WO2022197842A1

WO2022197842A1 - Methods of treating pulmonary fibrosis associated with viral infection using tissue differentiation factor related polypeptides (tdfrps)

Info

Publication number: WO2022197842A1
Application number: PCT/US2022/020610
Authority: WO
Inventors: William D. Carlson
Original assignee: Therapeutics By Design, LLC
Priority date: 2021-03-16
Filing date: 2022-03-16
Publication date: 2022-09-22

Abstract

The present disclosure relates generally to methods of treating or preventing pulmonary fibrosis associated with viral infection, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP), wherein the TDFRP is administered in an amount effective to treat or prevent the pulmonary fibrosis in the subject.

Description

METHODS OF TREATING PULMONARY FIBROSIS ASSOCIATED WITH VIRAL INFECTION USING TISSUE DIFFERENTIATION FACTOR RELATED

POLYPEPTIDES (TDFRPs)

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/161,610, filed on March 16, 2021, the contents of which is incorporated by reference in its entirety herein.

BACKGROUND OF THE INVENTION

Coronavimses, a genus in the family of Coronaviridae, are large, enveloped plus strand RNA viruses. Coronavimses are named for the crown-like spikes on their surface. The genomic RNA is 27 to 32 kb in size, capped and polyadenylated. Three serologically distinct groups of coronavimses have been identified. Within each group, vimses are identified by hosts range and genome sequence. Coronavimses have been identified in mice, rats, chickens, turkeys, swine, dogs; cats, rabbits, horses, cattle and humans (Guy et ah, J Clin Microbiol.

38, 4523-4526 (2000); Holmes & Lai, M. M. C. Fields Virology. Fields, B. N., Knipe, D. M., Howley, P. M. & et al (eds.), pp. 1075-1093 (Lippincott-Raven Publishers, Philadelphia, 1996)). Coronavimses are classified as a family within the Nidovirales order, vimses that replicate using a nested set of mRNAs ("nido-" for "nest"). The coronavims subfamily is further classified into four genera: alpha, beta, gamma, and delta coronavimses. The human coronavimses (HCoVs) are in two of these genera: alpha coronavimses (HCoV-229E and HCoV-NL63) and beta coronavimses (HCoV-HKUl, HCoV-OC43, Middle East respiratory syndrome coronavims [MERS-CoV], the severe acute respiratory syndrome coronavims [SARS-CoV]), and SARS-CoV-2 (the novel coronavims that causes coronavims disease 2019, or COVID-19). At the end of 2019, a novel coronavims was identified as the cause of a cluster of pneumonia cases in Wuhan, a city in the Hubei Province of China. It rapidly spread, resulting in an epidemic throughout China, with sporadic cases reported globally. In February 2020, the World Health Organization designated the disease COVID-19, which stands for coronavims disease 2019 (World Health Organization. Director-General's remarks at the media briefing on 2019-nCoV on 11 February 2020. https://www.who.int/dg/speeches/detail/who-director-general-s-remarks-at-the-media- briefing-on-2019-ncov-on-ll-febmary-2020). The vims that causes COVID-19 is designated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); previously, it was referred to as 2019-nCoV.

Following the 2003 epidemic of severe acute respiratory syndrome (SARS), it was noticed that many patients who survived the severe illness developed residual pulmonary fibrosis, as shown by clinical findings and radiography (Venkataraman and Frieman.

Antiviral Res. 2017 Jul;143:142-150. doi: 10.1016/j. antiviral.2017.03.022. Epub 2017 Apr 5). Fibrosis is characterized by excessive deposition of extracellular matrix components and overgrowth of fibroblasts. Fibrosis can occur in all tissues but is especially prevalent in organs with frequent exposure to chemical and biological insults including the lung, skin, digestive tract, kidney, and liver (Eddy, 1996, J Am Soc Nephrol, 7(12):2495-503; Dacic el al, 2003, Am J Respir Cell Mol Biol, 29S: S5-9; Wynn, 2004, Nat Rev Immunol, 4(8):583- 94).

Induction of fibrosis is not unique to SARS-CoV. A recent meta-analysis of global idiopathic pulmonary fibrosis (IPF) rate finds that from the year 2000 onwards, a conservative incidence range of 3-9 cases per 100,000 per year for Europe and North America (Hutchinson et al, Eur Respir J. 2015 Sep; 46(3):795-806). In the case of patients suffering from IPF, there is no known trigger for the onset of disease but viral infections are thought to be a co-factor (Naik and Moore, Expert Rev Respir Med. 2010 Dec; 4(6):759-71; Vannella and Moore, Fibrogenesis Tissue Repair. 2008 Oct 13; 1(1):2). Herpesviruses, such as Epstein-Barr virus (EBV) or human cytomegalovirus (HCMV), adenovirus, transfusion- transmitted virus (TTV, also known as Torque Teno Virus) and hepatitis C virus (HCV) have all been associated with IPF disease by detection of antibodies against viral proteins or viral gene products in lungs of IPF patients (Naik and Moore, 2010). Elevated serum levels of chemokines (such as transforming growth factor Beta 1 (TGF-bI)) and development of pulmonary fibrosis have also been reported in influenza A (H1N1) virus -infected patients (Wen et al, J Investig Allergol Clin Immunol. 2011; 21(l):44-50). Irrespective of the etiology, pulmonary fibrosis has been shown to develop after apparent recovery from the infection. This suggests that the some of the pathways induced by SARS-CoV infection that lead to the observed pulmonary fibrosis may be shared irrespective of the damage inducing factor.

The transforming growth factor beta (TGFP) protein family consists of three distinct isoforms found in mammals (TGFpi, TGFP2, and TGFp3). The TGFP proteins activate and regulate multiple gene responses that influence disease states, including cell proliferative, inflammatory, and cardiovascular conditions. TGFP is a multifunctional cytokine originally named for its ability to transform normal fibroblasts to cells capable of anchorage- independent growth. The TGFPs are known to be involved in many proliferative and non proliferative cellular processes such as cell proliferation and differentiation, embryonic development, extracellular matrix formation, bone development, wound healing, hematopoiesis, and immune and inflammatory responses.

The canonical TGF-b signal transduction is initiated with the ligand binding to its serine/threonine kinase receptors on the cell surface, which leads to the activation of downstream cytoplasmic effectors, the Smad proteins. The receptor- activated Smad2 and Smad3 are involved in TGF-b signaling. Once phosphorylated by the type I receptor, they form heteromeric complexes with Smad4, and the Smad heterocomplexes are accumulated in the nucleus, where they regulate target gene transcription in association with DNA binding partners.

TGF-b plays a pivotal role in pulmonary fibrosis (Roberts el al. (2006) Cytokine Growth Factor Rev. 17, 19-27, 9). It increases the production of extracellular matrix proteins, enhances the secretion of protease inhibitors, and reduces secretion of proteases, thus leading to deposition of extracellular matrix proteins. TGF-b can also induce pulmonary fibrosis directly through stimulation of fibroblast chemotactic migration and proliferation as well as fibroblast-myofibroblast transition.

Zhao et al. (J Biol Chem. 2008 Feb 8;283(6):3272-80. Epub 2007 Nov 30) have shown that SARS-associated coronavims (SARS-CoV) nucleocapsid (N) protein potentiates TGF^-induced expression of plasminogen activator inhibitor- 1 but attenuates Smad3/Smad4-mediated apoptosis of human peripheral lung epithelial HPL1 cells. The promoting effect of N protein on the transcriptional responses of TGF-b is Smad3- specific. N protein associates with Smad3 and promotes Smad3-p300 complex formation while it interferes with the complex formation between Smad3 and Smad4. These findings provide evidence of a novel mechanism whereby N protein modulates TGF-b signaling to block apoptosis of SARS-CoV-infected host cells and meanwhile promote tissue fibrosis.

The development of new treatment strategies for treating lung fibrosis associated with corona vims infection are needed that will reverse or stop the progression of disease.

SUMMARY

According to one aspect, the disclosure features a method of treating pulmonary fibrosis associated with viral infection, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP), wherein the TDFRP is administered in an amount effective to treat the pulmonary fibrosis in the subject. According to one aspect, the disclosure features a method of preventing pulmonary fibrosis associated with viral infection, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide

(TDFRP), wherein the TDFRP is administered in an amount effective to prevent the pulmonary fibrosis in the subject. According to one aspect, the disclosure features a method of reducing pulmonary fibrosis associated with viral infection, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP), wherein the TDFRP is administered in an amount effective to reduce the pulmonary fibrosis in the subject. According to some embodiments, the disclosure features a method of treating coronavims disease (COVID-19) in a subject, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP), wherein the TDFRP is administered in an amount effective to treat COVID-19 in the subject. According to some embodiments, the subject has been diagnosed with COVID-19. According to some embodiments of any of the above aspects, the TDFRP is administered by a pulmonary route. According to some embodiments of any of the above aspects, the TDFRP is administered using a nebulizer. According to some embodiments of any of the aspects or embodiments herein, the viral infection is caused by a virus from the Coronaviridae family. According to some embodiments, vims from the

Coronaviridae family belong to a genus selected from the group consisting of

Alphacoronavirus , Betacoronavirus, Gammacoronavirus , Deltacoronavirus, Torovirus, and

Bafinivirus. According to some embodiments, the Betacoronavirus is Middle East respiratory syndrome (MERS). According to some embodiments, the Betacoronavirus is severe acute respiratory syndrome (SARS). According to some embodiments, the Betacoronavirus is severe acute respiratory syndrome coronavims 2 (SARS-CoV-2). According to some embodiments of the embodiments and aspects herein, the TDFRP is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. According to some embodiments of the embodiments and aspects herein, the TDFRP is 90%, 91%, 92%, 93%,

94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 1. According to some embodiments of the embodiments and aspects herein, the TDFRP is 90%, 91%, 92%, 93%,

94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 2. According to some embodiments of the embodiments and aspects herein, the TDFRP is 90%, 91%, 92%, 93%,

94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 3. According to some embodiments of the embodiments and aspects herein, the TDFRP is a Multiple Domain TDFRP. According to some embodiments, the TDFPR is administered with an additional agent. According to one embodiment, the serum half-life of the TDFRP is increased when administered with the additional agent, compared to administration of the TDFRP alone. According to some embodiments, the additional agent is an angiotensin converting enzyme (ACE) inhibitor, a neprilysin inhibitor or an angiotensin receptor-neprilysin inhibitor. According to some embodiments, the angiotensin converting enzyme (ACE) inhibitor is selected from the group consisting of captopril, zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, trandolapril, fosinopril, moexipril, cilazapril, spirapril, temocapril, alacepril, ceronapril, delepril, moveltipril, and combinations thereof. According to some embodiments, the neprilysin inhibitor is selected from the group consisting of thiorphan, candoxatril, and candoxatrilat. According to some embodiments, the angiotensin receptor-neprilysin inhibitor is sacubitril/valsartan. According to some embodiments, the additional agent is selected from the group consisting of: anti-neoplastic agents, antibiotics, vaccines, immunosuppressive agents, anti-hypertensive agents and mediators of the hedgehog signaling pathway.

According to another aspect, the disclosure features a kit comprising a pharmaceutical composition comprising at least one TDFRP and instructions for use in treating pulmonary fibrosis associated with viral infection. According to another aspect, the disclosure features a kit comprising a pharmaceutical composition comprising at least one TDFRP and instructions for use in preventing pulmonary fibrosis associated with viral infection.

DETAILED DESCRIPTION

Provided herein are TDFRPs that can advantageously be used to treat or prevent pulmonary fibrosis associated with viral infection.

Definitions

As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise.

The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

As used herein, the term “about,” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or

±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. As used herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

As used herein, “comprise,” “comprising,” and “comprises” and “comprised of’ are meant to be synonymous with “include”, “including”, “includes” or “contain”, “containing”, “contains” and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

As used herein, the terms “such as”, “for example” and the like are intended to refer to exemplary embodiments and not to limit the scope of the present disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, preferred materials and methods are described herein.

As used herein, “administration,” “administering” and variants thereof refers to introducing a composition or agent into a subject and includes concurrent and sequential introduction of a composition or agent. "Administration" can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. "Administration" also encompasses in vitro and ex vivo treatments. The introduction of a composition or agent into a subject is by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, or topically. Administration includes self-administration and the administration by another. Administration can be carried out by any suitable route. A suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.

As used herein, the term “analog” is meant to refer to a composition that differs from the compound of the present disclosure but retains essential properties thereof. A non limiting example of this is a polypeptide or peptide or peptide fragment that includes non natural amino acids, peptidomimetics, unusual amino acids, amide bond isosteres.

As used herein, an "effective amount" of a compound is meant to refer to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, for example, an amount which results in the prevention of or a decrease in the symptoms associated with a disease that is being treated, e.g., lung fibrosis associated with viral infection. The amount of compound administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. Typically, an effective amount of the compounds of the present disclosure, sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day, to about 10,000 mg per kilogram body weight per day. Preferably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. The compounds of the present disclosure can also be administered in combination with each other, or with one or more additional therapeutic compounds.

As used herein, an "isolated" or "purified" polypeptide or polypeptide or biologically- active portion thereof is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the tissue differentiation factor-related polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.

As used herein, the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a natural or synthetic peptide containing two or more amino acids linked typically via the carboxy group of one amino acid and the amino group of another amino acid. As will be appreciated by those having skill in the art, the above definition is not absolute and polypeptides or peptides can include other examples where one or more amide bonds could be replaced by other bonds, for example, isosteric amide bonds.

The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms “polypeptide”, “peptide” and “protein” also are inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma- carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. It will be appreciated, as is well known and as noted above, that polypeptides may not be entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslational events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well.

As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compound.

As used herein, the term “ small molecule” is meant to refer to a composition that has a molecular weight of less than about 5 kDa and more preferably less than about 2 kDa.

Small molecules can be, e.g., nucleic acids, peptides, polypeptides, glycopeptides, peptidomimetics, carbohydrates, lipids, lipopolysaccharides, combinations of these, or other organic or inorganic molecules.

As used herein, the terms “subject,” “individual,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. The methods described herein are applicable to both human therapy and veterinary applications. According to some embodiments, the subject is a mammal, and in other embodiments the subject is a human. According to some embodiments, a “subject in need” is meant to refer to a subject that has been diagnosed with a viral infection. According to some embodiments, a “subject in need” is meant to refer to a subject that is susceptible to a viral infection. Non-limiting examples of a subject that is susceptible to a viral infection include immunocomprosmised subjects, elderly subjects and infants. .

As used herein, the terms “therapeutic amount”, "therapeutically effective amount", an "amount effective", or “pharmaceutically effective amount” of an active agent (e.g. a TDFRP), as described herein, are used interchangeably to refer to an amount that is sufficient to provide the intended benefit of treatment (e.g., reduction in lung fibrosis). However, dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular active agent employed. Thus the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods. Additionally, the terms “therapeutic amount”, “therapeutically effective amounts” and “pharmaceutically effective amounts” include prophylactic or preventative amounts of the compositions of the disclosure. In prophylactic or preventative applications of the disclosure, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including biochemical, histologic and/or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes presenting during development of the disease, disorder or condition. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to some medical judgment. The terms “dose” and “dosage” are used interchangeably herein.

As used herein, the term “Transforming Growth Factor- beta (TGF-b) superfamily of polypeptides,” is meant to refer to a superfamily of polypeptide factors with pleiotropic functions that is composed of many multifunctional cytokines which includes, but is not limited to, TGF-Bs, activins, inhibins, anti-miillerian hormone (AMH), mullerian inhibiting substance (MIS), bone morphogenetic proteins (BMPs), and myostatin. The highly similar TGF-b isoforms TGF-Bl, TGF-B2, and TGF-B3 potently inhibit cellular proliferation of many cell types, including those from epithelial origin. Most mesenchymal cells, however, are stimulated in their growth by TGF-b. In addition, TGF-Bs strongly induce extracellular matrix synthesis and integrin expression, and modulate immune responses. BMPs, also known as osteogenic proteins (OPs), are potent inducers of bone and cartilage formation and play important developmental roles in the induction of ventral mesoderm, differentiation of neural tissue, and organogenesis. Activins, named after their initial identification as activators of follicle- stimulating hormone (FSH) secretion from pituitary glands, are also known to promote erythropoiesis, mediate dorsal mesoderm induction, and contribute to survival of nerve cells. Several growth factors belonging to the TGF-b superfamily play important roles in embryonic patterning and tissue homeostasis. Their inappropriate functioning has been implicated in several pathological situations like fibrosis, rheumatoid arthritis, and carcinogenesis. The term, tissue differentiation factor (TDF), as used herein, includes, but is not limited to, all members of the TGF-beta superfamily of polypeptides. TGF-beta superfamily polypeptides can be antagonists or agonists of TGF-beta superfamily receptors. As used herein, the term “Transforming Growth Factor- beta (TGF-beta) superfamily receptors,” is meant to refer to polypeptide receptors that mediate the pleiotropic effects of transforming growth factor- B (TGF-B) superfamily polypeptides, as well as fragments, analogs and homologs thereof. Such receptors may include, but are not limited to, distinct combinations of Type I and Type II serine/threonine kinase receptors. The term, tissue differentiation factor receptor (TDF), as used herein, includes, but is not limited to, all members of the TGF-beta superfamily of receptors.

As used herein, the terms “treat,” “treating,” and/or “treatment” include abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms of a condition, obtaining beneficial or desired clinical results. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).

Beneficial or desired clinical results, such as pharmacologic and/or physiologic effects include, but are not limited to, preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabili ation (i.e., not worsening) of the disease, disorder or condition, preventing spread of the disease, disorder or condition, delaying or slowing of the disease, disorder or condition progression, amelioration or palliation of the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

As used herein the term “therapeutic effect” refers to a consequence of treatment, the results of which are judged to be desirable and beneficial. A therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation. A therapeutic effect can also include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.

For any therapeutic agent described herein the therapeutically effective amount may be initially determined from preliminary in vitro studies and/or animal models. A therapeutically effective dose may also be determined from human data. The applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan. General principles for determining therapeutic effectiveness, which may be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference, are summarized below.

Pharmacokinetic principles provide a basis for modifying a dosage regimen to obtain a desired degree of therapeutic efficacy with a minimum of unacceptable adverse effects. In situations where the drug's plasma concentration can be measured and related to the therapeutic window, additional guidance for dosage modification can be obtained.

Drug products are considered to be pharmaceutical equivalents if they contain the same active ingredients and are identical in strength or concentration, dosage form, and route of administration. Two pharmaceutically equivalent drug products are considered to be bioequivalent when the rates and extents of bioavailability of the active ingredient in the two products are not significantly different under suitable test conditions.

As used herein, the term “variant,” is meant to refer to a compound that differs from the compound of the present disclosure, but retains essential properties thereof. A non limiting example of this is a polynucleotide or polypeptide compound having conservative substitutions with respect to the reference compound, commonly known as degenerate variants. Another non-limiting example of a variant is a compound that is structurally different, but retains the same active domain of the compounds of the present disclosure. Variants include N-terminal or C-terminal extensions, capped amino acids, modifications of reactive amino acid side chain functional groups, e.g., branching from lysine residues, pegylation, and/or truncations of a polypeptide compound. Generally, variants are overall closely similar, and in many regions, identical to the compounds of the present disclosure. Accordingly, the variants may contain alterations in the coding regions, non-coding regions, or both.

COMPOSITIONS

According to one aspect, the present disclosure provides compositions comprising at least one TDFRP. The present disclosure provides in another aspect treating a subject with a composition comprising at least one TDFRP. Tissue Differentiation Factor Related Polypeptides (TDFRPs)

The present disclosure provides compounds that are functional analogs of tissue differentiation factors, i.e., compounds that functionally mimic TGF-beta superfamily proteins, for example by acting as TGF-beta superfamily receptor agonists, and preferentially bind to select AFK receptor(s). The present compounds are called TDFRPs, and include small molecules, more particularly polypeptides.

According to one embodiment, the TDFRP compound has the general structure identified as SEQ ID NOs: 1-208, disclosed in International Publication No.

WO/2003/106656, incorporated by reference in its entirety herein. According to one embodiment, a TDFRP compound includes an analog or homolog of SEQ ID NOs: 1-208. Compounds of the present disclosure include those with homology to SEQ ID NOs: 1-208, for example, preferably 50% or greater amino acid identity, more preferably 75% or greater amino acid identity, and even more preferably 90% or greater amino acid identity. The compounds of the present disclosure also include one or more polynucleotides encoding SEQ ID NOs: 1-208, including degenerate variants thereof. Accordingly, nucleic acid sequences capable of hybridizing at low stringency with any nucleic acid sequences encoding SEQ ID NOs: 1-208 are considered to be within the scope of the disclosure.

According to one embodiment, the TDFRP compound has the general structure identified as SEQ ID NOs: 1-347, disclosed in International Publication No.

WO/2007/035872, incorporated by reference in its entirety herein. According to one embodiment, a TDFRP compound includes an analog or homolog of SEQ ID NOs: 1-347. Compounds of the present disclosure include those with homology to SEQ ID NOs: 1-347, for example, preferably 50% or greater amino acid identity, more preferably 75% or greater amino acid identity, and even more preferably 90% or greater amino acid identity (90%,

91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% or greater). The compounds of the present disclosure also include one or more polynucleotides encoding SEQ ID NOs: 1-347, including degenerate variants thereof. Accordingly, nucleic acid sequences capable of hybridizing at low stringency with any nucleic acid sequences encoding SEQ ID NOs: 1-347 are considered to be within the scope of the disclosure.

According to one embodiment, the TDFRP compound has the general structure identified as SEQ ID NOs: 1-314, disclosed in International Publication No.

WO/2006/009836, incorporated by reference in its entirety herein. According to one embodiment, a TDFRP compound includes an analog or homolog of SEQ ID NOs: 1-314.

Compounds of the present disclosure include those with homology to SEQ ID Nos: 1-314, for example, preferably 50% or greater amino acid identity, more preferably 75% or greater amino acid identity, and even more preferably 90% or greater amino acid identity. The compounds of the present disclosure also include one or more polynucleotides encoding SEQ ID NOs:l-314, including degenerate variants thereof. Accordingly, nucleic acid sequences capable of hybridizing at low stringency with any nucleic acid sequences encoding SEQ ID NOs:l-314 are considered to be within the scope of the disclosure.

Sequence identity can be measured using sequence analysis software (Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), with the default parameters therein.

In the case of polypeptide sequences, which are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Thus, included in the disclosure are peptides having mutated sequences such that they remain homologous, e.g., in sequence, in structure, in function, and in antigenic character or other function, with a polypeptide having the corresponding parent sequence. Such mutations can, for example, be mutations involving conservative amino acid changes, e.g., changes between amino acids of broadly similar molecular properties. For example, interchanges within the aliphatic group alanine, valine, leucine and isoleucine can be considered as conservative. Sometimes substitution of glycine for one of these can also be considered conservative. Other conservative interchanges include those within the aliphatic group aspartate and glutamate; within the amide group asparagine and glutamine; within the hydroxyl group serine and threonine; within the aromatic group phenylalanine, tyrosine and tryptophan; within the basic group lysine, arginine and histidine; and within the sulfur-containing group methionine and cysteine. Sometimes substitution within the group methionine and leucine can also be considered conservative. Preferred conservative substitution groups are aspartate-glutamate; asparagine- glutamine; valine-leucine-isoleucine; alanine-valine; phenylalanine- tyrosine; and lysine- arginine.

The disclosure also provides for compounds having altered sequences including insertions such that the overall amino acid sequence is lengthened, while the compound still retains the appropriate TDF agonist or antagonist properties. Additionally, altered sequences may include random or designed internal deletions that truncate the overall amino acid sequence of the compound, however the compound still retains its TDF-like functional properties. According to certain embodiments, one or more amino acid residues within the sequence are replaced with other amino acid residues having physical and/or chemical properties similar to the residues they are replacing. Preferably, conservative amino acid substitutions are those wherein an amino acid is replaced with another amino acid encompassed within the same designated class, as will be described more thoroughly below. Insertions, deletions, and substitutions are appropriate where they do not abrogate the functional properties of the compound. Functionality of the altered compound can be assayed according to the in vitro and in vivo assays described below that are designed to assess the TDF-like properties of the altered compound.

According to some embodiments, particularly preferred peptides include but are not limited to, the following:

According to some embodiments, the particularly preferred peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 1.

According to some embodiments, the particularly preferred peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 3.

In yet another embodiment, the peptide of the invention can be: CYYDNSSSVLCKRXwRS (SEQ ID NO:4), wherein X14 is D-Tyr.

According to some embodiments, the particularly preferred peptide is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9% identical to SEQ ID NO: 4.

TDFRP Recombinant Expression Vectors

According to one aspect, the disclosure includes vectors containing one or more nucleic acid sequences encoding a TDFRP compound. For recombinant expression of one or more the polypeptides of the disclosure, the nucleic acid containing all or a portion of the nucleotide sequence encoding the polypeptide is inserted into an appropriate cloning vector, or an expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as detailed below.

In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the disclosure is intended to include such other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. Such viral vectors permit infection of a subject and expression in that subject of a compound.

The recombinant expression vectors of the disclosure comprise a nucleic acid encoding a compound with TDF-like properties in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g. , in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue- specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The expression vectors of the disclosure can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides, encoded by nucleic acids as described herein (e.g., TDFRP compounds and TDFRP-derived fusion polypeptides, etc.).

TDFRP -Expressing Host Cells

According to another aspect, the present disclosure pertains to TDFRP-expressing host cells, which contain a nucleic acid encoding one or more TDFRP compounds. The recombinant expression vectors of the disclosure can be designed for expression of TDFRP compounds in prokaryotic or eukaryotic cells. For example, TDFRP compounds can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), fungal cells, e.g., yeast, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, CA. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro , for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of polypeptides in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant polypeptide; (ii) to increase the solubility of the recombinant polypeptide; and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide.

Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET lid (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant polypeptide expression in E. coli is to express the polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, CA. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the expression host, e.g., E. coli (see, e.g., Wada, et ah, 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the disclosure can be carried out by standard DNA synthesis techniques.

In another embodiment, the TDFRP expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et al, 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, CA.), and picZ (Invitrogen Corp, San Diego, CA.). Alternatively, TDFRP can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of polypeptides in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

In yet another embodiment, a nucleic acid of the disclosure is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, el al. , 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector’s control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2^nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue- specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue- specific promoters include the albumin promoter (liver- specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid- specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983.

Cell 33: 741-748), neuron- specific promoters (e.g., the neurofilament promoter; Byme and

Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters

(Edlund, et al., 1985. Science 230: 912-916), and mammary gland- specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No.

264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).

The disclosure further provides a recombinant expression vector comprising a DNA molecule of the disclosure cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to a TDRFP mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see , e.g., Weintraub, el ah, “Antisense RNA as a molecular tool for genetic analysis,” Reviews-Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the disclosure pertains to host cells into which a recombinant expression vector of the disclosure has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, TDFRP can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and

“transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in

Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2^nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker ( e.g ., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding TDFRP or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

A host cell that includes a compound of the disclosure, such as a prokaryotic or eukaryotic host cell in culture can be used to produce (i.e., express) recombinant TDFRP. According to one embodiment, the method comprises culturing the host cell of disclosure (into which a recombinant expression vector encoding TDFRP has been introduced) in a suitable medium such that TDFRP is produced. According to another embodiment, the method further comprises the step of isolating TDFRP from the medium or the host cell. Purification of recombinant polypeptides is well-known in the art and include ion-exchange purification techniques, or affinity purification techniques, for example with an antibody to the compound.

TDFRP-derived Chimeric and Fusion Polypeptides

The present disclosure also provides for compounds that are TDFRP-derived chimeric or fusion polypeptides. As used herein, a TDFRP-derived “chimeric polypeptide” or “fusion polypeptide” comprises a TDFRP operatively-linked to a polypeptide having an amino acid sequence corresponding to a polypeptide that is not substantially homologous to the TDFRP, e.g., a polypeptide that is different from the TDFRP and that is derived from the same or a different organism (i.e., non-TDFRP). Within a TDFRP-derived fusion polypeptide, the

TDFRP can correspond to all or a portion of a TDFRP. According to one embodiment, a

TDFRP-derived fusion polypeptide comprises at least one biologically-active portion of a

TDFRP, for example a fragment of SEQ ID NOs: 1-347. According to another embodiment, a TDFRP-derived fusion polypeptide comprises at least two biologically active portions of a

TDFRP. In yet another embodiment, a TDFRP-derived fusion polypeptide comprises at least three biologically active portions of a TDFRP polypeptide. Within the fusion polypeptide, the term “operatively linked” is intended to indicate that the TDFRP polypeptide and the non- TDFRP polypeptide are fused in-frame with one another. The non-TDFRP polypeptide can be fused to the N-terminus or C-terminus of the TDFRP.

According to one embodiment, the fusion polypeptide is a GST-TDFRP fusion polypeptide in which the TDFRP sequences are fused to the N-or C-terminus of the GST (glutathione S -transferase) sequences. Such fusion polypeptides can facilitate the purification of recombinant TDFRP by affinity means.

According to another embodiment, the fusion polypeptide is a TDFRP polypeptide containing a heterologous signal sequence at its N-terminus. In certain host cells ( e.g ., mammalian host cells), expression and/or secretion of TDFRP can be increased through use of a heterologous signal sequence.

According to yet another embodiment, the fusion polypeptide is a TDFRP- immunoglobulin fusion polypeptide in which the TDFRP sequences are fused to sequences derived from a member of the immunoglobulin superfamily. The TDFRP-immunoglobulin fusion polypeptides of the disclosure can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a TDF and a TDF receptor polypeptide on the surface of a cell, to thereby suppress TDF-mediated signal transduction in vivo. The TDFRP-immunoglobulin fusion polypeptides can be used to affect the bioavailability of a TDFRP, for example to target the compound to a particular cell or tissue having the requisite antigen. Inhibition of the TDF/TDF receptor interaction can be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g., promoting or inhibiting) cell survival.

TDFRP 1 -linker-TDFRP2

The TDFRP compounds described herein contain multiple TDF-related polypeptides (i.e., multiple domain TDF-related polypeptide compounds, hereinafter “TDFRP”) with the general structure shown below:

TDFRP 1 - linker- TDFRP2

Where a first TDFRP domain (TDFRP 1, i.e., TDF-related polypeptide 1) is covalently linked via the C-terminus, N-terminus, or any position with a functionalizable side group, e.g., lysine or aspartic acid to a linker molecule, which, in turn, is covalently linked to the N- terminus of a second TDFRP domain (TDFRP2). The TDRFP domains are compounds that include small molecules. Variants, analogs, homologs, or fragments of these compounds, such as species homologs, are also included in the present disclosure, as well as degenerate forms thereof.

A first domain is linked to a second domain through a linker. The term “linker, “ as used herein, refers to an element capable of providing appropriate spacing or structural rigidity, or structural orientation, alone, or in combination, to a first and a second domain, e.g., TDFRP1 and TDFRP2, such that the biological activity of the TDFRP is preserved. For example, linkers may include, but are not limited to, a diamino alkane, a dicarboxylic acid, an amino carboxylic acid alkane, an amino acid sequence, e.g., glycine polypeptide, a disulfide linkage, a helical or sheet-like structural element or an alkyl chain. According to one aspect the linker is not inert, e.g., chemically or enzymatically cleavable in vivo or in vitro. In another aspect the linker is inert, i.e., substantially unreactive in vivo or in vitro, e.g., is not chemically or enzymatically degraded. Examples of inert groups which can serve as linking groups include aliphatic chains such as alkyl, alkenyl and alkynyl groups (e.g., C1-C20), cycloalkyl rings (e.g., C3-C10), aryl groups (carbocyclic aryl groups such as 1-naphthyl, 2- naphthyl, 1-anthracyl and 2-anthracyl and heteroaryl group such as /V-imidazolyl, 2- imidazole, 2-thienyl, 3 -thienyl, 2-furanyl, 3-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2- pyrimidy, 4-pyrimidyl, 2-pyranyl, 3-pyranyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 2- pyrazinyl, 2-thiazole, 4-thiazole, 5-thiazole, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2- benzothienyl, 3-benzothienyl, 2-benzofuranyl, 3-benzofuranyl, 2-indolyl, 3-indolyl, 2- quinolinyl, 3-quinolinyl, 2-benzothiazole, 2-benzooxazole, 2-benzimidazole, 2-quinolinyl, 3- quinolinyl, 1-isoquinolinyl, 3-quinolinyl, 1-isoindolyl, and 3-isoindolyl), non-aromatic heterocyclic groups (e.g., 2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahyrothiophenyl, 3- tetrahyrothiophenyl, 2-morpholino, 3-morpholino, 4-morpholino, 2-thiomorpholino, 3- thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1- piperazinyl, 2-piperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl and 4- thiazolidinyl) and aliphatic groups in which one, two or three methylenes have been replaced with -0-, -S-, -NH-, -SO2-, -SO- or -SO2NH-.

The TDFRP compounds include small molecules, more particularly TDFRP compound domains, with the general structure identified herein, as detailed below. The TDFRP compound domains disclosed herein may be present in an TDFRP compound in any combination or orientation. Variants, analogs, homologs, or fragments of these TDFRP compound domains, such as species homologs, are also included in the present disclosure, as well as degenerate forms thereof. The TDFRP compound domains of the present disclosure may be capped on the N-terminus, or the C-terminus, or on both the N-terminus and the C- terminus. The TDFRP compounds may be pegylated, or modified, e.g., branching, at any amino acid residue containing a reactive side chain, e.g., lysine residue, or chemically reactive group on the linker. The TDFRP compound of the present disclosure may be linear or cyclized. The tail sequence of the TDFRP or TDFRP domains may vary in length. According to one aspect of the present disclosure, the TDFRP compounds of the disclosure are prodmgs, i.e., the biological activity of the TDFRP compound is altered, e.g., increased, upon contacting a biological system in vivo or in vitro.

The TDFRP compounds can contain natural amino acids, non-natural amino acids, d- amino acids and 1-amino acids, and any combinations thereof. According to certain embodiments, the compounds of the disclosure can include commonly encountered amino acids, which are not genetically encoded. These non-genetically encoded amino acids include, but are not limited to, b-alanine (b-Ala) and other omega-amino acids such as 3- aminopropionic acid (Dap), 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; a-aminoisobutyric acid (Aib); e-aminohexanoic acid (Aha); d-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Om); citrulline (Cit); t-butylalanine (t- BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); 2-naphthylalanine (2-Nal); 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)); 4- fluorophenylalanine (Phe(4-F)); penicillamine (Pen); l,2,3,4-tetrahydroisoquinoline-3- carboxylic acid (Tic); b-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (hArg); N-acetyl lysine (AcLys); 2,3-diaminobutyric acid (Dab); 2,3-diaminobutyric acid (Dbu); p-aminophenylalanine (PhcipNfP)); N-methyl valine (MeVal); homocysteine (hCys) and homoserine (hSer). Non-naturally occurring variants of the compounds may be produced by mutagenesis techniques or by direct synthesis.

Measurement of TDFRP Biological Activity

The biological activity, namely the agonist or antagonist properties of TDF polypeptides or TDFRP compounds can be characterized using any conventional in vivo and in vitro assays that have been developed to measure the biological activity of the TDFRP compound, a TDF polypeptide or a TDF signaling pathway component.

TGF-b/BMPs Superfamily members are associated with a number of cellular activities involved in injury responses and regeneration. TDFRP compounds can be used as agonists of

BMPs or antagonists of TGF-b molecules to mediate activities that can prevent, repair or alleviate injurious responses in cells, tissues or organs. According to some embodiments, the anti-fibrotic properties of the TDFRPs is measured. According to some embodiments, the anti-fibrotic properties of TDFRP compounds can been demonstrated using in vitro and in vivo assays. High-resolution chest CT (HRCT) can be used to detect pulmonary fibrosis in patients. Furter, measuring the activity of extracellular matrix (ECM) deposition and remodeling (e.g., by longitudinal change in markers of ECM synthesis such as collagen synthesis neoepitopes, PRO-C3 and PRO-C6 (collagen type 3 and 6)).

METHODS

The present disclosure provides TDFRPs compounds that are functional analogs of tissue differentiation factors, i.e., compounds that functionally mimic TGF-beta superfamily proteins, for example by acting as TGF-beta superfamily receptor agonists.

The disclosure provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant TDF polypeptide or TDFRP compound target molecule expression or activity. TDF and TDFRP compound target molecules, such as TDF receptors, play a role in cell differentiation. Cell differentiation is the central characteristic of tissue morphogenesis. Tissue morphogenesis is a process involved in adult tissue repair and regeneration mechanisms. The degree of morphogenesis in adult tissue varies among different tissues and is related, among other things, to the degree of cell turnover in a given tissue.

The bone morphogenetic proteins are members of the transforming growth factor-beta superfamily. Ozkaynak et al. (EMBO J. 9: 2085-2093, 1990) purified a novel bovine osteogenic protein homolog, which they termed 'osteogenic protein- G (OP-1; a.k.a., BMP-7). The authors used peptide sequences to clone the human genomic and cDNA clones of OP-1, later named BMP-7. The BMP-7 cDNAs predicted a 431-amino acid polypeptide that includes a secretory signal sequence. The TDFRP compounds described herein are structural mimetics of the biologically active regions of bone morphogenic proteins, for example, but not limited to, BMP-7 (OP-1), and related peptides. Biologically active regions include, for example, the Finger 1 and Finger 2 regions of BMP-7. Groppe et al. (Nature 420: 636-642, 2002) reported the crystal structure of the antagonist Noggin (602991) bound to BMP-7.

TDFRP compounds are useful to treat diseases and disorders that are amenable to treatment with BMP polypeptides. According to some embodiments, the TDFRP compounds of the disclosure are useful to treat pulmonary fibrosis.

Fibrosis According to one aspect, the disclosure features a method of treating pulmonary fibrosis associated with viral infection, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP), wherein the TDFRP is administered in an amount effective to treat the pulmonary fibrosis in the subject. According to one aspect, the disclosure features a method of preventing pulmonary fibrosis associated with viral infection, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP), wherein the TDFRP is administered in an amount effective to prevent the pulmonary fibrosis in the subject. According to one aspect, the disclosure features a method of reducing pulmonary fibrosis associated with viral infection, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP), wherein the TDFRP is administered in an amount effective to reduce the pulmonary fibrosis in the subject. According to some embodiments of any of the above aspects, the TDFRP is administered by a pulmonary route. According to some embodiments of any of the above aspects, the TDFRP is administered using a nebulizer. According to some embodiments of any of the aspects or embodiments herein, the viral infection is caused by a virus from the Coronaviridae family. According to some embodiments, vims from the Coronaviridae family belong to a genus selected from the group consisting of Alphacoronavirus , Betacoronavirus, Gammacoronavirus , Deltacoronavirus, Torovirus, and Bafinivirus. According to some embodiments, the Betacoronavirus is Middle East respiratory syndrome (MERS). According to some embodiments, the Betacoronavirus is severe acute respiratory syndrome (SARS). According to some embodiments, the Betacoronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

As used herein, “fibrosis” refers to the formation of excess fibrous connective tissue as a result of the excess deposition of extracellular matrix components, for example collagen. Fibrous connective tissue is characterized by having extracellular matrix (ECM) with a high collagen content. The collagen may be provided in strands or fibers, which may be arranged irregularly or aligned. The ECM of fibrous connective tissue may also include glycosaminoglycans. According to some embodiments, the fibrosis is pulmonary fibrosis.

According to some embodiments, treating fibrosis refers to reversing fibrosis. As used herein, “reversing fibrosis” refers to where the fibrotic material or components under treatment in a target tissue or organ is decreased or eradicated. According to certain embodiments, reversing fibrosis refers to where least about 10%, or about 25%, or about

50%, or more preferably by at least about 75%, or more preferably by about 85%, or still more preferably by about 90%, or more preferably still about by 95%, or more preferably still by 99% or more of the fibrotic components or material has been removed as compared to pre treatment. According to other embodiments, treating fibrosis refers to inhibiting fibrosis. According to certain embodiments, “inhibiting fibrosis” refers to where the net amount or level of fibrosis at a desired target fibrotic site does not increase with time. According to some embodiments, treatment of fibrosis may be effective to prevent progression of the fibrosis, e.g. to prevent worsening of the condition or to slow the rate of development of the fibrosis. According to some embodiments treatment or alleviation may lead to an improvement in the fibrosis, e.g. a reduction in the amount of deposited collagen fibers. Prevention of fibrosis may refer to prevention of a worsening of the condition or prevention of the development of fibrosis, e.g. preventing an early stage fibrosis developing to a later, chronic, stage.

As used herein, "excess fibrous connective tissue" refers to an amount of connective tissue at a given location (e.g. a given tissue or organ, or part of a given tissue or organ) which is greater than the amount of connective tissue present at that location in the absence of fibrosis, e.g. under normal, non-pathological conditions. As used herein, "excess deposition of extracellular matrix components" refers to a level of deposition of one or more extracellular matrix components which is greater than the level of deposition in the absence of fibrosis, e.g. under normal, non-pathological conditions.

The cellular and molecular mechanisms of fibrosis are described in Wynn, J. Pathol. (2008) 214(2): 199-210, and Wynn and Ramalingam, Nature Medicine (2012) 18:1028-1040, which are hereby incorporated by reference in their entirety.

The main cellular effectors of fibrosis are myofibroblasts, which produce a collagen- rich extracellular matrix.

In response to tissue injury, damaged cells and leukocytes produce pro-fibrotic factors such as TGFp, IL-13 and PDGF, which activate fibroblasts to aSMA-expressing myofibroblasts, and recruit myofibroblasts to the site of injury. Myofibroblasts produce a large amount of extracellular matrix, and are important mediators in aiding contracture and closure of the wound. However, under conditions of persistent infection or during chronic inflammation there can be overactivation and recruitment of myofibroblasts, and thus over production of extracellular matrix components, resulting in the formation of excess fibrous connective tissue.

Diseases characterized by excessive fibrosis include but are not restricted to systemic sclerosis, scleroderma, hypertrophic cardiomyopathy, dilated cardiomyopathy (DCM), atrial fibrillation, ventricular fibrillation, myocarditis, liver cirrhosis, kidney diseases, diseases of the eye, asthma, cystic fibrosis, arthritis and idiopathic pulmonary fibrosis.

According to some embodiments, the fibrosis is pulmonary fibrosis. According to one embodiment, the pulmonary fibrosis is idiopathic pulmonary fibrosis. According to another embodiment, the fibrosis is associated with a viral infection. According to one embodiment, the disease or condition is a chronic disease or condition.

According to one embodiment, treating fibrosis comprises restoring function to the tissue that is affected. For example, according to one embodiment, treating the pulmonary fibrosis comprises restoring the function of the pulmonary tissue. Tests to measure lung volume, capacity, rates of flow, and gas exchange can be used to determine pulmonary function. According to some embodiments, the disease/condition associated with fibrosis is idiopathic pulmonary fibrosis (IPF). IPF is a debilitating and life-threatening lung disease characterized by a progressive scarring of the lungs that hinders oxygen uptake. The cause of IPF is not known. As scarring progresses, patients with IPF experience shortness of breath (dyspnea) and difficulty with performing routine functions, such as activities of daily living. According to the National Institutes of Health, about 100,000 people in the United States have IPF, and approximately 30,000 to 40,000 new cases are found each year. Worldwide, IPF affects 13 to 20 out of every 100,000 people. A similar prevalence exists for six other interstitial lung diseases and systemic sclerosis that may benefit from antifibrotic therapy. There are no FDA-approved treatments for IPF, and approximately two-thirds of patients die within five years after diagnosis. Patients are often treated with corticosteroids and immunosuppressive agents; however, none have been clinically proven to improve survival or quality of life. It is thought that stabilization or reversal of lung fibrosis could stabilize lung function and diminish the impact of this devastating disease. Thus, the TDFRP compounds in combination with an additional agent (e.g., an inhibitor of angiotensin converting enzyme (ACE), a neprilysin inhibitor and an angiotensin receptor-neprilysin inhibitor) of the present disclosure may be used to inhibit or reverse lung fibrosis associated with IPF.

According to some embodiments of the embodiments and aspects herein, the TDFRP is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. According to some embodiments of the embodiments and aspects herein, the TDFRP is a Multiple Domain TDFRP. According to some embodiments, the TDFPR is administered with an additional agent. According to one embodiment, the serum half-life of the TDFRP is increased when administered with the additional agent, compared to administration of the TDFRP alone.

Biological Effect

In various embodiments of the disclosure, suitable in vitro or in vivo assays are performed to determine the effect of a specific TDFRP-based therapeutic, and whether its administration is indicated for treatment of the affected tissue in a subject.

In various specific embodiments, in vitro assays can be performed with representative cells of the type(s) involved in the patient's disorder, to determine if a given TDFRP-based therapeutic exerts the desired effect upon the cell type(s). Compounds for use in therapy can be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model system known in the art can be used prior to administration to human subjects.

PHARMACEUTICAL COMPOSITIONS

The pharmaceutical compositions of the disclosure typically contain a therapeutically effective amount of a compound described herein. Those skilled in the art will recognize, however, that a pharmaceutical composition may contain more than a therapeutically effective amount, such as in bulk compositions, or less than a therapeutically effective amount, that is, individual unit doses designed for multiple administration to achieve a therapeutically effective amount. Typically, the composition will contain from about 0.01-95 wt % of active agent, including, from about 0.01-30 wt %, such as from about 0.01-10 wt %, with the actual amount depending upon the formulation itself, the route of administration, the frequency of dosing, and so forth. According to one embodiment, a composition suitable for an oral dosage form, for example, may contain about 5-70 wt %, or from about 10-60 wt % of active agent.

According to some aspects, the TDFRP compounds of the disclosure, and derivatives, fragments, analogs and homologs thereof, are be incorporated into pharmaceutical compositions suitable for administration.

According to some embodiments, the pharmaceutical composition includes one or more TDFPR compounds and an additional agent. According to some embodiments, the

TDFRP compositions of the disclosure may be physically mixed with the additional agent to form a composition containing both agents; or the TDFRP and additional agent each may be present in separate and distinct compositions which are administered to the patient simultaneously or at separate times. For example, a TDFRP compound of the disclosure can be combined with an additional agent using conventional procedures and equipment to form a combination of active agents comprising a TDFRP compound of the disclosure and an additional agent. Additionally, the additional agents may be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition comprising a compound of the disclosure, a second active agent and a pharmaceutically acceptable carrier. In this embodiment, the components of the composition are typically mixed or blended to create a physical mixture. The physical mixture is then administered in a therapeutically effective amount using any of the routes described herein.

Alternatively, the TDFRP compound and additional agent may remain separate and distinct before administration to the patient. In this embodiment, the TDFRP compound and additional agent are not physically mixed together before administration but are administered simultaneously or at separate times as separate compositions. Such compositions can be packaged separately or may be packaged together in a kit. When administered at separate times, the additional agent will typically be administered less than 24 hours after administration of the compound of the disclosure, ranging anywhere from concurrent with administration of the compound of the disclosure to about 24 hours post-dose. This is also referred to as sequential administration. Thus, a compound of the disclosure can be orally administered simultaneously or sequentially with the additional agent using two tablets, with one tablet for the TDFRP compound and one tablet for the additional agent, where sequential may mean being administered immediately after administration of the compound of the disclosure or at some predetermined time later (for example, one hour later or three hours later). It is also contemplated that the additional agent may be administered more than 24 hours after administration of the compound of the disclosure. Alternatively, the combination may be administered by different routes of administration, that is, one orally and the other by inhalation.

According to one embodiment, the TDFRP is provided in the same pharmaceutical composition as the additional agent. According to another embodiment, the TDFRP is provided in a separate composition as the additional agent.

As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration.

Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non- aqueous vehicles such as fixed oils may also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (/._<?., topical), transmucosal, and rectal administration. According to some embodiments, a pharmaceutical composition is formulated to be compatible with intravenous, intraperitoneal, intramuscular, subcutaneous, inhalation, transmucosal, and oral routes of administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF,

Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.

The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic compounds, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound, which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound ( e.g ., a TDFRP compound and/or additional agent) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding compounds, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate, or orange flavoring.

Release agents, wetting agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the pharmaceutical compositions. Exemplary coating agents for tablets, capsules, pills and like, include those used for enteric coatings, such as cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymers, cellulose acetate trimellitate, carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, and the like. Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, alpha-tocopherol, and the like; and metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid, phosphoric acid, and the like.

According to some embodiments, the present discosure is directed to methods and compositions for pulmonary delivery of TDFRP therapeutic compositions comprising penetration enhancers, carrier compounds and/or transfection agents. Methods of pukmonary delivery are disclosed in International Publication WO 99/60166, which is incorporated herein by reference in its entirety.

Briefly, the compounds and methods of the invention employ particles containing TDFRP therapeutics or diagnostics. The particles can be solid or liquid, and can be of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. In general, particles ranging from about 5 to 20 microns in size are respirable and are expected to reach the bronchioles (Allen, Secundum Artem, Vol. 6, No. 3, on-line publication updated May 8,

1998, and available at www.paddocklabs.com/secundum/secamdx.html). It is greatly desirable to avoid particles of non-respirable size, as these tend to deposit in the throat and be swallowed, thus reducing the quantity of TDFRP reaching the lung. Fiquid pharmaceutical compositions of TDFRPs can be prepared by combining the oligonucleotide with a suitable vehicle, for example sterile pyrogen free water, or saline solution. Other therapeutic compounds may optionally be included.

The present invention also contemplates the use of solid particulate compositions. Such compositions can comprise particles of TDFRPs that are of respirable size. Such particles can be prepared by, for example, grinding dry oligonucleotide by conventional means, for example with a mortar and pestle, and then passing the resulting powder composition through a 400 mesh screen to segregate large particles and agglomerates. A solid particulate composition comprised of a TDFRP can optionally contain a dispersant which serves to facilitate the formation of an aerosol, for example lactose. In accordance with the methods of the present invention, TDFRP compositions are aerosolized. Aerosolization of liquid particles can be produced by any suitable means, such as with a nebulizer. See, for example, U.S. Pat. No. 4,501,729. Nebulizers are commercially available devices which transform solutions or suspensions into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable nebulizers include those sold by Blairex® under the name PARI LC PLUS, PARI DURA-NEB 2000, PARI-BABY Size, PARI PRONEB Compressor with LC PLUS, PARI WALKHALER Compressor/Nebulizer System, PARI LC PLUS Reusable Nebulizer, and PARI LC Jet+ ©Ncbuli/cr.

Exemplary formulations for use in nebulizers comprise a TDFRP compound in a liquid, such as sterile, pyrogen free water, or saline solution, wherein the oligonucleotide comprises up to about 40% w/w of the formulation. If desired, further additives such as preservatives (for example, methyl hydroxybenzoate) antioxidants, and flavoring agents can be added to the composition.

TDFRP compounds can also be aerosolized using any solid particulate medicament aerosol generator known in the art. Such aerosol generators produce respirable particles, as described above, and further produce reproducible metered dose per unit volume of aerosol. Suitable solid particulate aerosol generators include insufflators and metered dose inhalers. Metered dose inhalers suitable for used in the art (along with the trade name, manufacturer and indication they are used for) and useful in the present invention include:

Delivery Device/Trade name/Manufacturer /Indication Metered Dose Inhaler (MDI)/Alupent/Boehringer Ingelheim/Beta-adrenergic bronchodilator

Metered Dose Inhaler (MDI)/Atrovent/Boehringer Ingelheim/ Anticholinergic bronchodilator

Aerobid/Aerobid-M/Forest/Steriodal Anti-inflammatory

Beclovent/Beconase/Glaxo Wellcome/S teriodal Anti-inflammatory

Flo vent/Glaxo Wellcome/S teriodal Anti-inflammatory

Ventolin/Glaxo Wellcome/Beta-adrenergic bronchodilator

Proventil/Key Pharm./Beta-adrenergic bronchodilator

Maxair/3M Pharm./Beta-adrenergic bronchodilator

Azmacort/Rhone-Poulenc Rorer/S teriodal Anti-inflammatory Tilade/Rhone-Poulenc Rorer/ Anti-inflammatory (inhibits release of inflammatory mediators)

Intal/Rhone-Poulenc Rorer/Inhibits mast cell degranulation (Asthma)

V anceril/Schering/Steriodal Anti-inflammatory Tornalate/Dura Pharm./B eta- adrenergic bronchodilator Solutions for Nebulization

Alupent/Boehringer Ingelheim/B eta- adrenergic bronchodilator Pulmozyme/Genetech/Recombinant human deoxyribonuclease I Ventolin/Glaxo Wellcome/Beta-adrenergic bronchodilator Tornalate/Dura Pharm./B eta- adrenergic bronchodilator Intal/Rhone-Poulenc Rorer/Inhibits mast cell degranulation (Asthma)

Capsules (Powder) for Inhalation

Ventolin/Glaxo Wellcome/(Rotocaps for use in Rotohaler device)/B eta- adrenergic bronchodilator

Powder for Inhalation

Pulmicort/Astra USA/(Turbuhaler device)/Steroidal Anti-inflammatory According to some embodiments, liquid or solid aerosols are produced at a rate of from about 10 to 150 liters per minute, from about 30 to 150 liters per minute, or about 60 liters per minute.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared as pharmaceutical compositions in the form of suppositories ( e.g ., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. The compounds can be prepared for use in conditioning or treatment of ex vivo explants or implants.

Compositions may also be formulated to provide slow or controlled release of the active agent using, by way of example, hydroxypropyl methyl cellulose in varying proportions or other polymer matrices, liposomes and/or microspheres. In addition, the pharmaceutical compositions of the disclosure may contain opacifying agents and may be formulated so that they release the active agent only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active agent can also be in micro-encapsulated form, optionally with one or more of the above-described excipients. According to one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811, incorporated by reference in its entirety herein.

According to some embodiments, it is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The nucleic acid molecules of the disclosure can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et ah, 1994. Proc. Natl. Acad. Sci. USA 91: 3054- 3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

All publications and patent applications cited in this specification are herein incorporated by reference in their entirety for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference for all purposes. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors described herein are not entitled to antedate such disclosure by virtue of prior disclosure or for any other reason.

Claims

CLAIMS We claim:

1. A method of treating pulmonary fibrosis associated with viral infection, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP), wherein the TDFRP is administered in an amount effective to treat the pulmonary fibrosis in the subject.

2. A method of preventing pulmonary fibrosis associated with viral infection, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP), wherein the TDFRP is administered in an amount effective to prevent the pulmonary fibrosis in the subject.

3. A method of reducing pulmonary fibrosis associated with viral infection, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP), wherein the TDFRP is administered in an amount effective to reduce the pulmonary fibrosis in the subject.

4. A method of treating coronavirus disease (COVID-19) in a subject, the method comprising administering to a subject in need of treatment at least one tissue differentiation factor related polypeptide (TDFRP), wherein the TDFRP is administered in an amount effective to treat COVID-19 in the subject.

5. The method of any one of claims 1-4, wherein the TDFRP is administered by a pulmonary route.

6. The method of any one of claims 1-4, wherein the viral infection is caused by a virus from the Coronaviridae family.

7. The method of claim 6, wherein the vims from the Coronaviridae family belong to a genus selected from the group consisting of Alphacoronavirus, Betacoronavirus, Gammacoronavirus , Deltacoronavirus, Torovirus, and Bafinivirus.

8. The method of claim 7, wherein the Betacoronavirus, is Middle East respiratory syndrome (MERS).

9. The method of claim 7, wherein the Betacoronavirus is severe acute respiratory syndrome (SARS).

10. The method of claim 7, wherein the Betacoronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

11. The method of any one of the preceding claims, wherein the TDFRP is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

12. The method of any one of the preceding claims, wherein the TDFRP is a Multiple Domain TDFRP.

13. The method of any one of the preceding claims, wherein the TDFPR is administered with an additional agent.

14. The method of any one of the preceding claims, wherein the serum half-life of the TDFRP is increased when administered with the additional agent, compared to administration of the TDFRP alone.

15. The method of claim 14, wherein the additional agent is an angiotensin converting enzyme (ACE) inhibitor, a neprilysin inhibitor or an angiotensin receptor- neprilysin inhibitor.

16. The method of claim 15, wherein the angiotensin converting enzyme (ACE) inhibitor is selected from the group consisting: of captopril, zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, trandolapril, fosinopril, moexipril, cilazapril, spirapril, temocapril, alacepril, ceronapril, delepril, moveltipril, and combinations thereof.

17. The method of claim 15, wherein the neprilysin inhibitor is selected from the group consisting of: thiorphan, candoxatril, and candoxatrilat.

18. The method of claim 15, wherein the angiotensin receptor- neprilysin inhibitor is sacubitril/valsartan.

19. The method of claim 13, wherein the additional agent is selected from the group consisting of: anti-neoplastic agents, antibiotics, vaccines, immunosuppressive agents, anti-hypertensive agents and mediators of the hedgehog signaling pathway.