Title:  Methods and compositions for inhibition of viral replication

United States Patent:  6,274,611

Assignee:  The United States of America as represented by the Department of Health and (Washington, DC)

Filed:  January 15, 1999

The present invention comprises methods and compositions for treating viral infection by inhibiting the activity of host cellular enzymes. More specifically, methods and compositions comprising casein kinase II inhibitors and various related compounds such as precursors, analogs, metabolites and hydrolysis products that inhibit cellular proteins and thus viral replication are provided.

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods for the treatment of viral disease that are mediated by inhibition of viral replication are provided. Particularly, methods and compositions of the present invention are directed to inhibition of protein kinase enzymes such as casein kinase II in mammalian cells. Inhibition of protein kinases inhibits the replication, including transcription and translation, of many types of viruses. Among other activities, such inhibition leads to the inability of the cell to support the replication of viruses including HIV-1 (Human Immunodeficiency Virus-1). Such compounds include casein kinase II inhibitors and various related compounds such as precursors, analogs, metabolites and hydrolysis products.

Inhibition of Viral Transcription and Replication

The identification of cellular factors that are required to complete various steps of a virus lifecycle is important for the development of new therapeutics. Herein, the lifecycle of HIV-1 is discussed, though it is intended as an example. The present invention is not limited to treatments for retroviral infections, but is also intended for treatments for other viruses, particularly RNA genome viruses.

One key step of the viral replication process for retroviruses, transcription from the integrated provirus, is inhibited by members of two structurally distinct classes of compounds, the flavonoids and the benzothiophenes. A marked specificity of these compounds toward inhibiting HIV-1 transcription, is evidenced by the ability of drug-treated cells to retain their proliferative and differentiation capabilities. In addition, flavonoids and benzothiophenes do not impede the activation and function of the transcriptional factor NF-.kappa.B. Chemical and immunologic analyses disclosed herein have identified the cellular factors targeted by the flavonoids and the benzothiophenes as the individual subunits of casein kinase II (CKII). Thus, the benzothiophenes and flavonoids specifically inhibit CKII, without interfering with cellular transcription factors such as NF-.kappa.B, and yield selective inhibition of HIV.

It is the inventor's surprising finding that selective inhibition of CKII by compositions, such as the benzothiophenes and flavonoids, allows for cessation of viral activities and yet continuation of host cellular activities. Other RNA genome viruses can also be treated using the methods and compositions described herein for HIV. Though not wishing to be bound by any particular theory, it is believed that CKII regulates HIV4 transcription by phosphorylating cellular proteins involved in HIV-1 transactivation containing multiple CKII phosphorylation consensus sequences. Though these two classes of compounds are structurally unrelated, both chrysin and benzothiophene selectively bind to CKII. Furthermore, both chrysin and benzothiophene inhibit human recombinant CKII enzymatic activity and show competitive kinetics with respect to ATP, analogous to the classic CKII inhibitor 5,6-dichloro-1-.beta.-D-ribofuranosylbenzimidazole (DRB). Moreover, DRB potently inhibits HIV1 expression in chronically infected cells.

Both flavonoids, chrysin in particular, and benzothiophenes act as potent inhibitors of HIV-1 transcription in chronically infected cells (Butera et al, Mol. Med. vol. 1, pp. 758-767 (1995); Critchfield et al., AIDS Res. Hum. Retr. vol. 12, pp. 39-46) (1996)). They block HIV-1 transcriptional activation in cells treated with tumor necrosis factor-.alpha. (TNF-.alpha.) or PMA. They also suppress HIV-1 replication in constitutively HIV-1 expressing 8 E5 cells and in OM-10.1 cultures under continued pressure (TNF-.alpha. treatment) to express virus. An especially unique feature of these compounds is that the activation and function of NF-.kappa.B is not affected. Furthermore, a specificity toward inhibiting HIV-1 transcription is evidenced by the ability of drug-treated cells to not only remain proliferative, but also to retain the capacity to differentiate.

Flavonoids represent a class of compounds of potential use in attenuating HIV activation. These naturally occurring compounds are ubiquitous in vascularized plants and possess a variety of cellular and biochemical effects in animals. For example, certain flavonoids are potent and reversible growth inhibitory agents for numerous human tumor cell lines and also have been found to modify a variety of immune cell responses, including the inhibition of lymphocyte proliferation. Flavonoids also inhibit a large array of mammalian enzymes, including, but not limited to, protein kinase C, cyclic mononucleotide phosphodiesterase, membrane ATPases, cytochrome P-450 enzymes, glutathione S-transferase, cyclooxygenase, and lipoxygenase.

With regard to HIV, some flavonoids have been characterized as inhibiting HIV-1 viral-coded proteins such as reverse transcriptase, protease, and integrase. It is the inventors' surprising finding that flavonoids can be used to inhibit viral replication, particularly HIV, by inhibiting cellular, not viral, proteins. In vitro studies showing inhibition of viral proteins in cultured cells have also shown that the flavonoid baicalin inhibits HIV replication in acutely infected H9 cells, CEM-ss cells, and primary human peripheral blood mononuclear cells (PBMCs). Other work using C81166 cultures indicates that several compounds from the flavan category of flavonoids are effective inhibitors of HIV-1 infection at concentrations where toxicity is very low. More recently, several studies have demonstrated that flavonoids, including chrysin and glycosides of acacetin and apigenin, show significant inhibitory activity in acutely HIV-infected H9 cells. These flavonoids were identified by isolation from particular plant extracts or by the testing of purified compounds.

Two benzothiophene derivative compounds designated PD121871 and PD144795 have been described as inhibiting HIV transcription at micromolar concentrations in models of latent and chronic infection. See S. Butera et al, "Compounds that Target Novel Cellular Components Involved in HIV-1 Transcription." Molecular Medicine, Vol. 1 (1995), incorporated herein by reference. These compounds induce a state of viral latency in cells actively expressing HIV, even when maintained under conditions of constant viral stimulation. The compounds selectively inhibited HIV transcription by an unknown mechanism not involving Tat function or NF-.kappa.B activation. It was the inventors' present discovery that benzothiophenes could be used to inhibit cellular proteins and thus effect viral replication.

The benzothiophene derivatives were characterized by an ability to block TNF-.alpha.-induced HWV activation. However, these agents did not appear to act primarily as TNF antagonists. They did not inhibit NF-.kappa.B activation or autocrine TNF-.alpha. transcription in response to TNF-.alpha. treatment of OM-10.1 cells. Also, they inhibited HIV-1 transcription in chronically infected 8 E5 cells that express HIV-1 independent of exogenous stimulation.

The benzothiophene derivative compounds accelerated the return to viral latency in OM-10.1 cultures when the extracellular stimulus was removed, and induced a state of viral latency in the presence of continued viral stimulation. They also severely restricted viral expression during an acute infection of MT-4 T cells possibly targeting post-integration events.

Cell Lines

Critical tools for studying the efferent phase of the HIV life cycle are the latently infected cell lines such as UI, ACH2, J1, and OM-10.1. The most recently developed of these is the OM-10.1 cell line which displays a rapid down modulation of cell surface CD4 on HIV activation in response to TNF-.alpha., along with the subsequent return of CD4 expression following removal of the HIV-inducing stimulus. HIV activation can also be achieved by phorbol esters such as PMA (phorbol -12-myristate-13-acetate).

The CD4 response is a direct consequence of viral activation and allows a rapid, convenient and highly reproducible assessment of treatments that modify TNF-.alpha. expression of HIV from its latent state. In addition, the down modulation of CD4 is not affected by inhibitors of virion assembly or release such as protease inhibitors. Thus, by measuring cell surface CD4 and a virion constituent such as reverse transcriptase (RT), it is possible to discriminate between inhibition of HIV assembly, release and earlier events. For example, in TNF-.alpha.-induced OM-10.1 cultures, the protease inhibitor Ro 31-48959 (0.1 .mu.M) completely inhibits RT but has no effect on the down modulation of CD4. In contrast, the antagonist of HIV transactivation, Ro 5-3335, inhibits both of these measures to a similar extent.

Casein Kinase

Human casein kinase II (CKII) is a multifunctional serine/threonine protein kinase whose catalytic subunits .alpha. and .alpha.' are about 40-44 kD in size, and regulatory subunit .beta. is approximately 29 kD in size. Unlike most serine/threonine kinases, CKII phosphorylates within acidic amino acid stretches. CKII has been proposed as an I.kappa.B.alpha. kinase, phosphorylating in the motif called the PEST region, in mouse pre-B cells. Multiple proteins which have short half-lives (less than 2 hours) have been shown to contain PEST sequences and to be phosphorylated by CKII. PEST sequences are amino acid sequences that are rich in the amino acids, P (proline), E (glutamic acid), S (serine) and T (threonine).

CKII may also interact with Vpu, an accessory protein encoded by HIV-1. The genome of HIV-1 codes for catalytic and structural proteins as well as for non structural proteins including Vpu. Vpu is present at low concentrations in HIV infected cells, but is absent from virus particles. Vpu is an 81-amino acid amphipathic integral membrane protein with at least two different biological functions: enhancement of virus particle release from the plasma membrane of HIV-1-infected cells, and degradation of the virus receptors CD4. Deletion of the Vpu gene leads to accumulation of viral structural proteins and intracellular budding of premature virus particles, accompanied by an increased cytopathogenicity.

The inventors surprisingly found that viral infections can be treated by specific inhibition of cellular enzymes such as CKII. The role of CKII is demonstrated in that CKII is directly inhibited by two chemically distinct classes of HIV-1 transcriptional inhibitors, the flavonoids and the benzothiophenes. In addition a third chemically distinct compound, the classic CKII inhibitor DRB, also mimics the antiviral properties of the flavonoids and benzothiophenes.

It would not be apparent to those skilled in the art that inhibition of CKII would have any effect on viral infections, because inhibitors of CKII do not effect the activation cycle of viruses such as HIV. Interaction by protein I-.kappa.B and Vpu with CKII are known and it is known that these proteins are important for HIV transcription, but surprisingly, inhibition of CKII has no effect on regulation of these proteins Vpu and I-.kappa.B.

Further support for the treatment of viral infection by inhibiting CKII while preserving normal host cell functions, is demonstrated by the interaction of CKII with inhibitor-kappa B (I-.kappa.B). I-.kappa.B undergoes phosphorylation and subsequent degradation in response to activating stimuli (including TNF-.alpha.). I-.kappa.B degradation results in the release of active nuclear factor-kappa B (NF-.kappa.B) that subsequently plays an important role in activating HIV-1 expression. Although some reports indicate that CKII is capable of phosphorylating I-.kappa.B on multiple sites, it has been shown that factors that inhibit CKII such as flavonoids and benzothiophenes do not impede either the degradation of I-.kappa.B nor the activation and function of NF-.kappa.B. Normal degradation of I-.kappa.B upon cellular activation is also observed in the presence of DRB.

The Vpu protein of HIV-1 has been shown to be phosphorylated on serine residues 52 and 56 by CKII. These phosphorylations are required for the ability of Vpu to accelerate the decay of CD4. However, Vpu has not been implicated in regulating virus transcription, and viral mutants completely lacking this gene are replication competent.

HIV1 infection is not the only viral infection to be treated by inhibition of CKII because other viruses require cellular CKII to replicate. The most extensively studied of these is vesicular stomatitis virus, the P protein of which must be phosphorylated by CKII to accomplish viral transcription. In addition, CKII is specifically packaged as part of the ribonucleoprotein complex within the vesicular stomatitis virus virion. Other RNA genome viruses also require CKII activity for phosphorylation of the P protein, including respiratory syncitial virus and measles. With regard to retroviruses, cells transformed by Abelson and Moloney murine leukemia viruses contain higher levels of CKII activity.

Compositions contemplated by the present invention include compounds capable of inhibiting CKII. Such compounds are capable of inhibiting CKII in vitro and in vivo and show antiviral activity both in vitro and in vivo. In addition, the compositions of the present invention are capable of inhibiting CKII without detrimentally effecting cellular viability.

Therapeutic induction of viral latency in cells actively expressing HIV would be a new approach to reduce the viral burden and slow disease progression. Therapeutic control of viral transcription in cells expressing HIV constitutes an appealing intervention and a potential supplement to other pharmacologic agents targeting viral-specific gene products. With the identification of relevant molecular targets, these experimental compounds may elucidate new cellular processes that prevent HIV expression and provide a means for prolonging the clinically asymptomatic phase that precedes AIDS.

Treatment of other RNA genome viruses are contemplated by the claimed methods and compositions. Such viruses include, but are not limited to, respiratory syncitial virus, measles virus, and vesicular stomatitis viruses. These viruses, and viruses taxonomically related to these viruses, cause a variety of illnesses in both humans, animals and plants.

Therapeutic Intervention

Compositions for inhibiting protein kinase enzymes such as casein kinase enzymes, can be provided as pharmaceutically acceptable formulations using formulation methods known to those of ordinary skill in the art. These formulations can be administered by standard routes. In general, the combinations may be administered by the topical, transdermal, oral, rectal or parenteral (e.g., intravenous, subcutaneous or intramuscular) route. In addition, the combinations may be incorporated into biodegradable polymers allowing for sustained release of the compound, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of infection.

The dosage of the compound will depend on the condition being treated and the extent of infection, the particular compound, and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound. It is to be understood that the present invention has application for both human and veterinary use. For administration to humans, a dosage of between approximately 5-75 mg/kg/day, preferably , a dosage of between approximately 10-50 mg/kg/day a dosage, most preferably, a dosage of between approximately 10-30 mg/kg/day. Depending on the route of administration, the compound administered and the toxicity of that compound, a preferable dosage would be one that would yield a blood level in the patient of approximately 1-50 .mu.molar, and more preferably, 1-30 .mu.molar, and most preferably, 3-10 .mu.molar.

The formulations include those suitable for oral, rectal, ophthalmic, (including intravitreal or intracameral) nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intratracheal, and epidural) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into associate the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil emulsion and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be optionally coated or scored and may be formulated so as to provide a slow or controlled release of the active ingredient therein.

Formulations suitable for topical administration in the mouth include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; pastiles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the ingredient to be administered in a suitable liquid carrier.

Formulations suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising the ingredient to be administered in a pharmaceutical acceptable carrier. A preferred topical delivery system is a transdermal patch containing the ingredient to be administered.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having an appropriate particle size, microns which is administered in the manner in which snuff is administered, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.

Formulations suitable for vaginal administration may be presented as pessaries, tamports, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) conditions requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient.

Claim 1 of 8 Claims

What is claimed is:

1. A method of treating measles virus infection in a human or animal in need thereof, comprising administering to the human or animal an effective amount of a composition comprising a compound selected from the group consisting of flavonoids, benzothiophenes, and 5,6-dichloro-1-.beta.-D-ribofuranosylbenzinidazole (DRB), and their precursors, analogs, metabolites and hydrolysis products, wherein casein kinase II is inhibited.

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