WO2021188854A1 - Ribonucleases for treating viral infections - Google Patents

Abstract

This disclosure is directed to compounds and pharmaceutical compositions for treating and preventing viral diseases, as Covid-19. Among others, the invention relates to the use of ranpirnase in the preparation and use of pharmaceutical formulations for the intravenous treatment of said disease.

Description

RIBONUCLEASES FOR TREATING VIRAL INFECTIONS

FIELD OF THE DISCLOSURE

[1] This disclosure is directed to compounds and pharmaceutical compositions for treating and preventing Covid-19. Among others, the invention relates to the use of ranpirnase in the preparation and use of pharmaceutical formulations for the intravenous treatment of said disease.

SEQUENCE LISTING STATEMENT

[001] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 19, 2021, is namedP-603950-PC-SQL-ST25.txt and is 2.15 Kbytes in size.

BACKGROUND

[2] Several human diseases are caused by viruses, as the common cold, influenza, chickenpox, cold sores, rabies, Ebola virus disease, AIDS (HIV), avian influenza, SARS, and Covid-19. These diseases are usually detected by clinical presentation, for instance severe muscle and joint pains preceding fever, or skin rash and swollen lymph glands.

[3] Coronaviruses (CoV) are a large family of viruses that cause illness ranging from the common cold to more severe diseases such as Middle East Respiratory Syndrome (MERS-CoV), Severe Acute Respiratory Syndrome (SARS-CoV), and Covid-19. Coronaviruses are in the subfamily Orthocoronavirinae in the family Coronaviridae, in the order Nidovirales. They are enveloped viruses with a positive-sense single- stranded RNA genome and a nucleocapsid of helical symmetry. The genome size of coronaviruses ranges from approximately 26 to 32 kilobases, the largest for an RNA virus. Coronaviruses are zoonotic, meaning they are transmitted between animals and people. Coronaviruses further cause colds with major symptoms, such as fever and sore throat from swollen adenoids, primarily in the winter and early spring seasons, pneumonia, and bronchitis, among others.

[4] The novel coronavirus SARS-CoV-2, informally known as the Wuhan coronavirus, is a contagious virus that causes acute respiratory diseases, and has been the cause of a major virus outbreak known as 2019-20 Wuhan coronavirus outbreak. The virus is thought to have a zoonotic origin, as suggested by its similarity to SARS-CoV and bat coronaviruses. However, human-to-human transmission of the virus has been confirmed, primarily through close contact, in particular through respiratory droplets from coughs and sneezes. Viral RNA has also been found in stool samples from infected patients.

[5] There are no vaccines or antiviral drugs to prevent or treat human coronavirus infections.

SUMMARY OF THE INVENTION

[6] In one aspect, disclosed herein is a pharmaceutical composition for treating or preventing Covid-19 in a subject, wherein said composition consists of ranpirnase as a single active agent and a pharmaceutical carrier, and is formulated for intravenous delivery.

BRIEF DESCRIPTION OF THE DRAWINGS

[7] The subject matter disclosed herein is particularly pointed out and distinctly claimed in the concluding portion of the specification. The compositions and methods for using thereof disclosed herein may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[8] Figure 1 shows an assay of ranpirnase and FDA-Approved Drugs efficacy against SARS-CoV-2 in vitro.

DETAILED DESCRIPTION

[9] In some embodiments, disclosed herein is a composition for treating or preventing a viral disease in a subject, said composition formulated for intravenous delivery and comprising ribonuclease as a single active agent. In some embodiments, disclosed herein is a composition for treating or preventing a Covid-19 in a subject, said composition formulated for intravenous delivery comprising ranpirnase as a single active agent.

[10] A skilled artisan would appreciate that Ranpirnase, called herein also “onconase”, “P-30”, “TMR004”, and “Pannon”, is a ribonuclease enzyme found in the oocytes of the Northern Leopard Frog (Rana pipiens). Ranpirnase is a member of the pancreatic ribonuclease (RNase A) protein superfamily and degrades RNA substrates with a sequence preference for uracil and guanine nucleotides. Ranpirnase has been studied as a potential cancer and antiviral treatment due to its unusual mechanism of cytotoxicity tested against transformed cells and antiviral activity. Ranpirnase UniProt identification number is P85073.

[11] In some embodiments, ranpirnase comprises an amino acid sequence comprising EDWLTFQKKHITNTRDVDCDNIMSTNLFHCKDKNTFIYSRPEPVKAICKGIIASKN VFTT S EF YES DCN VT S RPCKYKFKKS TNKFC VT CEN Q AP VHF V G V GS C (SEQ ID No.: 1). In some embodiments, ranpirnase comprises an amino acid sequence comprising at least 80%, 85%, 90%, 95%, or 99% homology to SEQ ID No.:l.

[12] In some embodiments, ranpirnase comprises an amino acid sequence comprising ED WLTF QKKH VTNTRD VDCNNIMS TNLFHC KDKNTFI Y S RPEP VKAICKGII AS K N VLTT S EF YLS DCN VT S RPCKYKLKKS TNKFC VT CEN Q AP VHF V G V GRC (SEQ ID No.: 2). In some embodiments, ranpirnase comprises an amino acid sequence comprising at least 80%, 85%, 90%, 95%, or 99% homology to SEQ ID No.:2.

[13] A skilled artisan would appreciate that different ribonucleases exert their antiviral activity by different mechanisms. All are relevant to the compositions and methods of the present disclosure. In some embodiments, a ribonuclease enters into the cells via receptor- mediated endocytosis and once internalized into the cytosol, selectively degrades tRNA, resulting in inhibition of protein synthesis and induction of cell apoptosis.

Viral Diseases

[14] In some embodiments, a viral disease comprises Covid-19. In some embodiments, a viral disease is caused by SARS-CoV-2. A skilled artisan will recognize that Covid-19, also termed “novel coronavirus pneumonia”, “NCP”, “SARS-CoV-2 acute respiratory disease”, and “COVID-19” comprises an infectious respiratory disease caused by the 2019 novel coronavirus (SARS-CoV-2), which was first detected during the 2019-20 Wuhan coronavirus outbreak. In some embodiments, SARS-CoV-2 is transmitted through human- to-human transmission, generally via respiratory droplets as sneeze, cough or exhalation. In some embodiments, NCP symptoms appear after an incubation period of between 2 to 14 days. In some embodiments, coronavirus primarily affects the lower respiratory tract. In some embodiments, coronavirus primarily affects the upper respiratory tract. In some embodiments, NCP symptoms comprise fever, coughing, shortness of breath, pain in the muscles, tiredness, pneumonia, acute respiratory distress syndrome, sepsis, septic shock, death, or any combination thereof. [15] A skilled artisan will recognize that SARS-CoV-2 belongs to the broad family of viruses known as coronaviruses. SARS-CoV-2 is a positive-sense single- stranded RNA (+ssRNA) virus. SARS-CoV-2 is a member of the subgenus Sarbecovirus (Beta-CoV lineage B), having an RNA sequence of approximately 30,000 bases in length.

[16] Eighty-one genomes of SARS-CoV-2 had been isolated and reported. The present disclosure comprises compositions and methods for treating these SARS-CoV-2 variants, or any further one.

[17] A skilled artisan will recognize that seven coronavirus types are known to affect humans. The compositions and methods disclosed herein are useful for treating any of them. In some embodiments, coronavirus comprises Human coronavirus 229E (HCoV- 229E). In some embodiments, coronavirus comprises Human coronavirus OC43 (HCoV- OC43). In some embodiments, coronavirus comprises Severe acute respiratory syndrome- related coronavirus (SARS-CoV). In some embodiments, coronavirus comprises Human coronavirus NL63 (HCoV-NL63, New Haven coronavirus). In some embodiments, coronavirus comprises Human coronavirus HKU1. In some embodiments, coronavirus comprises Middle East respiratory syndrome-related coronavirus (MERS-CoV), previously known as novel coronavirus 2012 and HCoV-EMC. In some embodiments, coronavirus comprises Novel coronavirus (SARS-CoV-2), also known as Wuhan coronavirus.

Methods of Treatment

[18] In one embodiment, the compositions of the invention are used in the methods of the invention described herein. In one embodiment, the invention provides a method of preventing or treating a viral disease, for example Covid-2019 in a subject, comprising any of the compositions disclosed herein. In one embodiment, the term “treatment” refers to any process, action, application, therapy, or the like, wherein a subject, including a human being, is subjected to medical aid with the object of improving the subject's condition, directly or indirectly. In another embodiment, the term “treating” refers to reducing incidence, or alleviating symptoms, eliminating recurrence, preventing recurrence, preventing incidence, improving symptoms, improving prognosis or combinations thereof in other embodiments. [19] “Treating” embraces in another embodiment, the amelioration of an existing condition. The skilled artisan would understand that treatment does not necessarily result in the complete absence or removal of symptoms. Treatment also embraces palliative effects: that is, those that reduce the likelihood of a subsequent medical condition. The alleviation of a condition that results in a more serious condition is encompassed by this term.

[20] As used herein, “subject” refers in one embodiment, to a human or any other animal which has been exposed to and is now immune to CoV related disease or Covid-2019. A subject refers to a human presenting to a medical provider for diagnosis or treatment of a disease, such as a CoV related disease or Covid-2019 in another embodiment. A human includes pre- and postnatal forms. In one embodiment, subjects are humans being treated for symptoms associated with a CoV related disease or Covid-2019.

[21] The term “therapeutically effective amount” or “effective amount” refers in one embodiment, to an amount of a monovalent or combination vaccine sufficient to elicit a protective immune response in the subject to which it is administered. The immune response may comprise, without limitation, induction of cellular and/or humoral immunity.

[22] The dosage regimen for treating a condition with the compositions of this invention is selected in one embodiment, in accordance with a variety of factors, such as the type, age, weight, ethnicity, sex and medical condition of the subject, the severity of the condition treated, and the particular compound employed, and thus may vary widely while still be in the scope of the invention.

Pharmaceutical Compositions

[23] In some embodiments, the compositions disclosed herein are formulated for intravenous (iv) delivery. In one embodiment, the pharmaceutical preparation of the invention, used in the methods of the invention comprise a carrier, excipient, flow agent, processing aid, a diluent, or a combination thereof.

[24] In one embodiment, the compositions used in the invention further comprise a carrier, or excipient, lubricant, flow aid, processing aid or diluent in other embodiments, wherein the carrier, excipient, lubricant, flow aid, processing aid or diluent is a gum, starch, a sugar, a cellulosic material, an acrylate, calcium carbonate, magnesium oxide, talc, lactose monohydrate, magnesium stearate, colloidal silicone dioxide or mixtures thereof. [25] In another embodiment, the composition further comprises a binder, a disintegrant, a buffer, a protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an emulsifier, a stabilizing agent, a viscosity increasing agent, a sweetener, a film forming agent, or any combination thereof.

[26] In one embodiment, the term “pharmaceutically acceptable carriers” includes, but is not limited to, may refer to 0.01-0.1M and preferably 0.05M phosphate buffer, or in another embodiment 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be in another embodiment aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.

[27] The pharmaceutical preparations of the invention can be prepared by known dissolving, mixing, granulating, or tablet-forming processes. In addition, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.

[28] In some embodiments, prior to administration, the lyophilizate is resuspended aseptically in a sterile buffer. In some embodiments, a physiologically tolerated buffer is added to facilitate pH control. In some embodiments, the formulations of the present invention have pH between about 6.8 and about 7.8. In some embodiments, buffers include phosphate buffers, sodium phosphate, or phosphate buffered saline (PBS). In some embodiments, the final product lyophilizate may include preformulated isotonicity agents as glycerin, stabilizers excipients, such as carbohydrates (trehaloze, sucrose), an antioxidant, a chelating agent, such as EDTA and EGTA, human serum albumin, or a combination thereof, which can optionally be added to the formulations or compositions to reduce aggregation. Surfactants additives are particularly useful if a pump or plastic container is used to administer the formulation. An optional carrier additive is human serum albumin, or an enhancer surfactant as described below. The presence of pharmaceutically acceptable surfactant mitigates the propensity of proteins to aggregate. [29] The term “about” as used herein means in quantitative terms plus or minus 5%, or in another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment plus or minus 20%.

[30] The term “subject” refers in one embodiment to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae. The subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice and humans. The term “subject” does not exclude an individual that is normal in all respects.

[31] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

Example 1

Pre- Clinical Evaluation

[32] The objective of this study is to test ranpirnase potency as IV monotherapy in a murine SARS-CoV virus infection in-vivo model.

[33] BALB/c male and/or female mice (6-8 weeks of age) will be allowed to habituate for 1 week prior to the study. The animals will be housed in climate-controlled quarters (24°C at 50% humidity) with 12 h light/dark cycles and had free access to food and water. All experimental protocols will be approved by an Institutional Animal Welfare Committee.

[34] A. Model establishment of Severe acute respiratory syndrome (SARS)-CoV infection.

Group allocation: a. Baseline group (without infection) (n=10) b. 1 x 102 PFU (n =10) c. 1 x 103 PFU (n =10) d. 1 x 105 PFU (n =10)

[35] B. Ranpirnase potency (0.1-5 pg/ml) as monotherapy (via IV) Group allocation: a. Baseline group (without infection) (n=10) b. Negative control group (infected, no active therapy) (n =10) c. Ranpirnase low dose IV administration (n =10) d. Ranpirnase medium dose IV administration (n =10) e. Ranpirnase high dose IV administration (n =10)

[36] SARS-Cov Model induction: Murine or human strain suitable to work up to BSL- 2 laboratories. Animals will be anesthetized with a mixture of ketamine-xylazine and infected intranasally either with the virus or with phosphate-buffered saline (PBS) in a dose of 50 pi. Virus doses ranged from 1 x 102 to 1 x 105 PFU/50-mI dose, diluted in PBS prior to intranasal administration (Day CW et al. Virology 2009).

[37] Study duration: 4 weeks (one-week STZ, 4 weeks treatment)

[38] Handling: 5 weeks (including one-week acclimation)

[39] Animals: BALB/c male and/or female mice (6-8 weeks of age)

[40] Examinations

[41] Body weight: 3 times a week.

[42] Morbidity & mortality check: Daily.

[43] Clinical observation: 3 times a week.

[44] Termination

[45] Animals will be sacrificed four weeks following treatment initiation, gross pathology will be performed, and lungs tissues will be collected.

[46] Lung samples from each test group will be pooled and homogenized in MEM solution and assayed in duplicate for infectious for virus yield assays using triplicate wells of Vero 76 cells.

[47] Lung tissues will be fixed in PBS-4% paraformaldehyde (pH 7.3); tissues will be embedded in paraffin; and 5-pm-thick sections will be prepared for histopathology. Extent of inflammation will be determined, sections will be stained with hematoxylin and eosin (H&E) and will be scored from 1 to 5 for overall inflammation, eosinophilia, neutrophilia, alveolitis, bronchiolar denudation, and edema. [48] Hyaline membrane formation will be evaluated in lungs in comparison to control groups.

[49] Blood will be harvested from all animals (by terminal bleeding), serum separation and complete blood count and biochemistry panel will be performed.

[50] Multiplex ELISA will be performed at different time (serum and lung samples) points following challenge and treatment to measure IL-la and IL-6, and chemokines MIP- la, MCP-1, and RANTES.

Example 2

Ranpirnase Efficacy in vitro

[51] The objective of this experiment was to compare the efficacy of ranpirnase and FDA approved drugs for reducing SARS-CoV-2 concentration in vitro.

[52] Ranpirnase was one of 2 drugs to reduce virus concentrations in the assay (Figure 1) Similarly, UTHSC tested ranpirnase in their high-throughput screening (HTS) method for SARS-CoV-2 with the 50% effective dose (EC50) ~6 mM.

[53] Based on the in vitro activity against SARS-CoV-2, ranpirnase is expected to be active against SARS-CoV-2 in a hamster infection study, in which hamsters will be challenged with SARS-CoV-2 with analysis of lung infection on days 3, 5, and 7 to evaluate virus reduction in these tissues.

Example 3

Clinical Study of Ranpirnase for Treating Covid-19

[54] The proposed clinical study is expected to present minimal risk to participants while contributing to development of a safe, efficacious COVID-19 therapeutic. The clinical research will be conducted in accordance with applicable International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines and FDA regulations and guidance. Ranpirnase will be tested in a human Phase II/III clinical trial to evaluate the technical feasibility of ranpirnase to reduce viral load and risk of an IL-6 cytokine storm in COVID-19 patients. Table 1 shows the Phase II/III protocol synopsis we are proposing to conduct. The study is designed for participation by adult (> 21 years old) COVID-19 patients. The clinical study will enroll up to 45 subjects with a 3:1 treatmenkplacebo (standard of care) ratio. All inclusion and exclusion criteria must be met for eligibility. Evaluation of this investigational therapeutic will include laboratory tests, medical history, physical assessment by clinicians, and subject self-assessment (if appropriate). The hypotheses are that ranpirnase is safe and reduces the viral burden in COVID-19 patients. Primary endpoints will be safety and efficacy. The assessment of product safety will include clinical observation and monitoring of hematological, and chemical parameters while efficacy assessment will evaluate virological measures.

[55] Table 1: Protocol Synopsis for Phase TT/TTT Clinical Study

[56] Having described preferred embodiments of the invention, it is to be understood that the invention is not limited to the precise embodiments, and that various changes and modifications may be affected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

Claims

1. A pharmaceutical composition for treating or preventing Covid-19 in a subject, wherein said composition consists of ranpirnase as a single active agent and a pharmaceutical carrier, and is formulated for intravenous delivery.