+

WO2008128299A1 - Protocole thérapeutique pour le traitement de l'hémoglobinopathie - Google Patents

Protocole thérapeutique pour le traitement de l'hémoglobinopathie Download PDF

Info

Publication number
WO2008128299A1
WO2008128299A1 PCT/AU2008/000569 AU2008000569W WO2008128299A1 WO 2008128299 A1 WO2008128299 A1 WO 2008128299A1 AU 2008000569 W AU2008000569 W AU 2008000569W WO 2008128299 A1 WO2008128299 A1 WO 2008128299A1
Authority
WO
WIPO (PCT)
Prior art keywords
subject
group
cells
agent
iii
Prior art date
Application number
PCT/AU2008/000569
Other languages
English (en)
Inventor
Dimitrios Vadolas
Hady Wardan
Original Assignee
Murdoch Childrens Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2007902177A external-priority patent/AU2007902177A0/en
Application filed by Murdoch Childrens Research Institute filed Critical Murdoch Childrens Research Institute
Publication of WO2008128299A1 publication Critical patent/WO2008128299A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/265—Esters, e.g. nitroglycerine, selenocyanates of carbonic, thiocarbonic, or thiocarboxylic acids, e.g. thioacetic acid, xanthogenic acid, trithiocarbonic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/409—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/664—Amides of phosphorus acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates generally to a protocol for treating hemoglobinopathy and agents useful for same.
  • ⁇ -Thalassemia is one of the most common genetic disorders affecting hemoglobin synthesis. Approximately 300,000 patients are born worldwide with ⁇ -thalassemia and related hemoglobinopathies. Because of mass migration of populations from endemic regions such as the Mediterranean, Middle East, African and Asian regions, ⁇ -thalassemia is being seen with increasing frequency in other parts of the world, including Australia.
  • Fetal hemoglobin is composed of two alpha chains coded by a gene located on chromosome 16 and two gamma chains coded by the A ⁇ and G ⁇ 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%.
  • HbF erythropoietin
  • 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.
  • 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.
  • ribavirin has toxic side effects such as causing hemolysis which makes it contraindicated in conditions such as ⁇ -thalassemia and infection with hepatitis C (Cohen et al, HematologyA4-34, 2004).
  • 5'-Azacytidine and 5'aza-2'-deoxycytidine 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).
  • 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).
  • 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.
  • 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 ⁇ -thalassemia or other hemoglobinopathies.
  • 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.
  • 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.
  • 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.
  • 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).
  • compounds which influence the activity of the ⁇ -globin gene promoter and thus expression of the ⁇ -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.
  • 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 G ⁇ -globin gene promoter in the human ⁇ -globin locus. Induction of EGFP expression driven by the G ⁇ -globin gene promoter parallels that of endogenous globin chain synthesis.
  • 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.
  • 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.
  • 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 5 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
  • the agent increases HbF in the cells of the subject by increasing promoter activity of the ⁇ -globin gene promoter.
  • Another aspect of the present invention is directed to a method of treating a condition selected from the group consisting of ⁇ — 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 pro
  • 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.
  • an agent
  • 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 invention contemplates a method of determining the ability of an agent to increase fetal hemoglobin levels in cells of a subject, the method comprising:
  • 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 ⁇ -thalassemia and non-sickle cell anemia.
  • HbF are conveniently increased by using agents which target the ⁇ -globin gene promoter, increasing its activity and level of ⁇ -globin gene expression.
  • 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).
  • Figure 1 is a diagrammatical representation showing the structures of compounds which were found to increase promoter activity of HhF genes.
  • 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 G ⁇ - A ⁇ 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.
  • Figure 3 is a graphical representation showing the percentage increase in promoter activity and percentage cell viability for K562 cells stably transfected with the G ⁇ - A ⁇ 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.
  • 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 G ⁇ - A ⁇ EGFP reporter construct. Cells were treated with specific concentrations of various compounds: cisplatin, isoliquiritigenin, melphalan, quercetin, thiotepa, hydroquinone, ribavirin and acyclovir.
  • a hemoglobinopathy includes a single hemoglobinopathy, as well as two or more hemoglobinopathies
  • 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.
  • HbF fetal hemoglobin
  • 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
  • a "hemoglobinopathy” includes any condition, the symptoms of which can be ameliorated by increasing HbF levels such as but not limited to ⁇ -thalassemia and non- sickle cell anemia. Generally, therefore, the agents of the present invention result in an increase in HbF levels.
  • 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 ⁇ -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 provis
  • a method for treating a condition selected from the group consisting of ⁇ -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
  • a method for treating hemoglobinopathy in a subject 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
  • 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.
  • the agent increases HbF levels in cells of the subject by increasing promoter activity of the ⁇ -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 ⁇ -globin gene expression in such cultures by the RNAse protection assay, globin chain biosynthesis, high performance liquid chromatography (HPLC), and staining by ⁇ -globin specific fluorescent antibodies have all been used to identify fetal globin gene inducers.
  • cells it is understood to mean the red blood cells, including erythrocytes and reticulocytes of the subject in which hemoglobin is normally present.
  • HbF fetal hemoglobin
  • 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
  • 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 hemo
  • 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.
  • the nucleoside analog increases HbF in the cells of the subject by increasing promoter activity of the ⁇ -globin gene promoter. More particularly, the nucleoside analog is not 5-azacytidine or 5'aza-2'-deoxycytidme (decitabine).
  • 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.
  • nucleoside analog is selected from the group consisting of acyclovir and zidovudine (AZT).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the agent is particularly provided in the form of a pharmaceutical composition
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the subject being treated is not infected with HIV or has a condition characterized by polymerization of hemoglobin in the cells of the subject.
  • the additional therapeutic compound in the pharmaceutical composition will be determined according to desired treatment.
  • the agent according to the present invention may further include a known compound, for example hydroxyurea for the treatment of ⁇ -thalassemia.
  • 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.
  • the subject is not infected with HIV or has a condition characterized by polymerization of hemoglobin in cells of the subject.
  • 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.
  • pharmaceutical additives for example, excipients, binders, preservatives, stabilisers, flavors, etc.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water or an oil medium for example peanut oil, liquid paraffin, or olive oil.
  • Aqueous suspensions containing the agent according to the invention are provided in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • 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,
  • 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.
  • preservatives for example ethyl, or n-propyl, p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl, p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl, p-hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl, p-hydroxybenzoate
  • sweetening agents such as sucrose or saccharin.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • 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.
  • 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.
  • 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.
  • creams, ointments, jellies, solutions or suspensions, etc., containing the agent(s) of the present invention are employed.
  • topical application shall include mouthwashes and gargles.
  • the agent of the present invention can also be administered in the form of liposomes.
  • 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.
  • 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.
  • 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 ⁇ -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.
  • 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.
  • 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).
  • the subject is selected from the group consisting of human, horse or dog. More particularly, the subject is a human.
  • the subject according to the invention may also be transgenic species encoding the human /3-globin locus.
  • 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.
  • 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.
  • 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.
  • the subject may be an altitude climber for whom the administration of the agent according to the invention would alleviate or prevent altitude sickness.
  • 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.
  • 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 methods of the invention can also be applied in the veterinary setting.
  • the subject according to the fourth aspect of the invention is a thoroughbred horse or a greyhound.
  • the subject is a human athlete.
  • 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:
  • 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 ⁇ -globin specific fluorescent antibodies or flow cytometry detection. Other methods have been described above.
  • 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 ⁇ -thalassemia and non-sickle cell anemia.
  • HbF are conveniently increased by using agents which target the ⁇ -globin gene promoter, increasing its activity and level of ⁇ -globin gene expression.
  • 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, hernatoporphyrin dihydrochloride and quercetin; and (iv) a combination of (i), (ii) and/or (iii).
  • 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 ⁇ g/ml streptomycin and supplemented with an antioxidant mix (1 mM sodium pyruvate, 50 ⁇ M a-thioglycerol and 2OnM bathocuprionedisulfonate). The cell density was maintained between 1-8 x 10 5 cells/ml and cultures were incubated at 37 0 C. Cells were grown in continuous culture and in the absence of hygromycin selection.
  • the PAC clone 148022 containing the /m ⁇ -globin locus in a 183 kb genomic fragment was first isolated from the RPCI I PAC library (http://www.chori,org/bacpac ⁇ and shown to contain the ⁇ -globin locus.
  • the 183 kb genomic fragment was retrofitted into the pEBAC140 cloning vector as a single Not I fragment to generate pEBAC/148 ⁇ (Narayanan et al, Gene Ther.
  • Escherichia c o li , EGFP reporter gene was introduced into the intact ⁇ -globin locus contained in a BAC vector.
  • the pEBAC/148:: G ⁇ - A ⁇ EGFP containing the EGFP-modified hu ⁇ -globin locus was purified from DHlOB cells by cesium chloride gradient ultracentrifugation.
  • the 183 kb genomic insert containing the EGFP-modified hu ⁇ -globin locus was released from the BAC vector by Not I digestion and separated by pulsed field gel electrophoresis (PFGE).
  • PFGE pulsed field gel electrophoresis
  • 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).
  • agarose buffer 10 mM Tris-HCl pH 7.5, 1 mM EDTA.
  • the agarose pieces were digested with 1 U of ⁇ -agarase I (New England Biolabs, MA, USA) per 100 mg of agarose for 2 h at 4O 0 C. 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 4 0 C using Millipore filter (0.05 ⁇ m pore size, Millipore Corporation, MA 5 USA). DNA quality and quantity were estimated by pulsed field gel analysis. The DNA concentration was adjusted to 0.4 ng/ ⁇ l using microinjection buffer and injected into fertilized mouse oocytes (C57BL/6).
  • 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), l ⁇ M Dexamathasone (Sigma), 40 ng/mL recombinant human IGF (Promega, Madison WI USA) .
  • Fetal livers were isolated from transgenic mice carrying the pEBAC/148 ⁇ :: G ⁇ - A ⁇ 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, NH 4 Cl 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 TAC (Tris base, NH 4 Cl 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 TAC (Tris base, NH 4 Cl pH 7.2) for 5 min at 37°C and washed 3 times in 5 ml PBS. The cells were re
  • 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).
  • Ganciclovir (0-4 ⁇ M) 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • Stavudine (0-1250 ⁇ M) 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.
  • 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.
  • 7,8Dimethoxyflavone (0-54 ⁇ M) Available from Sigma (50mg vial MW: 282.3g/mol). Prepare 4OmM stock by dissolving the 50mg in 4.5ml of DMSO.
  • Isoliquiritigenin (0-180 ⁇ M) (10mg vial MW: 256.3g/mol). Prepare 4OmM stock by dissolving the lOmg in 0.95ml of DMSO.
  • K562 cells and primary murine liver cells stably transfected with the G ⁇ - ⁇ ⁇ EGFP construct express low amounts of e-globin and ⁇ -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.
  • this cell line has been investigated as an in vitro model system to study globin gene regulation and to identify and evaluate ⁇ -globin inducers.
  • the G ⁇ - A ⁇ 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 ⁇ -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 ⁇ -globin genomic reporter cellular assay. The screen identified a small but distinct group of compounds that induced the expression of ⁇ - globin promoter in primary and secondary cultures.
  • the inventors routinely investigated the responsiveness of the G ⁇ - A ⁇ EGFP cell line and primary murine liver cells carrying the pEBAC/148 ⁇ :: G ⁇ - A ⁇ EGFP construct to hemin as a positive control, to assess the uniformity and reproducibility of the cellular assay.
  • Treatment with hemin (0-100 ⁇ M) 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 ⁇ M after 5 days.
  • the inventors next investigated the responsiveness of the G ⁇ - A ⁇ EGFP cell line and primary murine liver cells carrying the pEBAC/148 ⁇ :: G ⁇ - A ⁇ EGFP construct to several FDA approved drugs, which were either initial hits in the HTP screen or lead compounds.
  • ribavirin (0-2.2 mM) 100 ⁇ M 429% maximum level of induction
  • valacyclovir (0-2 mM) 1250 ⁇ M 551% maximum level of induction
  • 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.
  • 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.
  • mice and humans 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
  • FBE Blood Examination
  • Other tests include the reticulocyte count, the detection of Heinz bodies and sometimes, the Erythrocyte Sedimentation Rate (ESR).
  • ESR Erythrocyte Sedimentation Rate
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Emergency Medicine (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • Pain & Pain Management (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Diabetes (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention porte sur des composés qui influencent l'activité du promoteur de gène de g-globine et ainsi l'expression du gène de g-globine. De tels composés sont proposés pour être utiles dans des protocoles thérapeutiques pour le traitement de l'hémoglobinopathie par induction de production d'hémoglobine fœtale. Le composé est choisi dans le groupe constitué par (i) un analogue de nucléoside ; (ii) un analogue de nucléotide ; ou (iii) un composé choisi dans le groupe constitué par le 4-acétoxyphénol, l'acide pénicillique, l'hydroquinone, l'érysoline, la diaziquone, l'aclacinomycine A1, le thiotepa, la 4-hydroxychalcone, le melphalan, la 6,3-diméthoxyflavone, la 3-desméthyl-5-déshydroxyscléroïne, l'isoliquiritigénine, le pipobroman, le dichlorhydrate d'hématoporphyrine et la quercétine et (iv) une combinaison de (i), (ii) et/ou (iii), à la condition que le sujet ne soit pas infecté par le VIH ou présente une condition caractérisée par la polymérisation de l'hémoglobine dans les cellules du sujet.
PCT/AU2008/000569 2007-04-24 2008-04-23 Protocole thérapeutique pour le traitement de l'hémoglobinopathie WO2008128299A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2007902177A AU2007902177A0 (en) 2007-04-24 Inducers of fetal haemoglobin
AU2007902177 2007-04-24

Publications (1)

Publication Number Publication Date
WO2008128299A1 true WO2008128299A1 (fr) 2008-10-30

Family

ID=39874994

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2008/000569 WO2008128299A1 (fr) 2007-04-24 2008-04-23 Protocole thérapeutique pour le traitement de l'hémoglobinopathie

Country Status (1)

Country Link
WO (1) WO2008128299A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020198367A1 (fr) 2019-03-26 2020-10-01 Academia Sinica COMPOSÉS DESTINÉS À ÊTRE UTILISÉS DANS L'INDUCTION PHARMACOLOGIQUE DE HBF POUR LE TRAITEMENT DE LA DRÉPANOCYTOSE ET DE LA β-THALASSÉMIE
US11179379B2 (en) * 2017-01-30 2021-11-23 The Children's Hospital Of Philadelphia Compositions and methods for hemoglobin production
WO2022038490A1 (fr) * 2020-08-17 2022-02-24 Shorla Pharma Ltd Formulations stables comprenant du thiotépa
WO2022129536A1 (fr) * 2020-12-18 2022-06-23 Nuvamid Sa Dérivés de nicotinamide mononucléotide et leur utilisation dans la stimulation de l'expression de l'hémoglobine foetale
US11975013B2 (en) 2020-08-17 2024-05-07 Shorla Pharma Ltd. Stable formulations comprising thiotepa

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004909A1 (fr) * 1994-08-12 1996-02-22 Takeda Chemical Industries, Ltd. Utilisation de derives de quinone et d'hydroquinone pour traiter la cachexie
WO1999025367A2 (fr) * 1997-11-14 1999-05-27 The General Hospital Corporation Traitement de troubles hematologiques
US20040204430A1 (en) * 2001-04-06 2004-10-14 Debellis Robert H. Methods of treating sickle cell disease
WO2006053252A2 (fr) * 2004-11-12 2006-05-18 Northwestern University Methodes permettant de traiter des malignites hematologiques a l'aide de medicaments du type analogues nucleosidiques
EP1886677A1 (fr) * 1996-07-26 2008-02-13 Susan P. Perrine Composition comprenant un agent d'induction et agent antiviral pour le traitement de maladies sanguines, virales et cellulaires

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004909A1 (fr) * 1994-08-12 1996-02-22 Takeda Chemical Industries, Ltd. Utilisation de derives de quinone et d'hydroquinone pour traiter la cachexie
EP1886677A1 (fr) * 1996-07-26 2008-02-13 Susan P. Perrine Composition comprenant un agent d'induction et agent antiviral pour le traitement de maladies sanguines, virales et cellulaires
WO1999025367A2 (fr) * 1997-11-14 1999-05-27 The General Hospital Corporation Traitement de troubles hematologiques
US20040204430A1 (en) * 2001-04-06 2004-10-14 Debellis Robert H. Methods of treating sickle cell disease
WO2006053252A2 (fr) * 2004-11-12 2006-05-18 Northwestern University Methodes permettant de traiter des malignites hematologiques a l'aide de medicaments du type analogues nucleosidiques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DEBELLIS R.H., CHEN B.X., ERLANGER B.F.: "Inhibition of sickling in vitro by three purine-based antiviral agents: An approach to the treatment of sickle cell disease", BLOOD CELLS, MOLECULES AND DISEASES, vol. 31, 2003, pages 286 - 290 *
DESIMONE J. ET AL.: "Fetal Hemoglobin Production in Adult Baboons by 5-Azacytidine or by Phenylhydrazine-Induced Hemolysis is Associated With Hypomethylation of Globin Gene DNA", GLOBIN GENE EXPRESSION AND HEMATOPOIETIC DIFFERENTIATION, 1983, pages 489 - 500 *
STEINBERG M.H.: "Therapies to Increase Fetal Hemoglobin in Sickle Cell Disease", CURRENT HEMATOLOGY REPORTS, vol. 2, 2003, pages 95 - 101 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11179379B2 (en) * 2017-01-30 2021-11-23 The Children's Hospital Of Philadelphia Compositions and methods for hemoglobin production
US20220071981A1 (en) * 2017-01-30 2022-03-10 The Children's Hospital Of Philadelphia Compositions and methods for hemoglobin production
WO2020198367A1 (fr) 2019-03-26 2020-10-01 Academia Sinica COMPOSÉS DESTINÉS À ÊTRE UTILISÉS DANS L'INDUCTION PHARMACOLOGIQUE DE HBF POUR LE TRAITEMENT DE LA DRÉPANOCYTOSE ET DE LA β-THALASSÉMIE
CN114945572A (zh) * 2019-03-26 2022-08-26 刘扶东 用于治疗镰状血球贫血症及β-地中海型贫血的HBF药理诱导化合物
EP3947382A4 (fr) * 2019-03-26 2022-11-23 Academia Sinica Composés destinés à être utilisés dans l'induction pharmacologique de hbf pour le traitement de la drépanocytose et de la beta-thalassémie
US12246021B2 (en) 2019-03-26 2025-03-11 Academia Sinica Compounds for uses in pharmacological induction of HBF for treatment of sickle cell disease and ß-thalassemia
WO2022038490A1 (fr) * 2020-08-17 2022-02-24 Shorla Pharma Ltd Formulations stables comprenant du thiotépa
US11975013B2 (en) 2020-08-17 2024-05-07 Shorla Pharma Ltd. Stable formulations comprising thiotepa
WO2022129536A1 (fr) * 2020-12-18 2022-06-23 Nuvamid Sa Dérivés de nicotinamide mononucléotide et leur utilisation dans la stimulation de l'expression de l'hémoglobine foetale

Similar Documents

Publication Publication Date Title
Wang et al. Hydrogen sulfide alleviates the anxiety-like and depressive-like behaviors of type 1 diabetic mice via inhibiting inflammation and ferroptosis
Dover et al. Hydroxyurea induction of hemoglobin F production in sickle cell disease: relationship between cytotoxicity and F cell production
US6262116B1 (en) Transcription therapy for cancers
Gambari et al. Medicinal chemistry of fetal hemoglobin inducers for treatment of β-thalassemia
Fibach et al. Resveratrol: Antioxidant activity and induction of fetal hemoglobin in erythroid cells from normal donors and β-thalassemia patients
RU2288718C2 (ru) Лекарственные средства, содержащие ингибиторы человеческой h-sgk киназы, контролирующей клеточный объем
US20070042937A1 (en) Inhibitors of 2-oxoglutarate dioxygenase as gamma globin inducers
US20120134979A1 (en) Methods and compositions for the treatment of sickle cell disease
WO2008128299A1 (fr) Protocole thérapeutique pour le traitement de l'hémoglobinopathie
WO2003026576A2 (fr) Induction d'adipocytes bruns au moyen du facteur de transcription nfe2l2
US20170333378A1 (en) Methods of Treating Sickle Cell Disease and Related Disorders Using Fumaric Acid Esters
EP0371789B1 (fr) Médicament pour l'augmentation de l'hémoglobine foetale
Marianna et al. Valproic acid, trichostatin and their combination with hemin preferentially enhance gamma-globin gene expression in human erythroid liquid cultures
WO2021195325A1 (fr) Compositions anti-coronavirus, dosages et méthodes associés
US6566359B1 (en) 2,4,6-trimethyl-1,4-dihydro-pyridine-3,5-dicarboxylic acid esters as neuroprotective drugs
US20240216320A1 (en) Compositions and methods for treating sickle cell disease
Zhang et al. Effects of sodium metabisulfite on the expression of BKCa, KATP, and L-Ca2+ channels in rat aortas in vivo and in vitro
Stamatoyannopoulos Future prospects for treatment of hemoglobinopathies
US20210290571A1 (en) Compositions for the treatment of copper deficiency and methods of use
WO2008137533A1 (fr) Procédés et compositions pour la régulation à la hausse de l'activité de gata
KR102529484B1 (ko) 스타틴에 의한 고혈당 예방용 약학적 조성물
US20230151114A1 (en) Compositions and methods of treatment of sickle cell anemia and beta-thalassemia
UA66416C2 (en) Method for treating inflammatory and inflammatory-related disorders and normalizing metabolic parameters in blood with the aid of compound consisting of purified yeast rna
WO2024240822A1 (fr) Inhibiteurs de kinase zak alpha (map3k20) à utiliser dans le traitement de l'anémie
NO338411B1 (no) Anvendelse av én eller flere forbindelser for fremstilling av et medikament for å stimulere hematopoese, samt preparat.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08733396

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08733396

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载