reduced capacity for C activation.

In human monocytes a strong correlation exists between expression of the M3 antigen, Fc-mediated phagocytic activity and failure to participate in extracellular antibody-dependent cytotoxicity.

Human fresh blood monocytes can phagocytize and lyse antibody-coated target cells by contact with membrane Fc receptors. Recently, the monocyte differentiation antigen Leu M3 has been described to be associated with monocyte/macrophage maturation pathway and to be linked to functionally distinct monocyte subsets. In the present study peripheral blood monocytes were separated into M3+ and M3- subsets, and evaluated for their ability to mediate antibody-driven effector functions.