WO2017142807A1

WO2017142807A1 - Treatment or prevention of zika virus infections by administration of a ribonuclease

Info

Publication number: WO2017142807A1
Application number: PCT/US2017/017429
Authority: WO
Inventors: III Thomas W. HODGE
Original assignee: Tamir Biotechnology, Inc.
Priority date: 2016-02-16
Filing date: 2017-02-10
Publication date: 2017-08-24

Abstract

The invention relates to treatment or prevention of Zika virus infections by administration of a ribonuclease.

Description

TREATMENT OR PREVENTION OF ZIKA VIRUS INFECTIONS BY ADMINISTRATION OF A RIBONUCLEASE

Field of the Invention

Background of the Invention

Zika virus is a flavivirus which is transmitted by mosquitoes of the genus Aedes, such as Aedes aegypti and Aedes albopictus. The infection with Zika virus which is also called Zika fever or Zika virus disease causes symptoms such as mild fever, skin rash, conjunctivitis, muscle and joint pain, malaise and headache.

However, in rare cases Zika infection of an adult may result in Guillain-Barre syndrome. If Zika virus is transmitted from a pregnant woman to her fetus, it can cause microcephaly, severe brain malformations and other birth defects. In 2015 and 2016 there was a Zika virus epidemic in Latin America and the Caribbean. So far, there is no vaccine or treatment for Zika virus infections.

Hence, there is a need to identify substances which can be used to efficiently treat or prevent Zika virus infections.

Summary of the Invention

Recent experiments have now shown that ranpirnase is active against Zika virus in Huh-7 human liver carcinoma cells.

Accordingly, the present invention relates to an RNase of the RNase A superfamily or a functional derivative thereof for use in treating a Zika virus infection in a mammalian subject, wherein the RNase of the RNase A superfamily has an amino acid sequence selected from the group consisting of SEQ ID Nos. 1, 2, 3 and 4 and more preferably, it has the amino acid sequence according to SEQ ID No. 1 which is the amino acid sequence of ranpirnase.

The present invention also relates to an RNase of the RNase A superfamily or a functional derivative thereof for use in preventing a Zika virus infection in a mammalian subject, wherein the RNase of the RNase A superfamily has an amino acid sequence selected from the group consisting of SEQ ID Nos. 1, 2, 3 and 4 and more preferably, it has the amino acid sequence according to SEQ ID No. 1 which is the amino acid sequence of ranpirnase. In another preferred embodiment the mammalian subject is a human.

Also preferably, the RNase is administered systemically, more preferably it is administered by intravenous, intramuscular, oral, rectal or nasal administration. Brief Description of the Drawings

In the drawings, wherein

CC50 is the cytotoxic concentration (expressed in nM) of ranpirnase, i.e. the ranpirnase concentration that decreased cell viability by 50%, and

IC50 is the inhibitory concentration (expressed in nM) of ranpirnase, i.e. the ranpirnase concentration that inhibited replication of the virus under test by 50%,

SI, the selective index, is CC50/IC50. The higher the value of SI, the more active is the ranpirnase against the virus under test,

Figure 1 shows the results of testing the anti-viral activity of ranpirnase against Zika virus in Huh-7 human liver carcinoma cells.

Detailed Description of the Invention

Recent experiments have shown that ranpirnase demonstrates surprisingly strong antiviral effects against a surprisingly large number of different viruses, including viruses (e.g. MERS-CoV and EBOV) that are highly resistant to treatment and viruses for which no treatment is available (e.g. Zika virus).

It is believed that the surprisingly broad- spectrum activity of the invention comes from the ways in which ranpirnase degrades various forms of RNA. To date, three RNA-degrading mechanisms appear to be relevant to antiviral therapy using ranpirnase.

The first of these mechanisms is degradation of tRNA. Degrading tRNA inside a mammalian cell makes that cell resistant to some viral infections. This is because some viruses replicate by protein synthesis using the ribosome, and protein synthesis cannot occur unless transfer RNAs enter the ribosome to deliver the amino acids needed to synthesize the protein. Thus, a systemic application of an agent that degrades tRNA will prevent or at least substantially impede some viruses from spreading to uninfected cells. If this application occurs before the virus has spread widely enough to endanger the host mammal, the virus will eventually die.

The second mechanism is degradation of viral double-stranded RNA. Some viruses produce double-stranded RNA as part of their process of proliferation in mammalian cells, and destroying that double- stranded RNA can prevent or at least substantially impede replication of such viruses.

The third mechanism is degradation of microRNA and siRNA. In certain viruses that proliferate using double-stranded RNA, that double-stranded RNA is produced by the interaction of microRNA or siRNA with single-stranded RNA. Destroying the microRNA or siRNA can prevent the formation of the viral double- stranded RNA by which the virus replicates.

RNases of the RNase A superfamily are pyrimidine- specific endonucleases found in high quantity in the pancreas of certain mammals and of some reptiles. They are involved in endonucleolytic cleavage of 3'-phosphomononucleotides and 3'- phosphooligonucleotides ending in C-P or U-P with 2',3'-cyclic phosphate intermediates. Members of this superfamily include ranpirnase and variants thereof, amphinase, r-Amphinase-2, bovine seminal vesicle and brain ribonucleases; kidney non-secretory ribonucleases; liver-type ribonucleases, angiogenin; eosinophil cationic protein, and pancreatic ribonucleases from different species including human and bovine pancreatic ribonucleases.

Ranpirnase is an RNase isolated from oocytes of the leopard frog Rana pipiens which is disclosed in U.S. Pat. No. 5,559,212, and is also known as Onconase^®. The amino acid sequence of ranpirnase is provided in SEQ ID NO: 1. Ranpirnase has been tested and found to be cytotoxic to cancer cells because of its enzymatic activity against RNA.

A variant of ranpirnase (hereinafter, the "'805 variant") is disclosed in U.S. Pat. No. 5,728,805. The '805 variant is also an RNase, and has likewise been found to be cytotoxic to certain cancer cells. The '805 variant is a close variant of ranpirnase; its amino acid sequence is identical to that of ranpirnase except that it has valine instead of isoleucine at position 11 , asparagine instead of aspartic acid at position 20, and arginine instead of serine at position 103 of the ranpirnase amino acid sequence. In some embodiments, the '805 variant is referred to as "Vall l, Asn20, Argl03- Ranpirnase". The amino acid sequence of the '805 variant is provided in SEQ ID NO:2. Amphinase 2 is also an RNase. It is the protein identified as 2325p4 in U.S. Pat. No. 6,239,257 and it too has been found to be cytotoxic to cancer cells. The amino acid sequence of Amphinase 2 is provided in SEQ ID NO: 3.

Recombinant Amphinase 2 ("rAmphinase 2") is similar to Amphinase 2, but has a Met residue at position -1 and lacks glycan moieties that are located in Amphinase 2 at positions 27 and 91. rAmphinase 2 is described in U.S. Pat. No. 7,229,824. The amino acid sequence of rAmphinase 2 is provided in SEQ ID NO: 4.

The term "functionally equivalent thereof is intended to comprise proteins which differ from naturally occurring RNases by one or more amino acids, but retain RNase activity. For example, the '805 variant discussed above may be considered as a functional derivative of ranpirnase.

In one embodiment the term " RNase of the RNase A superfamily" includes a fusion protein of an RNase as described above with another functional moiety such as an immunoglobulin moiety. Such a fusion protein is described in WO 2005/080586. In another embodiment the term " RNase of the RNase A superfamily" does not include a fusion protein of an RNase as described above with another functional moiety such as an immunoglobulin moiety as described for example in

WO 2005/080586.

U.S. Pat. Nos. 5,559,212, 5,728,805, 6,239,257, 7,229,824, 8,518,399, 8,663,964, and U.S. Publication Nos. 2012-0003266 and 2014-0037610 are all incorporated by reference herein in their entireties for all purposes.

Ranpirnase is known to degrade tRNA very effectively (see Lin et al., Biochemical and Biophysical Research Communications 201 (1), 156 - 162 (1994)). Because normal mammalian cells degrade approximately 80% of their tRNA as a natural process, this degradation causes little if any harm to the cells themselves. As a result, except in instances where a viral infection has spread too far to be effectively controlled, a systemic application of ranpirnase, particularly before the viral infection has spread too far, causes the virus to die out without killing the normal cells that the virus infects. The present inventors have found that ranpirnase is effective against Zika virus. Zika virus is a flavivirus, i.e. a single-stranded RNA virus with positive sense RNA which is classified in Baltimore Classification Group IV. A Zika virus infection is usually diagnosed by detecting Zika virus RNA in a patient's serum or by detecting anti-Zika IgM antibodies in the patient's serum or cerebrospinal fluid.

In one aspect, the present invention relates to an Rnase of the Rnase A superfamily for use in inhibiting the growth or replication of Zika virus, or for reducing the ability of Zikavirus to infect cells, comprising contacting cells or tissues with said RNase.

The anti- viral effect of each tested compound may be assessed as a 50% effective concentration (EC50) value, which is the concentration at which the amount of viral transcript is reduced by 50% when compared to cultures infected only with the virus and not treated with an RNase. The term EC50 is used interchangeably herein with AC50 (50% active concentration).

The cytotoxic effect of each tested compound is assessed as a 50% cytotoxic concentration (CC50), which is the concentration that results in the death of 50% of the host cells, as measured in a standard cytoxicity assay such as, for example, trypan blue exclusion or mitochondrial function (e.g., MTT) assay.

The National Institute of Allergy and Infectious Diseases (NIAID) (a component of the National Institute of Health) uses a Selectivity Index (SI) ratio as a common indicator to assess the potency of a test compound. The SI, which equals CC50/EC50 (or CC50/AC50), measures the ability of the tested RNase to inhibit replication of a viral infection without killing the infected cells. Where the SI is greater than 1, the RNase under test is active against the virus indicated, and increasing values of SI indicate increasing activity. Because SI measures the ability of a substance under test to inhibit replication of a particular virus without killing the infected cells themselves, it is reasonably correlated with usefulness of the substance in treating a living subject that is infected with the virus. Accordingly, test results in which SIM indicate that living subjects infected with the virus tested can be treated by administration of an appropriate dose of the corresponding RNase.

In some aspects, the lower range of SI of the ranpirnase is about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 4.0, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, about 10, about 15, about 20. In some aspects, the upper range of SI of the ranpimase is about 2.0, about 2.5, about 3.0, about 4.0, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, about 10, about 15, about 20 or about 25.

In particular aspects, the SI of the ranpirnase ranges from about 1.0 to about 25, about 2.0 to about 24, about 3.0 to about 23, about 4.0 to about 22, about 5.0 to about 21, about 10 to about 20 or about 12 to about 18.

As used herein, the term "about" refers to an amount or number that one of skill in the art would understand is close to the stated amount or number. For example, the term "about" herein refers to an amount that is within 10% above or below the stated amount or number.

The terms "treating" and "treatment," as used herein, refer to administering to a subject infected with Zika virus a therapeutically effective amount of an RNase such as ranpirnase, a ranpirnase variant such as the '805 variant, Amphinase 2, or rAmphinase 2. As used herein, the term "treating" covers any treatment of a viral disease which results in a desired pharmacologic and/or physiologic effect, including arresting disease development, causing regression of the disease, limiting spread of the virus from one cell to another within an individual, limiting replication of a virus in an individual, limiting entry of a virus into the cell of an individual and reducing the number of viruses in an individual or a tissue of this individual. The terms "prevention" and "preventing", as used herein, refer to preventing the disease or a symptom thereof from occuring in a subject which is at risk of developing the disease or symptom, but has not yet been diagnosed as having it.

The term "therapeutically effective amount" is used interchangeably herein with the term "therapeutically effective dose" and refers to an amount of an RNase that results in an improvement or remediation of the symptoms of a disease or condition to be treated. A therapeutically effective amount of an RNase such as ranpirnase, '805 variant, Amphinase 2, or rAmphinase 2, in one embodiment, delays or minimizes the onset of, or hastens or increases recovery of a subject from, a Zika virus infection. In another embodiment, an RNase such as ranpirnase, '805 variant, Amphinase 2, or rAmphinase 2 reduces the overall mortality rate of a disease mediated by a Zika virus infection. In one embodiment, the RNase reduces the viral titer of the infected subject. In another embodiment, the RNase prevents the viral titer of the infected subject from increasing. In one embodiment, a therapeutically effective amount of an RNase provides a therapeutic benefit in the treatment or management of a virus infection or virus -mediated disease. In one embodiment, a therapeutically effective amount of an RNase reduces the spread of the virus from one cell to another. In one embodiment, a therapeutically effective amount of an RNase reduces morbidity or mortality. A therapeutically effective amount may also prevent disease and/or reduce the severity of symptoms.

A therapeutically effective amount can be determined by the skilled person as a matter of routine experimentation. The therapeutically effective dosage of the pharmaceutical composition can be determined readily by the skilled artisan, for example, from animal studies. In addition, human clinical studies can be performed to determine the preferred effective dose for humans by a skilled artisan. Such clinical studies are routine and well known in the art. The precise dose to be employed will also depend on the route of administration. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal test systems. The RNase may be administered to a subject in need thereof in a single dose or in multiple doses. In one embodiment, the RNase is administered to a subject in need thereof once per day, or in multiple doses per day. In one embodiment, the RNase is administered to the subject until symptoms resolve and/or until the subject is no longer at risk of a virus infection.

In some embodiments, the administration of a therapeutically effective amount of the RNase, in particular ranpirnase, reduces the virus titer compared to a control not treated with the RNase, but infected with the virus by at least 10%, preferably by at least 15%, ore preferably by at least 20% and most preferably by at least 25%. Determination of virus titers is discussed in Reischl, Front Biosci. 1996 Aug 1, 1 :e 72-7, Application of molecular biology-based methods to the diagnosis of infectious diseases.

In some embodiments, the administration of a therapeutically effective amount of the RNase, in particular ranpirnase, leads to a reduction of the virus titer below the detection level.

In one embodiment, a therapeutically effective dose may be based on the body weight of the subject in need thereof. In one embodiment, a therapeutically effective dose may be in the range of about 0.001 mg kg to about 1 mg/kg, or about 0.004 to about 0.5 mg kg, or about 0.02 to about 0.1 mg/kg. In one embodiment, a therapeutically effective dose may be about 0.02 mg kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, or about 0.5 mg/kg. However, it is apparent that the dosage may vary depending on the compound, the disease and its severity, as well as the age and the weight of the subject to be treated.

In some embodiments, the RNase, in particular ranpirnase, may be administered within 48 hours after exposure to virus. In other embodiments, the RNase may be administered within 72 hours, four days, five, days, six days, seven days, ten days, 14 days, three weeks, four weeks or two months after exposure to virus.

In some embodiments, multiple doses of the RNase, in particular ranpirnase, are administered. The frequency of administration of these multiple doses may vary, depending on factors such as the severity of symptoms. For example, the RNase may be administered once per month, twice per month, every other week, once per week, twice per week, three times per week, four times per week, every other day, once per day, twice per day or three times a day.

The duration of the administration of the RNase, in particular ranpirnase, i.e. the period over which the RNase is administered, can vary depending on factors such as the severity of symptoms, patient response, etc. For example, the RNase can be administered over a period ranging from one day, three days, seven days, two weeks, four weeks, two months, three months, four months, five months or six months or longer.

As used herein, the term "subject" or "patient" refers to any mammal, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species. Farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats (including cotton rats) and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like are also non-limiting examples. Both adult and newborn individuals are intended to be covered. In particular, the present invention relates to the treatment or prevention of virus infections in human subjects.

The term "systemic administration" is intended to comprise any route of administration of a medication, nutrition or other substance such that it enters into the circulatory system so that the entire body is affected by the administration. In contrast, topical administration acts only locally at the site of administration.

Generally, a person of ordinary skill in this art would conclude that any route by which the RNase, in particular ranpirnase, is systemically administered will be adequate to treat virus infection (although one route may be more effective than another in any particular instance). Thus, enteral administration (including without limitation oral administration and rectal administration) and parenteral administration (including without limitation intravenous administration, intramuscular

administration, and aerosol delivery) are appropriate methods for administration of the RNase, in particular ranpirnase. Additional exemplary appropriate methods for administration of ranpirnase include nasal, buccal, vaginal, ophthalmic, subcutaneous, intraperitoneal, intraarterial, spinal, intrathecal, intra-articular, intra-arterial, sub-arachnoid, sublingual, oral mucosal, bronchial, lymphatic, intra-uterine, integrated on an implantable device such as a suture or in an implantable device such as an implantable polymer, intradural, intracortical, or dermal. Such compositions would normally be administered as pharmaceutically acceptable compositions as described herein.

The term "pharmaceutical composition" as used herein encompasses a composition suitable for administration to a subject, such as a mammal, especially a human. In general a "pharmaceutical composition" is sterile, and free of contaminants that are capable of eliciting an undesirable response within the subject

The term "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. These agents are generally safe, non-toxic and neither biologically nor otherwise undesirable.

Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. The RNase, in particular ranpirnase and ranpirnase variants provided herein may be administered together with other biologically active agents.

A wide variety of pharmaceutically acceptable excipients is known in the art and therefore is not discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al, eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.

The pharmaceutical composition may comprise one or more additional antiviral agents. These antiviral agents inhibit viral replication. They may be specific to the virus to be treated, such as Zika virus, and may include antibodies directed against viral proteins or RNA which interacts with the viral RNA.

The pharmaceutical composition may also comprise compounds which act as immunomodulators to activate the host's defense. Such immunomodulators include cytokines such as interferons and interleukins and toll-like receptor agonists such as imiquimod and resiquimod. In an alternative embodiment the pharmaceutical composition comprises only the RNase as therapeutically active agent.

The RNase, in particular ranpirnase, may be administered to the subject using any convenient means capable of resulting in the desired reduction in viral titers, symptoms of viral infection, etc. Thus, the RNase can be incorporated into a variety of formulations for therapeutic administration. More particularly, the RNase can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, solutions, suppositories, injections, inhalants and aerosols. Aspects of the invention are further illustrated by the following example that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are incorporated herein by reference for all purposes.

Example 1: Zika Virus in Huh-7 Liver Carcinoma Cells

The antiviral activity of ranpirnase against Zika virus strain Uganda MR 766 in Huh-7 human liver carcinoma cells was assessed. Interferon (which is known to be active against this Zika virus strain) was run in parallel as a control.

The ranpirnase and the control were serially diluted to produce eight half- log dilutions in MEM medium. The diluent for ranpirnase was 50 μg/mL gentamicin and serum; the diluent for interferon was 50 μg/mL gentamicin and serum and trypsin. Each dilution was added to 5 wells of a 96-well plate with 80% - 100% confluent cells, and three wells of each dilution were then infected. Two wells remained uninfected as toxicity controls.

The virus was incubated for 4 days at 37 °C and 5% CO2. After cytopathic effect (CPE) was observed microscopically, plates were scored for degree of CPE and then stained with neutral red dye for approximately 2 hours, then supernatant dye was washed from the wells and the incorporated dye was extracted in 50:50 Sorensen citrate buffer/ethanol and read on a spectrophotometer. The optical density of test wells was converted to percent of cell and virus controls, then the concentration of ranpirnase required to inhibit CPE by 50% (IC50) was calculated by regression analysis. The concentration of ranpirnase that would cause 50% CPE in the absence of virus (CC50) was similarly calculated, as was the selectivity index SI.

The results of this experiment are shown in Fig. 1. Ranpirnase demonstrated a selectivity index of 21, indicating activity against the Uganda MR 766 strain of Zika virus.

The selectivity index SI is an accepted measurement of the ability of a drug under test to inhibit replication of a viral infection without killing the infected cells. Where SI in the accompanying Figure is greater than 1, ranpirnase is active against the virus indicated, and increasing values of SI indicate increasing activity. Thus, as can be seen in Fig. 1, ranpirnase is active against the Uganda MR766 strain of Zika virus in the Huh-7 human liver carcinoma.

Because SI measures the ability of a substance under test to inhibit replication of a particular virus without killing the infected cells themselves, it is reasonably correlated with usefulness of the substance in treating a mammalian subject that is infected with the virus. Accordingly, test results in which SI > 1 indicate that mammalian subjects infected with with Uganda MR 766 strain of Zika virus can be treated by systemic administration of an appropriate dose of ranpirnase. Additionally, activity against the Uganda MR 766 strain of the Zika virus is reasonably correlated with activity against all strains of the Zika virus because such strains are similar and behave similarly. Furthermore, other below-disclosed experimental results in VEEV, CHIV, and RV -14 indicate that it should be possible to use ranpirnase as a prophylactic to prevent Zika viral infection.

Claims

1. RNase of the RNase A superfamily or a functional derivative thereof for use in treating a Zika virus infection in a mammalian subject, wherein the RNase has an amino acid sequence selected from the group consisting of SEQ ID Nos. 1, 2, 3 and 4.

2. The RNase of the RNase A superfamily or functional derivative thereof for the use of claim 1, wherein the subject is a human being.

3. The RNase of the RNase A superfamily or functional derivative thereof for the use of any of claims 1 or 2, wherein the RNase is administered systemically.

4. RNase of the RNase A superfamily or a functional derivative thereof for use in preventing a Zika virus infection in a mammalian subject, wherein the RNase has an amino acid sequence selected from the group consisting of SEQ ID Nos. 1, 2, 3 and 4.

5. The RNase of the RNase A superfamily or functional derivative thereof for the use of claim 4, wherein the subject is a human being.

6. The RNase of the RNase A superfamily or functional derivative thereof for the use of any of claims 4 or 5, wherein the RNase is administered systemically.