WO2008128299A1

WO2008128299A1 - Therapeutic protocol for treating hemoglobinopathy

Info

Publication number: WO2008128299A1
Application number: PCT/AU2008/000569
Authority: WO
Inventors: Dimitrios Vadolas; Hady Wardan
Original assignee: Murdoch Childrens Research Institute
Priority date: 2007-04-24
Filing date: 2008-04-23
Publication date: 2008-10-30

Abstract

The invention identifies compounds which influence the activity of the γ-globin gene promoter and thus expression of the γ-globin gene. Such compounds are proposed to be useful in therapeutic protocols for treating hemoglobinopathy by induction of fetal hemoglobin production. The compound is selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin and (iv) a combination of (i), (ii) and/or (iii) wit the proviso that the subject is not infected with HIV or has a condition characterised by polymerization of hemoglobin in the cells of the subject.

Description

THERAPEUTIC PROTOCOL FOR TREATING HEMOGLOBINOPATHY

FIELD

[0001] The present invention relates generally to a protocol for treating hemoglobinopathy and agents useful for same.

BACKGROUND

[0002] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[0003] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0004] β-Thalassemia is one of the most common genetic disorders affecting hemoglobin synthesis. Approximately 300,000 patients are born worldwide with β-thalassemia and related hemoglobinopathies. Because of mass migration of populations from endemic regions such as the Mediterranean, Middle East, African and Asian regions, β-thalassemia is being seen with increasing frequency in other parts of the world, including Australia.

[0005] Treatment requires regular blood transfusions for life. However, blood turnover and the lack of natural mechanisms for iron excretion result in iron overload, requiring patients to undergo intense chelation therapy to remove the excess iron. The financial burden of thalassemia is also substantial due to the high cost of chelation therapy and the increasing range of life-threatening complications as patients get older. The only curative therapy is bone marrow transplantation, but only a small proportion of patients can benefit from this procedure due to a lack of compatible donors and the associated costs. [0006] An alternative therapeutic approach to the treatment of β-thalassemia is the reactivation of fetal hemoglobin (HbF). This is based on observations that a number of point mutations in the promoter regions of the γ-globin genes result in persistent HbF expression in adults that can compensate fully for defective γ-globin synthesis resulting in a lack of disease in these patients. Fetal hemoglobin is composed of two alpha chains coded by a gene located on chromosome 16 and two gamma chains coded by the ^Aγ and ^Gγ genes in the beta gene cluster located on the chromosome 11. Fetal hemoglobin is the major hemoglobin component during embryonic and fetal development and at birth represents about 70% of all hemoglobins. It decays rapidly throughout the first months after birth and by the first year its concentration stabilises at 1%.

[0007] Several chemical agents have been identified to induce HbF. A variety of compounds such as erythropoietin, and the antineoplastic agents 5-azacytidine, ribavirin, arabinosylcytosine (cytarabine), 5'aza-2'-deoxycytidine (decitabine) and hydroxyurea (HU) have been reported to increase HbF concentration in red cells. A number of these agents have been reported to induce differentiation of erythroid cells including the human leukemia K562 cell line (Gambari et al, Cell Differentiation 14(2):%1-91, 1984; Yu et al, Cancer Research ¥9:5555-5560, 1989). Erythroid induction is associated with (a) an increase of the overall globin synthesis and globin mRNA accumulation, (b) a relative increase of fetal globins with respect to embryonic globin, and (c) a decrease of the proliferative capacity of hemoglobin-containing cells. The induction of erythroid differentiation and fetal hemoglobin accumulation seen in this cell line with agents such as ribavirin and 5~azacytidine is not a consequence of a direct action on promoter activity of the fetal hemoglobin genes. Importantly, ribavirin has toxic side effects such as causing hemolysis which makes it contraindicated in conditions such as β-thalassemia and infection with hepatitis C (Cohen et al, HematologyA4-34, 2004).

[0008] 5'-Azacytidine and 5'aza-2'-deoxycytidine (decitabine) can affect DNA hypomethylation induced chromatin restructuring and inhibition of DNA methylase which may alter promoter activity (Keefer et al, Experimental Hematology 34:1150-1160, 2006; reviewed in Pace and Zein, Dynamics 235:1727-1737, 2006). [0009] Recent clinical trials have shown that HU can significantly reduce the need for blood transfusion in some thalassemia patients while in sickle cell disease patients it can ameliorate the clinical symptoms, owing to the ability of HbF to inhibit polymerization of sickle cell hemoglobin (Keefer et al, 2006 supra).

[0010] US Patent No. 6,946,457 describes the treatment of subjects with sickle cell disease with certain anti-viral agents which inhibit polymerization of hemoglobin S in the cell. However, the treatment is only limited to diseases characterized by cell sickling, and is no suggestion that the anti- viral agents used would be effective in the treatment of conditions such as β-thalassemia or other hemoglobinopathies. Moreover, there is nothing taught concerning induction of HbF via an increase in promoter activity and the consequent benefit of an increase in HbF levels for treating a condition such as β-thalassemia.

[0011] HIV infected patients treated with zidovudine and a protease inhibitor exhibit increases in HbF levels, however, the reasons for the hemoglobin increase observed in HIV infected patients are not resolved (Poli-Neto et al, Brazilian Journal of Medical and Biological Research 33:1313-1315, 2000). It has been postulated that this may be due to a direct viral action on the expression of the gamma chain gene. Interestingly, the increase in HbF appeared to be attributed to an increased F cell clone proliferation rather than an increase in hemoglobin F level in every cell.

[0012] A separate group of compounds also under evaluation are the short chain fatty acids such as butyrate which have been shown to up-regulate HbF in experimental in vitro models and in β-thalassemia and sickle cell anemia patients. Histone deacetylase inhibitors include sodium butyrate, trichostatin, adicipin and scriptaid (Reviewed in Pace and Zein, 2006 supra). The modification of histones can increase promoter activity and transcription of HbF. Mechanistically, butyrate has been shown to inhibit histone deacetylase, thus affecting nucleosome stability, chromatin structure and histone binding to DNA. Such pleiotropic effects appear to be important in the transcriptional regulation of genes in general. Despite a number of clinical trials investigating the potential of HbF-inducing chemical agents, many of these drags have low efficacy, specificity, and associated cytotoxicity, while some are potentially carcinogenic. There is, therefore, a need to identify pharmacological agents which lead to increased levels of HbF for use in treating hemoglobinopathy.

[0013] Agents which alter the intracellular cyclic nucleotide pathways of cAMP or cGMP may also affect induction of HbF by HU, or 5'-azacytodine or sodium butyrate. These include Zaprimast, Forskolin, 1H-[1,2,4] oxadiazolo[4,3-a]quinoxaline-l-one (OQD), rolipram, and SQ22536 (Keefer et al, 2006 supra). It has been demonstrated that the intracellular induction of HbF production by altering cGMP may be working through a nitric oxide 20 signaling mechanism (Haynes et al, Blood 705:3945-3950, 2004).

[0014] Without a clear understanding of the underlying mechanism(s) involved in the induction of HbF, it is virtually impossible to focus on any molecular target. A two-phase liquid culture assay using primary human erytliroid cells has been commonly used to evaluate potential inducers of HbF. Measurement of γ-globin gene expression in such cultures by the RNAse protection assays, globin chain biosynthesis, high performance liquid chromatography (HPLC) and staining by γ-globin specific fluorescent antibodies have all previously been used to identify HbF gene inducers. Such assays, however, are not suitable for screening large numbers of candidates.

SUMMARY

[0015] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0016] hi accordance with the present invention compounds are identified which influence the activity of the γ-globin gene promoter and thus expression of the γ-globin gene. Such compounds are proposed to be useful in therapeutic protocols for treating hemoglobinopathy by induction of fetal hemoglobin production. The compounds have been identified from chemical libraries in a high throughput (HTP) screen. In particular, candidate compounds are screened via a cellular genomic reporter assay that employs human erythroleukemic cells stably transfected with constructs that contain the EGFP reporter gene under the control of the ^Gγ-globin gene promoter in the human β-globin locus. Induction of EGFP expression driven by the ^Gγ-globin gene promoter parallels that of endogenous globin chain synthesis.

[0017] The identification of suitable agents which increase HbF production via an increase in promoter activity with greater efficacy and less toxicity enables the treatment of hemoglobinopathic conditions such as β-thalassemia and non-sickle cell anemia.

Furthermore, the ability to increase HbF production is of benefit to subjects in circumstances which may necessitate an increase in the oxygen carrying capacity of cells, for example in athletic events such as cycling or marathon running, deep sea diving, altitude climbing or pilots, particularly those of light aircraft or in a subject undergoing a blood transfusion. Additionally, agents which increase HbF production would also have veterinary applications for example, in horse racing or greyhound racing, or for the treatment of a condition for which an increase in the oxygen carrying capacity of the animal's cells is required.

[0018] Accordingly the present invention contemplates a method of treating a hemoglobinopathy in a subject, the method comprising administration of an effective amount of an agent which increases fetal hemoglobin levels, the agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al₅ thiotepa, 4-hydroxychalcone, melphalan, 6,3- dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i), (ii) and/or (iii) with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

[0019] In a particular embodiment, the agent increases HbF in the cells of the subject by increasing promoter activity of the γ-globin gene promoter.

[0020] The present invention provides, therefore, a method for increasing fetal hemoglobin in cells of a subject, the method comprising administration of an effective amount of an agent which increases the level of expression of the γ-globin gene via its promoter, the agent being selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, A- hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i), (ii) and/or (iii) with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

[0021] Another aspect of the present invention is directed to a method of treating a condition selected from the group consisting of β— thalassemia and non-sickle-cell hemoglobinopathy in a subject, the method comprising administering to the subject an effective amount of an agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; (iii) a compound selected from the group consisting of 4- acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5- deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochlori de and quercetin; and (iv) a combination of (i), (ii) and/or (iii) with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in cells of the subject.

[0022] The present invention further provides for the use of an agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3- dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) combination of (i), (ii) and/or (iii) in the manufacture of a medicament for the treatment of a hemoglobinopathy in a subject, with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

[0023] The present invention is also directed to a method of enhancing the endurance or performance of a subject, the method comprising administering to the subject an effective amount of an agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4- hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) or-a combination of (i), (ii) and/or (iii) with proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

[0024] In another embodiment, the invention contemplates a method of determining the ability of an agent to increase fetal hemoglobin levels in cells of a subject, the method comprising:

(a) selecting an agent which increases promoter activity of the γ-globin gene promoter and administering the agent to a subject;

(b) obtaining a sample of blood cells from the subject ;

(c) assaying the sample for the presence of fetal hemoglobin in the cells;

(d) comparing the content of fetal hemoglobin in the cells of the sample following administration of the agent with the level of fetal hemoglobin in the cells of the sample prior to administration of the agent.

[0025] The present invention provides, therefore, a therapeutic protocol for the treatment of a hemoglobinopathy. Particular hemoglobinopathies are those whose symptoms can be ameliorated by increasing levels of HbF. Such hemoglobinopathies include β-thalassemia and non-sickle cell anemia. HbF are conveniently increased by using agents which target the γ-globin gene promoter, increasing its activity and level of γ-globin gene expression.

[0026] A therapeutic kit is also provided herein comprising an agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3- dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i), (ii) and/or (iii).

BRIEF DESCRIPTION OF THE FIGURES

[0027] Figure 1 is a diagrammatical representation showing the structures of compounds which were found to increase promoter activity of HhF genes.

[0028] Figure 2 is a graphical representation showing a representation of the percentage increase in promoter activity and percentage cell viability for K562 cells stably transfected with the ^Gγ-^Aγ EGFP reporter construct and treated with specific concentrations of various nucleoside analogs (as detailed in the methods section); acyclovir, ribavirin famciclovir, ganciclovir, valciclovir, valganciclovir and stavudine.

[0029] Figure 3 is a graphical representation showing the percentage increase in promoter activity and percentage cell viability for K562 cells stably transfected with the ^Gγ-^Aγ EGFP reporter construct and treated with specific concentrations of various compounds (as detailed in the methods section); cisplatin, isoliquiritigenin, melphalan, quercetin, thiotepa, hydroquinone, ribavirin and acyclovir.

[0030] Figure 4 is a graphical representation showing the percentage increase in promoter activity and percentage cell viability for primary murine fetal liver cell derived from transgenic mice containing the ^Gγ-^Aγ EGFP reporter construct. Cells were treated with specific concentrations of various compounds: cisplatin, isoliquiritigenin, melphalan, quercetin, thiotepa, hydroquinone, ribavirin and acyclovir.

DETAILED DESCRIPTION

[0031] All scientific citations, patents, patent applications and manufacturer's technical specifications referred to hereinafter are incorporated herein by reference in their entirety.

[0032] It is to be understood that unless otherwise indicated, the subject invention is not limited to specific formulation components, manufacturing methods, dosage regimens, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0033] It must be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a hemoglobinopathy" includes a single hemoglobinopathy, as well as two or more hemoglobinopathies; reference to "an agent" includes a single agent, as well as two or more agents; reference to "the invention" includes a single for multiple aspects of an invention.;

[0034] In an effort to obtain compounds which increase HbF (fetal hemoglobin) compounds were identified selected from nucleoside analogs, nucleotide analogs and antineoplastic agents that elevated activity of the γ-globin gene. These compounds were identified using a genomic reporter assay (Vadolas et al, Human Molecular Genetics 13(2):223-233, 2004), the contents of which are incorporated herein by reference.

[0035] Accordingly the present invention contemplates a method of treating a hemoglobinopathy in a subject, the method comprising administration of an effective amount of an agent which increases fetal hemoglobin levels, the agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3- dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i), (ii) and/or (iii) with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

[0036] A "hemoglobinopathy" includes any condition, the symptoms of which can be ameliorated by increasing HbF levels such as but not limited to β -thalassemia and non- sickle cell anemia. Generally, therefore, the agents of the present invention result in an increase in HbF levels.

[0037] Accordingly, the present invention provides, therefore, a method for increasing fetal hemoglobin in cells of a subject, the method comprising administration of an effective amount of an agent which increases the level of expression of the γ-globin gene via its promoter, the agent being selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of A- acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5- deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i), (ii) and/or (iii) with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

[0038] In another embodiment, a method is contemplated for treating a condition selected from the group consisting of β-thalassemia and non-sickle-cell hemoglobinopathy in a subject, the method comprising administering to the subject an effective amount of an agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochlori de and quercetin; and (iv) a combination of (i), (ii) and/or (iii) with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in cells of the subject. [0039] The agents, in one embodiment, are selective for the γ-globin gene promoter and do not substantially results in an increase in adult hemoglobin levels. Notwithstanding, agents which elevate fetal and adult hemoglobin levels are encompassed by the present invention.

[0040] In relation to this aspect, there is provided a method for treating hemoglobinopathy in a subject, the method comprising administration of an effective amount of an agent which selectively or preferentially increases fetal hemoglobin levels compared to adult hemoglobin levels, the agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of 4- acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5- deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i), (ii) and/or (iii) with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

[0041] A "condition characterized by polymerization of hemoglobin in the cell of a subject" means a condition in which polymerization of hemoglobin S is present in at least one of the subject's cells. Polymerization of hemoglobin S results in the characteristic crescent shape of erythrocytes referred to as sickling. A condition characterized by polymerization of hemoglobin S includes sickle cell anemia, sickle cell /3-thalassemia, sickle cell hemoglobin C disease and any other sickle hemoglobinopathy in which hemoglobin S interacts with a hemoglobin other than hemoglobin S.

[0042] As indicated above, the agent increases HbF levels in cells of the subject by increasing promoter activity of the γ-globin gene promoter. Methods for determining whether an agent increases the promoter activity are known to persons skilled in the art. A two-phase liquid culture assay using primary human erythroid cells has been commonly used to evaluate potential inducers of HbF. Measurement of γ-globin gene expression in such cultures by the RNAse protection assay, globin chain biosynthesis, high performance liquid chromatography (HPLC), and staining by γ-globin specific fluorescent antibodies have all been used to identify fetal globin gene inducers.

[0043] By "cells", it is understood to mean the red blood cells, including erythrocytes and reticulocytes of the subject in which hemoglobin is normally present.

[0044] By "increasing fetal hemoglobin" or "HbF" it is meant that the content of HbF in cells of the subject is greater following administration of the agent compared with the level of HbF in the absence of the agent. Quantitative methods for determining HbF content are known to persons skilled in the art. For example, the quantitative biochemical hemoglobin F alkaline denaturation method of Betke et al, Nature 754:1877-1878, 1959 or the semiquantitative acid elution method of Kleihauer et al, Klimische Wochenschrift 35:637-638, 1957 maybe employed.

[0045] The present invention further contemplates a method of an agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3- dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) combination of (i), (ii) and/or (iii) in the manufacture of a medicament for the treatment of a hemoglobinopathy in a subject, with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

[0046] The present invention is also directed to a method of enhancing the endurance or performance of a subject, the method comprising administering to the subject an effective amount of an agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4- hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desrnethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) or-a combination of (i), (ii) and/or (iii) with proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

[0047] The nucleoside analog according to the invention is understood to include both purine and pyrimidine analogs. The nucleoside analog may or may not have antiviral activity. Particularly, the nucleoside analog increases HbF in the cells of the subject by increasing promoter activity of the γ-globin gene promoter. More particularly, the nucleoside analog is not 5-azacytidine or 5'aza-2'-deoxycytidme (decitabine).

[0048] Particulary, the nucleoside analog agent according to the invention is selected from the group consisting of 3-a-hydroxydeoxygedinin, a modified ribavirin with reduced toxic side effects (such as inducing hemolysis), acyclovir, zidovudine (AZT), valacyclovir, penciclovir, ganciclovir, stavudine, abacavir and valganciclovir.

[0049] More particularly, the nucleoside analog is selected from the group consisting of acyclovir and zidovudine (AZT).

[0050] Accordingly the present invention contemplates a method of treating a hemoglobinopathy in a subject, the method comprising administration of an effective amount of an agent which increases fetal hemoglobin levels which agent is a nucleoside analog with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

[0051] The nucleotide analog according to the present invention is understood to include both purine and pyrimidine analogs. The nucleotide analog may or may not have antiviral activity. Particularly, the nucleotide analog is 2-(6-aminopurin-9- yl)ethoxymethylphosphonic acid (PMEA or adefovir) or l-(6-aminopurin-9-yl) propan-2- yloxymethylphosphonic acid (PMPA or tenofovir).

[0052] Accordingly the present invention contemplates a method of treating a hemoglobinopathy in a subject, the method comprising administration of an effective amount of an agent which increases fetal hemoglobin levels, which agent is a nucleotide analog with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

[0053] Administration of the agent according to the present invention may be effected or performed using methods known to persons skilled in the art. Various methods of delivery are contemplated including intravenous, intranasal (such as by inhalation spray), intramuscular, intralesional, subcutaneous, intraperitoneal, liposome mediated, transmucosal, intestinal, oral (such as in the form of tablets, capsules, granules, or powders), topical (such as in the form of a cream or ointment), anal (such an in the form of suppositories), ocular, or otic.

[0054] The agent is particularly provided in the form of a pharmaceutical composition comprising at least one agent according to the invention or combinations thereof, in an amount effective therefore, together with a pharmaceutically acceptable carrier or diluent.

[0055] The pharmaceutical composition according to the subject invention may comprise combinations of the agents herein described or may include other additional known therapeutic compounds which have known efficacy in the methods of the present invention.

[0056] For example, in one embodiment, the pharmaceutical composition comprises a nucleoside analog in combination with a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5- deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin, together with a pharmaceutically acceptable carrier or diluent.

[0057] In another embodiment, the pharmaceutical composition comprises a nucleoside analog or nucleotide analog together with a compound or combination of compounds selected from the group consisting of 5-azacytidine, cytarabine, hydroxyurea, cisplatin, or short chain fatty acids such as butyrate and its derivatives, together with a pharmaceutically acceptable carrier or diluent.

[0058] In a further embodiment, the pharmaceutical composition comprises a nucleotide analog together with a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4- hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin, together with a pharmaceutically acceptable carrier or diluent.

[0059] In a still further embodiment, the pharmaceutical composition comprises a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin A 1, thiotepa, 4-hydroxychalcone, melphalan, 6,3dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin together with a compound or combination of compounds selected from the group consisting of 5-azacytidine, cytarabine, hydroxyurea, cisplatin, or short chain fatty acids such as butyrate and its derivatives, together with a pharmaceutically acceptable carrier or diluent.

[0060] The present invention further contemplates a method of treating a hemoglobinopathy in a subject, the method comprising administration of an effective amount of an agent which increases fetal hemoglobin levels, which agent is a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3- desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin. In an embodiment, the subject being treated is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

[0061] The additional therapeutic compound in the pharmaceutical composition will be determined according to desired treatment. For example, the agent according to the present invention may further include a known compound, for example hydroxyurea for the treatment of β-thalassemia.

[0062] Selection of an appropriate compound for use in combination therapy, additionally to those described above may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic compounds may act synergistically to effect the treatment or prevention of the condition. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

[0063] Hence, another aspect of the present invention provides a method for treating a hemoglobinopathy in a subject, the method comprising administration of an effective amount of two or more agents selected from the list consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of A- acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5- deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin. In an embodiment, the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in cells of the subject.

[0064] The pharmaceutical compositions may be formulated, for example, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preservatives, stabilisers, flavors, etc.) according to techniques such as those well known in the art of pharmaceutical formulation.

[0065] By "pharmaceutically acceptable" it is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0066] The pharmaceutical compositions of the present invention may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets are generally provided together with one or more agents according to the invention. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated to form osmotic therapeutic tablets for control release.

[0067] Formulations for oral use may also be presented as hard gelatin capsules wherein the active agent is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active agent is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

[0068] Aqueous suspensions containing the agent according to the invention are provided in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methyicellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0069] Oily suspensions may be formulated by suspending the active agent in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

[0070] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active agent in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

[0071] The pharmaceutical compositions of the invention may also be in the form of oil- in- water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally- occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents. [0072] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.

[0073] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0074] The pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

[0075] For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the agent(s) of the present invention are employed. (For purposes of this application, topical application shall include mouthwashes and gargles.)

[0076] The agent of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to the agent of the present invention, stabilisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and phosphatidyl cholines, both natural and synthetic. Methods to form liposomes are known in the art.

[0077] The pharmaceutical compositions according to the invention may be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved by the use of suitable pharmaceutical compositions comprising the present agent, or particularly, in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps.

[0078] The term "an effective amount of an agent" means the amount of the nucleoside analog, nucleotide analog or compound selected from the group consisting of 4- acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5- deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin that will elicit the biological or medical response in the cells of the subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The biological response elicited is the increase in fetal hemoglobin in the subject's cells as described above, hi one embodiment, the amount is effective to increase expression of the γ-globin gene promoter, hi another embodiment, the amount is effective to increase HbF levels. In a further embodiment, the amount is effective to increase HbF levels compared to adult hemoglobin levels.

[0079] The amount of agent according to the invention to be administered to the subject will be dependent upon such factors as the subject's weight, and the particular route of administration used. A person of ordinary skill in the art can perform simple titration experiments to determine what amount is required to be given to the subject to achieve the desired result. The amount can be delivered continuously, such as by continuous pump, or at periodic intervals. For example, on one or more separate occasions. Desired time intervals of multiple amounts of a particular antiviral agent can be determined without undue experimentation by a person skilled in the art. Typically, such dose/response determinations are conducted in a clinical trial setting.

[0080] The subject according to the invention may be a human, primate, or mammal including, but not limited to horse, dog, cow, sheep, goat, cat or other bovine, ovine, equine, canine, feline, or murine species, it may also include fish species, preferably but not limited to zebra fish (Danio rerio). Particularly, the subject is selected from the group consisting of human, horse or dog. More particularly, the subject is a human.

[0081] The subject according to the invention may also be transgenic species encoding the human /3-globin locus. These transgenic species include non-human primates, or mammals including, but not limited to horse, dog, cow, sheep, goat, cat or other bovine, ovine, equine, canine, feline, aquatic or murine species.

[0082] It will be appreciated by persons skilled in the art of the present invention that the ability to elicit an increase in the HbF content in a subject's cells would be of benefit in enhancing the subject's performance or endurance. For example, the subject may be an athlete, more particularly a marathon runner or a long distance cyclist for whom an increase in the oxygen carrying capacity of the cells of the subject will enhance their endurance in the event. Alternatively, the subject may be an altitude climber for whom the administration of the agent according to the invention would alleviate or prevent altitude sickness. Otherwise, the subject may be a pilot of an ultra light aircraft, where the oxygen concentration at altitude can be subject to fluctuation. Other applications of the invention would be apparent to persons skilled in the art of the invention.

[0083] The present invention is also directed to a method of enhancing the endurance or performance of a subject, the method comprising administering to the subject an effective amount of an agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4- hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) or-a combination of (i), (ii) and/or (iii) with proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

[0084] It will be appreciated by persons skilled in the art that the methods of the invention can also be applied in the veterinary setting. For example, in one embodiment the subject according to the fourth aspect of the invention is a thoroughbred horse or a greyhound.

[0085] Alternatively, the subject is a human athlete.

[0086] In another embodiment, the invention contemplates a method of determining the ability of an agent to increase fetal hemoglobin levels in cells of a subject, the method comprising:

(b) obtaining a sample of blood cells from the subject ;

(c) assaying the sample for the presence of fetal hemoglobin in the cells;

[0087] Methods for assaying the content of fetal hemoglobin in a blood sample are familiar to persons skilled in the art. Such methods include histochemistry by staining by γ-globin specific fluorescent antibodies or flow cytometry detection. Other methods have been described above.

[0088] The present invention provides, therefore, a therapeutic protocol for the treatment of a hemoglobinopathy. Particular hemoglobinopathies are those whose symptoms can be ameliorated by increasing levels of HbF. Such hemoglobinopathies include β-thalassemia and non-sickle cell anemia. HbF are conveniently increased by using agents which target the γ-globin gene promoter, increasing its activity and level of γ-globin gene expression.

[0089] A therapeutic kit is also provided herein comprising an agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3- dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hernatoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i), (ii) and/or (iii).

[0090] The present invention is further described by the following non-limiting Examples. In these Examples, various materials and methods are employed as described below.

Analysis of EGFP expression

[0091] The generation of stable K562 cells that constitutively express EBNAl (KEB cells) and stably transfected with the pEBAC/148β:^Gγ-^AY EGFP (^Gγ-^Aγ EGFP cell line) has previously been reported (Vadolas et al, 2004 supra). The start codon of the EGFP gene was targeted in frame with the start codon of the ^Gγ-globin gene, while the stop codon of the Neo/Kan gene in the EGFP-Neo/Kan cassette was placed at the termination codons of the ^A γ- globin gene respectively. KEB cells and derivative cell lines were maintained in continuous culture in Dulbecco's modified Eagles medium (Sigma, St Louis, MO) supplemented with 20% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and supplemented with an antioxidant mix (1 mM sodium pyruvate, 50 μM a-thioglycerol and 2OnM bathocuprionedisulfonate). The cell density was maintained between 1-8 x 10⁵ cells/ml and cultures were incubated at 37⁰C. Cells were grown in continuous culture and in the absence of hygromycin selection.

[0092] The percentage increase in MPF in response to various inducers was calculated by taking the ratio of MPF between induced and non-induced cells and subtracting the value corresponding to non-induced cells (100%). Creation and Characterization of transgenic mice carrying the EGFP-modified ''"β- globin locus

Characterization of the 183 kb genomic fragment containing the '" ' β-globin locus

[0093] The PAC clone 148022 containing the ^/mβ-globin locus in a 183 kb genomic fragment was first isolated from the RPCI I PAC library (http://www.chori,org/bacpacΛ and shown to contain the β-globin locus. The 183 kb genomic fragment was retrofitted into the pEBAC140 cloning vector as a single Not I fragment to generate pEBAC/148β (Narayanan et al, Gene Ther. 6 (3) :442-447 ^', 1999) Sequencing of the 5¹ and 3' ends of the genomic insert and alignment with the human genome sequence (GenBank Accession number NT_028310.10) revealed that the genomic insert is 183,039 bp long, with 122,076 bp upstream of the start codon of the e-globin gene and 17,672 bp downstream of the stop codon of the β-globin gene.

Introduction of the EGFP cassette into ^hu β-globin lcus

[0094] Using GET recombination, an inducible homologous recombination system for

Escherichia c o li , EGFP reporter gene was introduced into the intact β-globin locus contained in a BAC vector. The pEBAC/148:: ^Gγ-^Aγ EGFP containing the EGFP-modified ^huβ-globin locus was purified from DHlOB cells by cesium chloride gradient ultracentrifugation. The 183 kb genomic insert containing the EGFP-modified ^huβ-globin locus was released from the BAC vector by Not I digestion and separated by pulsed field gel electrophoresis (PFGE). The 183 kb band was excised from the agarose gel, cut into small pieces, and equilibrated with agarose buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA). The agarose pieces were digested with 1 U of β-agarase I (New England Biolabs, MA, USA) per 100 mg of agarose for 2 h at 4O⁰C. Undigested agarose was removed by centrifugation at 12,500 rpm for 15 min at room temperature. The DNA was concentrated by microdialysis in microinjection buffer (10 mM Tris-HCI pH 7.4, 0.2 mM EDTA, 100 mM NaCl) overnight at 4⁰C using Millipore filter (0.05 μm pore size, Millipore Corporation, MA₅ USA). DNA quality and quantity were estimated by pulsed field gel analysis. The DNA concentration was adjusted to 0.4 ng/μl using microinjection buffer and injected into fertilized mouse oocytes (C57BL/6).

Isolation and in vitro expansion of primary murine fetal liver cells

[0095] The erythroid-specific liquid culture media was made up with the following components: Stem Pro-34 SFM Complete Medium (Invitrogen), 200 mM L-glutamine, Pen/Step, 2 U/mL rh EPO (Stem Cell Technologies), 100 ng/mL recombinant murine SCF (Walter and Eliza Hall Institute), lμM Dexamathasone (Sigma), 40 ng/mL recombinant human IGF (Promega, Madison WI USA) .

[0096] Fetal livers were isolated from transgenic mice carrying the pEBAC/148β::^Gγ-^Aγ EGFP construct. The fetal livers were dissected away from surrounding tissue on days 13- 14 of pregnancy and placed into cold PBS. A single cell suspension was made by gentle pipetting. Mature red blood cells were removed by treating cells with TAC (Tris base, NH₄Cl pH 7.2) for 5 min at 37°C and washed 3 times in 5 ml PBS. The cells were resuspended in Stem Pro-34 SFM Complete Medium at a final cell concentration of

10⁷/ml. The appropriate dilutions were calculated to seed 4 x 10⁶ cells/ml into each well in a 6-well plate. Cells were expanded by daily partial medium changes and addition of fresh factors, keeping the cell density between 1.5-4.0 x 10⁶ cells/ml for 4 days followed by treatment with inducers of globin gene expression for 3-5 days. Aliquots of treated expanded erythroid progenitor cells were analyzed by flow cytometry.

Method of fetal liver cell analysis [0097] The percentage of EGFP positive cells and relative levels of EGFP expression, media peak fluorescence (MPF) were measured by flow cytometry. The argon laser was set at 488 nm, and fluorescent cells were analyzed with a 525 nm band-pass filter. Data acquisition and analysis was performed using WinMDI software (http://pingu.salk.edu/software.html). The percentage increase in MPF in response to various inducers was calculated by taking the ration of MPF between induced and non- induced cells and subtracting the value corresponding to non-induced cells (100%). [0098] MPF and percentage of viable EGFP-expressing cells were measured by flow cytometry after 5 days. Quantitative and qualitative differences in the induction of γ-globin gene expression were examined by measuring the percentage of EGFP-expressing cells and MPF by flow cytometry. Each compound was tested 3 to 5 times and the results were statistically compared with appropriate groups.

EXAMPLE 1

Preparation of compounds for testing

[0099] Acyclovir (0-6.6mM) available as IV acyclovir from Mayne Pharma Limited. Original stock at 101.1 mM (250 mg in 10 ml at MW: 247.2 g/mol). Working stock of 20 mM 30 prepared by dilution in phosphate buffered saline (PBS).

[0100] Ganciclovir (0-4μM) available as Cymevene from Roche as a powder (500 mg vial MW: 255.2 g/mol). Working stock of 20OmM prepared in 9.796 ml of dMQ filtered water.

[0101] Ribavirin (0-2.2mM) available as IV virazole from ICN Switzerland AG (0.1 g/ml with MW:244.21) at 410 mM. Working stock of 20 mM prepared by dilution in PBS.

[0102] Valacyclovir (0-2 mM) available as Valtrex from Glaxo SmithKline in 500 mg tablets with MW:360.797 g/mol. Working stock of 400 mM prepared in 6.929 ml of dMQ filtered water.

[0103] Valganciclovir (0-4 mM) available as valcyte in 450 mg tablets with MW: 390.83 g/mol. Prepare a 200 mM stock in 5.7569 ml of dMQ filtered water. Working stock of 20 mM 10 prepared by dilution in PBS .

[0104] Zidovudine (0-ImM) Available as Retrovir aka AZT. Purchased from Advanced Melocular Technologies as 3'-Deaza~3-deoxythymidine (120mg vial MW: 267.24g/mol). Prepare a 18OmM stock by dissolving the 120mg in 2.5ml of PBS containing 20% Acetonitrile. Prepare 2mM working stock by diluting with Media.

[0105] Stavudine (0-1250μM) Available as Zerit from Roche Products (202mg oral solution MW: 224.2g/mol). Prepare 2OmM stock, solution very high in sugar and is very syrupy. [0106] Hydroquinone (0-6.6mM) Available from Sigma as a powder (10Og vial MW 110.1 g/mol) Prepare a 2OmM stock by dissolving 1 l.Olmg in 5ml of DMSO.

[0107] Thiotepa (0-180μM) Available from Sigma Pharmaceuticals (MW: 189.21 g/mol). Prepare 20 2OmM stock by dissolving 18.9 mg in 5ml of DMSO.

[0108] Melphalan (0-18OLtM) Available as Alkeran from GSK in Lyophilized powder (50mg vial MW: 305.2g/mol). Prepare 2OmM stock by dissolving the 50mg in 8ml of DMSO.

[0109] 7,8Dimethoxyflavone (0-54μM) Available from Sigma (50mg vial MW: 282.3g/mol). Prepare 4OmM stock by dissolving the 50mg in 4.5ml of DMSO.

[0110] Isoliquiritigenin (0-180μM) (10mg vial MW: 256.3g/mol). Prepare 4OmM stock by dissolving the lOmg in 0.95ml of DMSO.

[0111] Quercetin (0-180μM) Available from Sigma (1Og vial MW: 338.26g/mol). Prepare 2OmM stock by dissolving 33.826mg in 5ml of DMSO.

EXAMPLE 2 Characterization of the ^Qγ-^AγEGFP GRA

[0112] Under normal growth conditions, K562 cells and primary murine liver cells stably transfected with the ^Gγ-^Λγ EGFP construct express low amounts of e-globin and γ-globin. Hemin, as well as a variety of other compounds induce erythroid differentiation, which results in a sharp increase in embryonic and fetal globin gene expression. Hence this cell line has been investigated as an in vitro model system to study globin gene regulation and to identify and evaluate γ-globin inducers.

[0113] Since the ^Gγ-^Aγ EGFP clonal cell line maintained uniform basal levels of EGFP expression and a very high percentage of EGFP expressing cells for over 1 year of continuous culture, the cell line was used to screen for novel γ-globin gene inducers. A particularly promising approach for drug discovery is the use of chemical libraries in a high-throughput (HTP) screening. The inventors conducted a HTP screening of 2000 FDA-approved compounds using their γ-globin genomic reporter cellular assay. The screen identified a small but distinct group of compounds that induced the expression of γ- globin promoter in primary and secondary cultures.

Evaluation of fetal hemoglobin gene inducers using the ^G^-^Ay EGFP GRA

[0114] The inventors routinely investigated the responsiveness of the ^Gγ-^Aγ EGFP cell line and primary murine liver cells carrying the pEBAC/148β::^Gγ-^Aγ EGFP construct to hemin as a positive control, to assess the uniformity and reproducibility of the cellular assay. Treatment with hemin (0-100 μM) for up to 5 days followed by flow cytometry analysis caused a large dose-dependent shift in the MPF of EGFP-expressing cells, reaching a maximum 764±145% increase with hemin at 100 μM after 5 days. This increase is generally in line with the increase in EGFP expression seen directly by protein analysis and is quantitatively and qualitatively in general accordance with the reported increase in the expression of the γ-globin genes that occurs in K562 cells or primary murine liver cells carrying the ρEBAC/148β::^Gγ-^AY EGFP construct following hemin induction. The inventors also routinely investigated hydroxyurea and butyrate in their cellular assay. Maximum induction (75±10%) with hydroxyurea was observed at the highest dose tested (100 μM) after five days of induction. Similarly, maximal induction with butyrate (82+30%) was observed after five days with the highest dose tested (1000 μM).

[0115] The inventors next investigated the responsiveness of the ^Gγ-^AγEGFP cell line and primary murine liver cells carrying the pEBAC/148β::^Gγ-^Aγ EGFP construct to several FDA approved drugs, which were either initial hits in the HTP screen or lead compounds.

[0116] Compounds that were identified to give a positive induction are shown in Figure 1 and are as follows:

1) 4-acetoxyphenol;

2) Penicillic Acid;

3) Hydroquinone;

4) Erysolin;

5) Diaziquone;

6) Ribavirin;

7) Aclacinomycin Al;

8) Thiotepa;

9) Acyclovir;

10) 4'-Hydroxychalcone;

11) Malphalan; 12) 6,3'Dimethoxyflavone;

13) 3-Desmethyl-5-deshydroxyscleroin;

14) Isoliquiritigenin; 15) Pipobroman;

16) 3-a-hydroxydeoxygedinin;

17) Hematoporphyrin dihydrochloride;

18) Quercetin;

19) Abacavir;

20) Zidovidine;

21) Stavudine;

22) Valganciclovir;

23) Valacyclovir;

24) Ganciclovir 25) Adefovir;

26) Tenofovir;

27) Emtricitabine; and

28) Lamivudine.

[0117] The EGFP -induction potency of several FDA-approved drugs at concentrations with maximum activity were compared (Figure 2, 3 and 4):

1) acyclovir (0-6.6 mM) 1100 μm with 893% maximum level of induction;

2) ganciclovir (0-4 μM) 2 pM with 829% maximum level of induction;

3) ribavirin (0-2.2 mM) 100 μM 429% maximum level of induction; 4) valacyclovir (0-2 mM) 1250 μM 551% maximum level of induction;

5) valganciclovir (0-4 mM) 2 pM gave 998% maximum level of induction;

6) Zidovudine (0-2.OmM) 950 pM 150% maximum level of induction; and

7) Stavudine (0- 2.0Mm) 250 μM gave a 200% increase. EXAMPLE 3 Administration of a FDA approved drug to humans, mice and other subjects

[0118] Administration of fetal hemoglobin gene inducers to subjects is performed using methods already established for FDA approved drugs. Subjects may include human patients infected with Herpes Simplex Virus, Hepatitis B virus, Hepatitis C virus and /S- thalassaemic and non-sickle cell anaemic patients. All drugs are administered at concentrations determined to produce no side effects in Phase I /II studies.

[0119] Blood from these patients are analyzed pre- and post-treatment using methods described in Example 5.

[0120] A syringe and needle is used to take a blood specimen from a patient (the use of butterfly clips allows large volumes to be removed more easily). Retro orbital eye bleeding is performed periodically on mice treated with fetal hemoglobin gene inducers.

EXAMPLE 4 Administration of a FDA approved drug to cells isolated from humans, mice and other subjects [0121] Blood is collected from /3-thalassaemic and non-sickle cell anaemic subjects. The isolated erythroid cells is cultured in a two phase liquid culture system and induced with the identified globin gene inducers at concentrations determined to produce no side effects in Phase I /II studies.

[0122] The cultured and treated cells are analyzed pre- and post-treatment using methods described in Example 5. EXAMPLE 5 Analysis of hemtological parameters including levels of HbF in subjects

[0123] The following methods are used to determine the levels of HbF in subjects (both mice and humans). Analysis of hematological parameters is performed on mice and humans using Routine Hematology Profile (also called the Full Blood Count (FBC) or Full

Blood Examination (FBE)), which consists of a qualitative and quantitative study of the patients blood sample. Other tests include the reticulocyte count, the detection of Heinz bodies and sometimes, the Erythrocyte Sedimentation Rate (ESR). In addition, analysis of red blood cell morphology, osmotic globular resistance in NaCl solution at 0.36% (w/v), cellulose acetate gel electrophoresis and high pressure liquid chromatography (HPLC) are used to monitor activity of fetal hemoglobin gene inducers.

[0124] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

Betke et al, Nature 184:1877-1878, 1959

Cohen et al, Hematology: 14-34, 2004

Gambari et al, Cell Differentiation 14(2):87-97, 1984

Haynes et al, Blood 103:3945-3950, 2004

Keefer et al, Experimental Hematology 34: 1150-1160, 2006

Kleihauer et al, Klimische Wochenschrift 35:637-638, 1957

Narayanan et al, Gene Ther. 6 (3). -442-447 , 1999

Pace and Zein, Dynamics 235:1727-1737, 2006

Poli-Neto et al, Brazilian Journal of Medical and Biological Research 33:1313-1315, 2000

Vadolas et al, Human Molecular Genetics 13(2):223-233, 2004

Yu et al, Cancer Research 49:5555-5560, 1989

Claims

CLAIMS:

1. A method of treating a hemoglobinopathy in a subject, said method comprising administration of an effective amount of an agent which increases fetal hemoglobin levels, said agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of 4- acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3desmethyly-5- deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) or a combination of (i), (ii) and/or (iii) with the proviso that the subject is not infected with HIV or having a condition characterized by polymerization of hemoglobin in the cells of the subject.

2. A method of increasing fetal hemoglobin in cells of a subject, the method comprising administration of an effective amount of an agent which increases the level of expression of the γ-globin gene via its promoter, the agent being selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3- dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i), (ii) and/or (iii) with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

3. The method of Claim 1 or 2, wherein the agent increases fetal hemoglobin levels in cells of the subject by increasing promoter activity of the γ-globin gene promoter.

4. The method of Claim 3, wherein the cells are erythrocytes or reticulocytes.

5. The method of any one of Claims 1 to 4, wherein the nucleoside analog is selected from the group consisting of 3-a-hydroxydeoxygedinin, a modified ribavirin with reduced toxic side effects, acyclovir, zidovudine (AZT), valacyclovir, penciclovir, ganciclovir, stavudine, abacavir and valganciclovir.

6. The method according to Claim 5, wherein the nucleoside analog is acyclovir or zidovudine (AZT).

7. The method of any one of Claims 1 to 4, wherein the nucleoside analog is not 5- azacytidine or 5'aza-2'-deoxycytidme (decitabine).

8. The method of any one of Claims 1 to 4, wherein the nucleotide analog is 2-(6- aminopurin-9-yl)ethoxymethylphosphonic acid (PMEA or adefovir) or l-(6-aminopurin-9- yl) propan-2-yloxymethylphosphonic acid (PMPA or tenofovir).

9. The method of Claim 1 or 2 wherein the agent selectively or preferentially elevates HbF levels compared to adult hemoglobin levels.

10. A method of treating a condition selected from the group consisting of (β- thalassemia and non sickle-cell hemoglobinopathy in a subject, said method comprising administering to the subject an effective amount of an agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3-5 dimethoxyflavone, S-desmethyly-S-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i), (ii) and/or (iii) with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

11. The method of Claim 10, wherein the nucleoside analog is selected from the group consisting of 3-a-hydroxydeoxygedinin, a modified ribavirin with reduced toxic side effects, acyclovir, zidovudine (AZT), valacyclovir, penciclovir, ganciclovir, stavudine, abacavir and valganciclovir.

12. The method of Claim 11, wherein the nucleoside analog is acyclovir or zidovudine (AZT).

13. The method of Claim 10, wherein the nucleoside analog is not 5azacytidine or 5 'aza-2'-deoxycytidine (decitabine) .

14. The method of Claim 10, wherein the nucleotide analog is 2-(6aminopurin-9- yl)ethoxymethylphosphonic acid (PMEA or adefovir) or l-(6-aminopurin-9yl) propan-2- yloxymethylphosphonic acid (PMPA or tenofovir).

15. The method of Claim 10 wherein the agent selectively or preferentially elevates HbF levels compared to adult hemoglobin levels.

16. The use of an agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5- deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i), (ii) and/or (iii) in the manufacture of a medicament for the treatment of β-thalassemia and non sickle-cell hemoglobinopathies in a subject, with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in cells of the subject.

17. The method of any one of Claims 1 to 15 or use according to Claim 16, wherein the agent is provided in the form of a pharmaceutical composition comprising at least one agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4- hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i), (ii) and/or (iii), in an amount effective therefore, together with a pharmaceutically acceptable carrier or diluent.

18. The method of any one of Claims 1 to 15, wherein the subject is selected from the group consisting of human, primate, mammal including horse, dog, cow, sheep, goat, cat or other bovine, ovine, equine, canine, feline, aquatic or murine species.

19. The method of Claim 18, wherein the aquatic species is zebrafish.

20. The method of any one of Claims 1 to 19, wherein the subject is a non human transgenic species encoding the human β-globin locus.

21. A method of enhancing the endurance or performance of a subject, said method comprising administering to the subject an effective amount of an agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3- dimethoxyflavone, S-desmethyly-S-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i)₅ (ii) and/or (iii) with proviso that the subject is not infected with HIV or having a condition characterized by polymerization of hemoglobin in the cells of the subject.

22. A method of Claim 21 wherein the subject is a thoroughbred horse or a greyhound.

23. A method of Claim 21 wherein the subject is a human athlete.

24. A method of determining the ability of an agent to increase fetal hemoglobin levels in the cells of the subject, said method comprising:

(a) selecting an agent which increases promoter activity of the γ-globin gene promoter and administering the agent to a subject; (b) obtaining a sample of blood cells from the subject;

(c) assaying the sample for the presence of fetal hemoglobin in the cells; and

25. A method for treating hemoglobinopathy in a subject, the method comprising administration of an effective amount of an agent which selectively or preferentially increases fetal hemoglobin levels compared to adult hemoglobin levels, the agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3-desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i), (ii) and/or (iii) with the proviso that the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.

26. A therapeutic kit comprising an agent selected from the group consisting of (i) a nucleoside analog; (ii) a nucleotide analog; or (iii) a compound selected from the group consisting of 4-acetoxyphenol, penicillic acid, hydroquinone, erysolin, diaziquone, aclacinomycin Al, thiotepa, 4-hydroxychalcone, melphalan, 6,3-dimethoxyflavone, 3- desmethyly-5-deshydroxyscleroin, isoliquiritigenin, pipobroman, hematoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i), (ii) and/or (iii).