WO2001027314A1 - Inhibiteurs d'histone acetyle transferase antiprotozoaire - Google Patents

Inhibiteurs d'histone acetyle transferase antiprotozoaire Download PDF

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Publication number
WO2001027314A1
WO2001027314A1 PCT/US2000/027337 US0027337W WO0127314A1 WO 2001027314 A1 WO2001027314 A1 WO 2001027314A1 US 0027337 W US0027337 W US 0027337W WO 0127314 A1 WO0127314 A1 WO 0127314A1
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Prior art keywords
histone
acetyl transferase
level
amount
histone acetyl
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PCT/US2000/027337
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English (en)
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Dennis M. Schmatz
Anne M. Gurnett
Samantha D. Samaras
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Merck & Co., Inc.
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Priority to AU79926/00A priority Critical patent/AU7992600A/en
Priority to CA002386299A priority patent/CA2386299A1/fr
Priority to EP00970565A priority patent/EP1222308A4/fr
Priority to JP2001529446A priority patent/JP2003511054A/ja
Publication of WO2001027314A1 publication Critical patent/WO2001027314A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa

Definitions

  • This invention is related to a method to identify pharmaceutical antiprotozoal compositions
  • this invention is directed to a method to identify antiprotozoal compositions that inhibit histone acetyl transferase
  • Parasitic protozoa are responsible for a wide variety of infections in man and animals Many protozoan infections are life threatening to the host Consequently, such infections pose significant health and economic threats
  • Health threats caused by protozoa include, for example, mala ⁇ a and trypanosomiasis Malaria is caused by protozoa of the genus Plasmodium such as P vivax, P falciparum, P ovale and P malariae Trypanosomiasis includes Chagas disease caused by Trypanosoma cruzi and Af ⁇ can sleeping sickness caused by T brucei
  • protozoa such as Pneumocystis carinu, Toxoplasma gondu, and
  • Cryptosporidium sp can cause opportunistic infections, especially in people with compromised immune systems
  • DNA has the basic structure of a double helix Instead of being meiely a dangling string, however, the DNA double helical structure is further organized by having portions of it wrapped about a core - much as thread is wrapped about a bobbin Histones form the core about which the DNA is wrapped.
  • the DNA wrapped/hi stone structure is known as a nucleosome or chromatm The nucleosomes can be further organized to form higher order structures.
  • the core histones have amino terminal regions, ⁇ ch in lysine, that are ready targets for acetylation.
  • DNA has lower affinity to acetylated histone than to deacetylated histone. As a result, the DNA is held less tightly to the acetylated histone core and the acetylated histone terminal regions are less rigidly held in relation to the DNA. Thus, the local chromatin environment is dramatically changed by acetylation, thereby affecting biological activity.
  • Acetylation of histone results from the action of the enzyme histone acetyl transferase ("HAT") Substantially acetylated histone is described as being in a state of "hyperacetylation " Deacetylation of histone results from the action of the enzyme histone deacetylase (“HDA"). Histone that is substantially without acetylation is desc ⁇ bed as being in a state of "hypoacetylation.”
  • apicidin can inhibit the action of HDA.
  • the HDA inhibiting action of apicidin is desc ⁇ bed in S. Darkin-Rattray et al., Proc. Natl. Acad. Sci. USA, 93:13143-13147 (1996). Trapoxin is desc ⁇ bed in H. Itazaki et al., J. Antibiotics, 43: 1524 (1990).
  • t ⁇ chostatin A and trapoxin A have been reported as reversible and irreversible inhibitors, respectively, of mammalian HDA (see e.g., Yoshida et al., BioAssavs, 17(5):423-430 (1995)). T ⁇ chostatin A has also been reported to inhibit partially pu ⁇ fied yeast HDA (Sanchez del Pino et al., Biochem. J., 303:723-729 (1994)).
  • T ⁇ chostatin A is an antifungal antibiotic and has been shown l) to have anti-t ⁇ chomonal activity as well as cell differentiating activity in mu ⁇ ne erythroleukemia cells, and n) the ability to induce phenotypic reversion in sis- transformed fibroblast cells (see e.g., U.S. Patent No. 4,218,478; and Yoshida et al., BioAssays, 17(5):423-430 (1995); and references cited therein).
  • Trapoxin A a cyclic tetrapeptide, induces mo ⁇ hological reversion of v-si ' s-transformed NIH3T3 cells (Yoshida and Sugita, Jap. J. Cancer Res., 83(4):324-328 (1992).
  • HDA inhibition as a target for cancer research is desc ⁇ bed in Saito et al, Proc. Natl Acad Sci USA, 96:4592-4597(1999); Bernardi et al., Ammo Acids 6:315-318 (1994), and R.E. Shute et al., J. Med. Chem 30:71-78 (1987). Utilizing HDA as a target for antiprotozoal agents is described in U.S.
  • U.S Patent No 4,218,478 describes the use of t ⁇ chostatin as an antiprotozoal agent
  • Antiprotozoal cyclic tetrapeptides, including the HDA inhibitor apicidin, are desc ⁇ bed in U.S Patent No 5,620,953, inco ⁇ orated herein by reference.
  • Apicidm [cvc/o(N-O-methyl-L-T ⁇ -L-Ile-D-P ⁇ p-L-2-am ⁇ no-8-oxo-decanoyl)] is a broad- spectrum antiprotozoal, antifungal and antineoplastic agent isolated from the fermentation culture of Fusa ⁇ um fungus The structure of apicidin is shown below:
  • This invention provides a method for identifying compounds having antiprotozoal activity
  • the method includes the steps of contacting an histone acetyl transferase with (l) an identifiably labeled compound that interacts with histone acetyl transferases, and (n) a test compound or composition, and quantitating the inhibition of the interaction of the labeled compound, wherein that inhibition is induced by the test compound or composition.
  • this invention identifies compounds having histone acetyl transferase inhibiting activity by the steps of l) determining a basal histone acetylation level in a histone/histone acetyl transferase/histone deacetylase system; ⁇ ) determining a refeience histone hyperacetylation level in a histone/histone acetyl transferase/histone deacetylase/histone deacetylase inhibitor system; in) determining a test histone acetylation level in a histone/histone acetyl transferase/histone deacetylase/histone deacetylase inhibitoi/test composition system; and IV) comparing the test level to the basal and reference levels.
  • Fig la is a schematic diagram of the action of uninhibited HAT and uninhibited HDA on the acetylation of histone.
  • Fig. lb is a schematic diagram of the action of inhibited HDA and uninhibited HAT on the acetylation of histone
  • Fig lc is a schematic diagram of the action of inhibited HDA and inhibited HAT on the acetylation of histone.
  • the action of HDA and HAT together control the net level of acetylation of histones
  • Inhibition of the action of HDA results in the accumulation of hyperacetylated histones from the unopposed action of HAT
  • hyperacetylation accumulation can be measured.
  • an ELISA Enzyme-linked immunosorbent assay
  • apicidin treated HeLa cells can directly quantify hyperacetylated histone buildup.
  • the ELISA can utilize antibodies such as, for example, one raised against an acetylated peptide which has a sequence identical to the amino terminus of human histone H3 or H4
  • Such ELISA serves as a control expe ⁇ ment that provides a baseline against which test compounds can be screened for each compound's effectiveness at inhibiting the action of HAT.
  • An inhibition of the action of HAT would result in a lessening of the accumulation of hyperacetylated histones That is, the inhibition of the action of HDA in the baseline control is counterbalanced by any inhibition of the action of HAT provided by the test candidate.
  • the present invention provides a method for identifying compounds having HAT inhibition activity by adding a test compound to the above- desc ⁇ bed control expe ⁇ ment. After a specific duration of time, the amount of hyperacetylation is compared to the control. The lower the build-up of hyperacetylated histone, the more effective is the inhibiting action of the test compound to the action of HAT
  • Fig la the basal condition of a histone/HAT/HDA system is shown in which there is an equihbnum level of acetylated histone on the right and deacetylated histone on the left as a result of the actions of HAT and HDA
  • This equilibrium level of acetylation can be measured as a basal level
  • an amount of an HDA inhibitor refer ⁇ ng to Fig lb
  • an augmented concentration of acetylated histone is caused by the decreased action of the inhibited HDA
  • a condition of hyperacetylation exists
  • This hyperacetylation level of acetylation can be measured as a reference or control level of acetylation (hyperacetylation)
  • an active test candidate one that effectively inhibits HAT
  • the present invention generally provides for l) measu ⁇ ng a basal acetylation condition, n) measu ⁇ ng an inhibited histone deacetylase reference condition, in) measu ⁇ ng a test acetylation condition of histone deacetylase inhibition in combination with histone acetyl transferase inhibition from a HAT inhibition candidate, and IV) compa ⁇ ng the test measurement to the other two (basal and reference) measurements
  • An active HAT inhibiting candidate will provide a measurement substantially similar to the basal condition
  • the present invention identifies compounds having histone acetyl transferase inhibiting activity by a method that includes the steps of I) determining a basal histone acetylation level in a histone/histone acetyl transferase/histone deacetylase system, n) determining a reference histone hyperacetylation level in a histone/histone acetyl transferase/histone deacetylase/histone deacetylase inhibitor system, in) determining a test histone acetylation level in a histone/histone acetyl transferase/histone deacetylase/histone deacetylase inhibitor/test composition system, and iv) compa ⁇ ng the test level to the basal and reference levels
  • a method of this invention includes the steps of
  • test compound or composition by affecting the action of HAT, affects the quantifiable amount of the labeled compound
  • quantification of the labeled compound serves as a diagnostic surrogate for determining any inhibiting of HAT by the test compound.
  • One embodiment of the present invention provides a method for identifying compounds having HAT inhibition activity comp ⁇ sing the steps of a) contacting with histone l) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, and in) a known amount of a labeled compound that interacts with histone acetyl transferase; b) measu ⁇ ng the level of histone acetylation and setting the measured level as a baseline (zero, basal or background) level; c) contacting with histone l) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, in) a known amount of a labeled compound that interacts with histone acetyl transferase, and iv) a known amount of an histone deacetylase inhibitor, wherein (c)( ⁇ ), (c)(n), and (c)(n ⁇ )
  • (e)( ⁇ ), (e)(n), (e)(u ⁇ ), and (e)( ⁇ v) are substantially in the same proportions to each other as (c)( ⁇ ), (c)(n), (c)( ⁇ u), and (c)( ⁇ v) above respectively, f) quantitating the affect of the test compound by measu ⁇ ng the level of histone acetylation after a set duration of time substantially the same duration as that of (d) and setting the measured level as a test level; and g) quantitating the HAT inhibiting effect of the test compound by compa ⁇ ng the results of (f) with (d) and (b).
  • the present invention provides a method for identifying compounds having HAT inhibition activity comp ⁇ sing the steps of aa) contacting with histone 1) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, in) a known amount of a labeled compound that interacts with histone acetyl transferase, and iv) a known amount of an histone deacetylase inhibitor, bb) quantitating the inhibition of the HDA by measu ⁇ ng the level of histone acetylation (hyperacetylation) after a set duration of time and setting the measured level as a reference level; cc) contacting with histone l) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, in) a known amount of a labeled compound that interacts with histone acetyl transferase, iv) a
  • Another method of this invention includes the steps of: A) contacting histone with l) a histone acetyl transferase, (n) a histone deacetylase, (in) a histone deacetylase inhibitor, (iv) a test compound or composition, and (in) an antibody that interacts with acetylated histone; and B) quantitating the antibody to determine a level of acetylated histone induced by the test compound.
  • the present invention provides a method for identifying compounds having HAT inhibition activity comp ⁇ sing the steps of aaa) contacting with histone 1) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, and in) a known amount of an antibody that interacts with acetylated histone; bbb) measuring the level of histone acetylation and setting the measured level as a baseline (zero, basal or background) level; ccc) contacting with histone 1) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, in) a known amount of an antibody that interacts
  • the present invention provides a method for identifying compounds having HAT inhibition activity comp ⁇ sing the steps of:
  • BB quantitating the inhibition of the HDA by measuring the level of histone acetylation (hyperacetylation) after a set duration of time and setting the measured level as a hyperacetylation or reference level;
  • CC contacting with histone 1) a known amount of histone deacetylase, n) a known amount of histone acetyl transferase, in) a known amount of an antibody that interacts with acetylated histone, iv) a known amount of an histone deacetylase inhibitor, and v) a known amount of a test compound, wherein (CC)( ⁇ ), (CC)(n), (CC)( ⁇ n), and (CC)( ⁇ v) are substantially in the same proportions to each other as (AA)( ⁇ ), (AA)(n), (AA)(n ⁇ ), and (AA)( ⁇ v) above respectively;
  • DD quantitating the affect of the test compound by measuring the level of histone acetylation after a set duration of time substantially the same duration as that of (BB) and setting the measured level as a test level; and EE) quantitating the HAT inhibiting effect of the test compound by compa ⁇ ng the results of (DD) with (BB).
  • the amount of histone deacetylase and histone acetyl transferase above can be those amounts found in a specific cell line, as well as extracellular known amounts. That is, the enzyme amounts can be that found intracellularly. The enzyme amounts can also be that amount mixed extracellularly.
  • the basal acetylation of histone is first measured by collecting subject cells, extracting their histones, placing the extractions on AUT gels, followed by quantitating by Western blots or with protein stains the amount of histone acetylation in a first measurement
  • the control amount of histone hyperacetylation is measured by dosing the same type subject cells with a control amount of a control HDA inhibitor, collecting the cells, extracting their histones, placing the extractions on AUT gels, and quantitating by Western blots or with protein stains the amount of acetylation (hyperacetylation) in a second measurement
  • a known amount of a test compound is added together with the control amount of the control HDA inhibitor, and the amount of hyperacetylation of histone is quantitated by Western blot or with protein stains in a third measurement The third measurement is compared to the first and second measurements to quantitate the inhibit
  • test candidates that more potently inhibit protozoal HAT, compared to their potency at inhibiting host HAT are likely effective antiprotozoal compounds
  • the reproductive rate of protozoa being usually much greater than that rate of the host cells causes protozoa to be more susceptible to HAT inhibition Therefore, compounds showing any substantial protozoal HAT inhibition properties are antiprotozoal compounds
  • the present invention provides a method for identifying compounds having antiprotozoal activity, by HAT inhibition, comp ⁇ sing the steps of:
  • the viability of the host is determined after a specified amount of time and compared to the viability of the parasite after the specified amount of time;
  • a good selective candidate is one that kills the parasite and not the host.
  • this invention provides facile and specific assays to screen compounds as HAT inhibitors and as potential antiprotozoal drugs.
  • the HDA can be any convenient protein or mixture of proteins known in the art such as, for example, a native protein in whole cells, a purified or partially purified native enzyme, a cloned histone deacetylase or an engineered va ⁇ ant thereof, a crude preparation of the enzyme, or an extract containing histone deacetylase activity
  • the enzyme can be from a mammalian (e g. human cervical carcinoma, HeLa cell), avian (e.g chicken liver or erythrocyte nuclei) or protozoal (e.g. Eimeria tenella or P. Berghei) source.
  • avian e.g chicken liver or erythrocyte nuclei
  • protozoal histone deacetylase is used. Fragments of histone deacetylase that retain the desired enzyme activity are also within the scope of this invention.
  • the HAT can be any convenient protein or mixture of proteins known in the art such as, for example, a native protein in whole cells, a pu ⁇ fied or partially pu ⁇ fied native enzyme, a cloned histone acetyl transferase or an engineered va ⁇ ant thereof, a crude preparation of the enzyme, or an extract containing histone acetyl transferase activity. Fragments of histone acetyl transferase that retain the desired enzyme activity are also within the scope of this invention.
  • the HDA inhibitor can be any convenient compound known in the art including those that act as substrates for the histone deacetylase enzyme, those that bind the enzyme at its active site, or those that otherwise act to alter enzyme activity by binding to an alternate site.
  • the test compound can be a synthetic compound, a pu ⁇ fied preparation, crude preparation, or an initial extract of a natural product obtained from plant, microorganism, or animal sources.
  • One embodiment of the present method is based on test compound induced inhibition of histone acetyl transferase activity as measured by scintillation counting to determine the histone acetylation level.
  • Compounds that inhibit histone acetyl transferase induced inco ⁇ oration of 3H acetyl CoA into histones - using any enzyme source (protozoan/mammahan/avian/etc — would be considered histone acetyl transferase inhibitors
  • the level of inco ⁇ oration can also be determined using fluorography or autoradiography following SDS polyacryl amide gel electrophoresis
  • the method of the present invention utilizes a histone acetyl transferase or an extract containing histone acetyl transferase obtained from a protozoal source, such as Eimeria or Plasmodium sp
  • Another method determines the test compound's inhibiting properties directed selectively to the parasite Compounds that inhibit parasite histone acetyl transferase induced inco ⁇ oration of H acetyl CoA into histones — with little or no effect on chicken or mammalian histone acetyl transferase induced inco ⁇ oration of H acetyl CoA into histones — would be considered selective or specific parasitic histone acetyl transferase inhibitors Similarly, the level of inco ⁇ oration can also be determined using fluorography or autoradiography following SDS polyacrylamide gel electrophoresis
  • the method of the present invention further comprises determining the IC50 of test compounds against host histone acetyl transferase in the enzyme inhibition assay as desc ⁇ bed above, to identify those compounds that have selectivity for parasitic histone acetyl transferase over that of a host
  • the assays are the same as previously described, with the histone acetyl transferase activity obtained from a host of protozoa, for example the host histone acetyl transferase may be obtained from a mammalian source, e g human, or an avian source, e g chicken
  • the target of the test compound may be ve ⁇ fied by examining the level of histone acetylation
  • the Eime ⁇ a infected host cell containing the enzyme is treated with Apicidin (to induce hyperacetylation of histones) and the test compound
  • the cells are lysed and the level of histone acetylation is determined using an antibody raised against an acetylated peptide the sequence of which is identical to amino terminus of human histone H3 or H4
  • a histone acetyl transferase inhibitor will cause inhibition of apicidin induced hyperacetylation of histones Since this method uses intact cells treated with the test compound, this technique may also be used to identify prodrugs that may be converted to histone acetyl transferase inhibitor within the cellular environment, but may not be so identified by assay
  • Cells are treated as desc ⁇ bed in preparation for the ELISA assay but the level of hyperacetylation is determined by any convenient method such as, for example, by protein stains or on AUT gels.
  • AUT the histones of host and parasite can be separated and can be detected, following transfer to nitrocellulose, using an antibody which is specific for certain acetylated lysme residues in the amino terminal region of histone H3 or H4 as described in B.M. Turner, et al , FEBS Lett., 253, 141-145 (1989).
  • These antibodies were raised against an acetylated peptide the sequence of which is identical to the amino terminus of human histone H3 or H4.
  • the present invention provides a method for the treatment of protozoal infections comp ⁇ sing administe ⁇ ng to a host suffenng from a protozoal infection a therapeutically effective amount of a compound which inhibits histone acetyl transferase
  • a therapeutically effective amount may be one that is sufficient to inhibit histone acetyl transferase of the causative protozoa
  • Compound I can be prepared using techniques desc ⁇ bed in V.I. Saloutin, et al., J. Fluorine Chemistry, 56(3 ⁇ .325-334 (1992); K.C. Joshi, et al., Indian J. Chem , 14: 1004 (1976); V.Y. Sosnovskykh, et al., Geterotskikl Soedin, 6.847-849 (1998), Q -F Wang, et al , Tetrahedron Lett., 39(16).2377-2380 (1998); and S. Eguchi, et al., Heterocvcles, 42(l).333-339 (1996).
  • Active HDA inhibiting compounds include TAN- 1746, HC-toxin, chlamydocin, WF-3161, trapoxin A, Cly-2 and analogues thereof. However, analogues of such compounds are good sources of test compounds for screening by the methods of this invention.
  • Apicidin la, lb, Ic are desc ⁇ bed in pending applications USSN 08/281,325 filed July 27, 1994 and 08/447,664 filed May 23, 1995. They are produced from a strain of Fusanum as disclosed in the above mentioned applications. Histone acetyl transferase inhibitors are useful as antiprotozoal agents. As such, they can be used in the treatment and prevention of protozoal diseases in human and animals, including poultry.
  • Examples of protozoal diseases against which histone acetyl transferase inhibitors may be used, and their respective causative pathogens, include 1) amoebiasis (Dientamoeba sp., Entamoeba histolytica); 2) giardiasis (Giardia lambha), 3) malaria (Plasmodium species including P vivax, P falciparum, P malanae and P. ovale); 4) leishmaniasis (Leishmania species including L. donovam, L tropica, L mexicana, and L.
  • brazihensis 5) trypanosomiasis and Chagas disease (Trypanosoma species including T brucei, T theile , T. rhodesiense, T gambiense, T. evansi, T. equiperdum, T. equinwn, T. congolense, T.
  • vivax and T cnizi 6) toxoplasmosis (Toxoplasma gondu); 7) neosporosis (Neospora caninwn); 8) babesiosis (Babesia sp.); 9) cryptospo ⁇ diosis (Cryptosporidium sp ); 10) dysentary (Balantidium coli); 1 1) vaginitis (Trichomonas species including T.vaginitis, and T. foetus); 12) coccidiosis (Eimeria species including E. tenella, E. necatrix, E. acervuhna, E. maxima and E. brunetti, E.
  • Histone acetyl transferase inhibitors are preferably used in the treatment or prevention of protozoal infections caused by a member of the sub- phylum Apicomplexans. More preferably histone acetyl transferase inhibitors are preferably used in the treatment or prevention of malaria, toxoplasmosis, and cryptospo ⁇ diosis in humans and animals; and in the management of coccidiosis, particularly in poultry, either to treat coccidial infection or to prevent the occurrence of such infection. Further, although not caused by an Apicomplexan, trypanosomiasis may be treated by histone acetyl transferase inhibitors.
  • the histone acetyl transferase inhibitor in the case that a histone acetyl transferase inhibitor is expected to be administered on a chronic basis, such as in the prevention of coccidiosis in poultry, the histone acetyl transferase inhibitor preferably is selective for protozoal over the host histone acetyl transferase. Long term administration of such a selective inhibitor would minimize adverse effects to the host due to histone acetyl transferase inhibition.
  • histone acetyl transferase inhibitors Two specific examples of using histone acetyl transferase inhibitors to prevent the establishment of parasitic infections in humans and animals are 1) the prevention of Plasmodium (mala ⁇ a) infection in humans in endemic areas and 2) the prevention of coccidiosis in poultry by administe ⁇ ng the compound continuously in the feed or drinking water Mala ⁇ a is the number one cause of death in the world. The disease is transmitted by mosquitoes in endemic areas and can very rapidly progress to a life threatening infection. Therefore, individuals living in or visiting areas where mala ⁇ a carrying mosquitoes are present routinely take prophylactic drugs to prevent infection The histone acetyl transferase inhibitor would be administered oi ally or parenterally one or more t ⁇ me(s) a day.
  • Coccidiosis is a disease that can occur in animals and is caused by several genera of coccidia
  • the most economically important occurrence of coccidiosis is the disease in poultry Coccidiosis in poultry is caused by protozoan parasites of the genus Eimeria.
  • the disease can spread quite rapidly throughout flocks of birds via contaminated feces. The parasites destroy gut tissue, thereby impai ⁇ ng nut ⁇ ent abso ⁇ tion.
  • a histone acetyl transferase inhibitor would be administered in the feed or drinking water for, a portion of, or the entire life of the birds.
  • the dose would range from about O.lppm to about 500ppm in the feed or water.
  • the histone acetyl transferase inhibitor could be administered orally or parenterally once the infection is suspected or diagnosed.
  • the treatment period would vary according to the specific parasitic disease and the seventy of the infection. In general the treatment would be continued until the parasites were eradicated and/or the symptoms of the disease were resolved.
  • Two specific examples are the treatment of a 1) Cryptosporidium parvum infection in an animal or human and treatment of acute Plasmodium falciparum mala ⁇ a in humans
  • Cryptosporidium parvum is a protozoan parasite that infects and destroys cells lining the intestinal tract of humans and animals. The infection establishes quite rapidly and has acute effects on the patient.
  • C. parvum infection In the case of humans, patients get severe dysentery for a period of 5-7 days. In immune compromised patients C. parvum infections can persist and can be life threatening. In animals C. parvum infection is the number one cause of death in young dairy calves. A C. parvum infection can be easily diagnosed by symptoms and examination of a stool sample. Once the disease is suspected and/or diagnosed treatment with a histone acetyl transferase inhibitor can be initiated. The dose would vary from about O.Olmg/kg to about 500mg/kg.
  • Treatments would be one or more t ⁇ me(s) a day, orally or parenterally until the infection is eliminated Routinely this dosing pe ⁇ od would be 1-3 weeks P falciparum causes acute life threatening malarial infections in humans The infection if left untreated can quite often result in death of the patient.
  • a malaria infection can be easily diagnosed by symptoms and/or examination of a blood sample from the patient Treatment would be initiated following diagnosis.
  • a histone acetyl transferase inhibitor would be administered one or more t ⁇ me(s) a day, orally or parenterally, until the infection was eliminated. The dose would range from about 0 01 mg/kg to about 200mg/kg.
  • Histone acetyl transferase inhibitors may be administered to a host in need of treatment in a manner similar to that used for other antiprotozoal agents; for example, they may be administered parenterally, orally, topically, or rectally.
  • the dosage to be administered will vary according to the particular compound used, the infectious organism involved, the particular host, the seventy of the disease, physical condition of the host, and the selected route of administration; the appropriate dosage can be readily determined by a person skilled in the art For the treatment of protozoal diseases in human and animals, the dosage may range from about
  • the dosage may range from about O.Olmg kg to about lOOmg/kg.
  • the compound is preferably administered in the animals' feed or dnnkmg water.
  • the dosage ranges from about O.lppm to about 500ppm.
  • compositions of the present invention compnse a histone acetyl transferase inhibitor and an inert earner.
  • the compositions may be in the form of pharmaceutical compositions for human and vetennary usage, or in the form of feed composition for the control of coccidiosis in poultry.
  • composition is intended to encompass a product comp ⁇ sing the active ⁇ ngred ⁇ ent(s), and the inert ⁇ ngred ⁇ ent(s) that make up the earner, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions of one or more of the ingredients.
  • the composition of the present invention thus includes a composition when made by admixing a histone acetyl transferase inhibitor and inert earner.
  • compositions of the present invention comprise a histone acetyl transferase inhibitor as an active ingredient, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients
  • the compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administrations, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered
  • the pharmaceutical compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.
  • a histone acetyl transferase inhibitor can be combined as the active ingredient in intimate admixture with a pharmaceutical earner according to conventional pharmaceutical compounding techniques
  • the earner may take a wide va ⁇ ety of forms depending on the form of preparation desired for administration, e.g , oral or parenteral (including intravenous).
  • any of the usual pharmaceutical media may be employed.
  • oral liquid preparations such as suspensions, elixirs and solutions
  • water, glycols, oils, alcohols, flavoring agents, preservatives, colo ⁇ ng agents and the like may be used
  • oral solid preparations such as powders, capsules and tablets
  • earners such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lub ⁇ cants, binders, disintegrating agents, and the like may be included.
  • tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical earners are obviously employed.
  • tablets may be coated by standard aqueous or nonaqueous techniques.
  • histone acetyl transferase inhibitors may also be administered by controlled release means and/or delivery devices.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion.
  • Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of b ⁇ nging into association the active ingredient with the earner, which constitutes one or more necessary ingredients.
  • compositions are prepared by uniformly and intimately admixing the active ingredient with liquid earners or finely divided solid earners or both, and then, if necessary, shaping the product into the desired presentation
  • a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be piepared by compressing, in a suitable machine, the active ingredient in a free- flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent
  • each tablet contains from about lmg to about 500mg of the active ingredient and each cachet or capsule contains from about lmg to about 500mg of the active ingredient.
  • compositions of the present invention suitable for parenteral administration may be prepared as solutions or suspensions of these active compounds in water suitably mixed with a surfactant such as hydroxypropylcellulose Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms
  • the pharmaceutical forms suitable for injectable use include ste ⁇ le aqueous solutions or dispersions and ste ⁇ le powders for the extemporaneous preparation of ste ⁇ le injectable solutions or dispersions
  • the form must be stenle and must be fluid to the extent that easy synngabihty exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacte ⁇ a and fungi.
  • the earner can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
  • Suitable topical formulations include transdermal devices, aerosols, creams, ointments, lotions, dusting powders, and the like These formulations may be prepared via conventional methods containing the active ingredient.
  • a cream or ointment is prepared by mixing sufficient quantities of hydrophilic matenal and water, containing from about 5-10% by weight of the compound, in sufficient quantities to produce a cream or ointment having the desired consistency.
  • compositions suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose supposito ⁇ es.
  • Suitable earners include cocoa butter and other mate ⁇ als commonly used in the art, and the suppositones may be conveniently formed by admixture of the combination with the softened or melted car ⁇ er(s) followed by chilling and shaping moulds.
  • the pharmaceutical formulations described above may include, as appropnate, one or more additional earner ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the pu ⁇ ose of rendering the formulation isotonic with the blood of the intended recipient.
  • additional earner ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the pu ⁇ ose of rendering the formulation isotonic with the blood of the intended recipient.
  • a histone acetyl transferase inhibitor may be conveniently administered as a component of a feed composition.
  • Suitable poultry feed composition will typically contain from about lppm to about lOOOppm, preferably from about 0.01% to about 0.1% percent, by weight of a histone acetyl transferase inhibitor.
  • the optimum levels will naturally vary with the species of Eimeria involved, and can be readily determined by one skilled in the art.
  • Levels of in poultry feed of from about 0.01% to about 0.1% by weight of the diet are especially useful in controlling the pathology associated with E.
  • the preferred concentration for similar control of mtestinal-dwellmg species is from about 0.01% to about 0.1% by weight of the diet Amounts of about 0.01% to about 0.1% by weight are advantageous in reducing the pathogenic effects of both cecal and intestinal coccidiosis.
  • a histone acetyl transferase inhibitor may be readily dispersed by mechanically mixing the same in finely ground form with the poultry feedstuff, or with an intermediate formulation (premix) that is subsequently blended with other components to prepare the final poultry feedstuff that is fed to the poultry.
  • Typical components of poultry feedstuff include molasses, fermentation residues, corn meal, ground and rolled oats, wheat shorts and middlings, alfalfa, clover and meat scraps, together with mineral supplements such as bone meal, calcium carbonate and vitamins.
  • compositions containing a compound of formula I may also be prepared in powder or liquid concentrate form.
  • conventional water soluble excipients such as lactose or sucrose, may be inco ⁇ orated in the powders to improve their physical properties.
  • suitable powders of this invention comp ⁇ se about 50 to 100% w/w, and preferably 60% to 80% w/w of the combination together with 0 to 50% w/w and preferably 20% to 40% w/w of conventional vetennary excipients.
  • These powders may either be added to animal feedstuff, for example by way of an intermediate premix, or diluted in animal d ⁇ nking water.
  • Liquid concentrates of this invention suitably contain a water-soluble compound combination and may optionally include a vete ⁇ nanly acceptable water miscible solvent, for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol.
  • a vete ⁇ nanly acceptable water miscible solvent for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol.
  • anti-histone H4 sera were prepared according to B.M Turner, et al., FEBS Lett., 253, 141-145 (1989).
  • Apicidin was prepared according to the procedure descnbed in U.S. Patent No 5,620,953. All temperatures expressed herein are in °C unless specifically stated otherwise. All percentages expressed herein are in percent by weight unless specifically stated otherwise.
  • the cells were treated with a mixture of 250ng/mL Apicidin and the test compound in a suitable solvent such as DMSO.
  • a suitable solvent such as DMSO.
  • the resulting treated cells were then incubated at 41°C for 5 hours, and washed with PBS (phosphate buffered saline) 3 times.
  • the washed cells were lysed by 3 successive rounds of freeze thawing followed by being blocked with 5% fat free d ⁇ ed milk in PBS with 0.5% Tween® 20 (polyoxyethylene (20) sorbitan monolaurate) at 200 ⁇ L/well for 1 hour at 37°C with shaking.
  • the blocking solution was removed and primary antibodies added (anti-hyperacetylated Histone H4 diluted 1:250 in 1% milk/PBS with 0.5% Tween® 20 at lOO ⁇ L ⁇ vell) for 1 hour at 37°C with shaking.
  • the wells were then washed 6 times with a solution of 0.5% Tween® 20 in PBS and a secondary antibody added
  • the secondary antibody was donkey anti-rabbit conjugated to hoiseradish peroxidase, diluted at 1 10,000 in 1% milk in PBS containing 0.5% Tween® 20 lOO ⁇ L of the diluted secondary antibody was added per well followed by incubation for 1 hour at 37°C with shaking Cells were then washed 3 times with a 0 5% Tween® 20 solution in PBS, followed by 3 more washings with PBS alone
  • To each plate was added 150 ⁇ L of a K-blue substrate solution available from Ehsa Technologies. The plates were then incubated for 30 minutes at room temperature. The optical density at 570n
  • E tenella oocysts Approximately 2 x 109 E tenella oocysts were suspended in 5mL of 50 mM H ⁇ P ⁇ S pH 7 4. Next, 4mL of an equal mixture (vol/vol) of 4.0mm and 1.0mm glass beads was added The resulting glass bead oocyst mixture was then shaken for 20 minutes to cause disruption of the oocysts. The efficiency of breakage was checked microscopically.
  • the resulting homogenate was separated from the glass beads and centnfuged at 3000 x g for lOmin
  • the pellet was then mixed with 20mL 50mM T ⁇ s (Tr ⁇ s(hydroxymethyl)am ⁇ nomethane) pH 8, 0 25M sucrose, 0 5M NaCl, ImM dithiothreitol (DTT), 15mM MgCl2, and 0 ImM phenylmethylsulfonyl fluoride (PMSF).
  • the resulting mixture was stirred for 30m ⁇ n on ice, centnfuged at 100,000 x g for lhr at 4°C and the supernatant dialyzed overnight at 4°C against 50mM T ⁇ s pH 8, ImM DTT, 15mM MgCl2, and O.lmM PMSF.
  • the dialysate was made 10% in glycerol, ahquoted, and was then frozen at -80°C or used immediately.
  • the sample was mixed with an equal volume of Tns-Glycine SDS Sample buffer available from Novex and analyzed by SDS gel electrophoresis followed by detection of labeled histones by fluorography or autoradiography, as known in the art Inhibition of the histone acetyl transferase was measured by a reduction in the number of counts per minute associated with histones either trapped on the filter or in a protein of approp ⁇ ate molecular weight.
  • Acid urea tnton (AUT) polyacryl amide gels were performed according to the methods of Alfageme, et al., J. Bwl Chem. 249, 3729, (1974) with modifications as desc ⁇ bed in Lennox and Cohen, Methods in Enzymology, 170, 532- 549 (1989). For optimal resolution of MDBK cell and E.
  • the separation gel contained I) 7.5M urea, n) 12% acrylamide, in) 0.38% Triton® X 100 available from Aldnch Chemical Company, Milwaukee, Wisconsin, iv) 0.8% bis acrylamide, and v) 0 87M acetic acid
  • the loading gels contained I) 7.5M urea, n) 0.37% Tnton® X 100, in) 6% acrylamide, iv) 0 04% bis acrylamide and v) 0.87M acetic acid Gels were run m 1M acetic acid Gels were pre-electrophoresed at 350V for 1 hour and run at 450V for 3.5 hours.

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Abstract

Selon l'invention, l'inhibition de l'histone acétyle transférase offre une cible permettant d'identifier des composés antiprotozoaires potentiels. Les inhibiteurs d'histone acétyle transférase antiprotozoaires sont utiles comme agents thérapeutiques contre les infections causées par des protozoaires. Un procédé permettant d'identifier des inhibiteurs d'histone acétyle transférase compare i) le niveau d'acétylation d'une combinaison d'un composé test avec un inhibiteur d'histone désacétylase à ii) un niveau de base d'acétylation.
PCT/US2000/027337 1999-10-08 2000-10-04 Inhibiteurs d'histone acetyle transferase antiprotozoaire WO2001027314A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU79926/00A AU7992600A (en) 1999-10-08 2000-10-04 Antiprotozoal histone acetyl transferase inhibitors
CA002386299A CA2386299A1 (fr) 1999-10-08 2000-10-04 Inhibiteurs d'histone acetyle transferase antiprotozoaire
EP00970565A EP1222308A4 (fr) 1999-10-08 2000-10-04 Inhibiteurs d'histone acetyle transferase antiprotozoaire
JP2001529446A JP2003511054A (ja) 1999-10-08 2000-10-04 抗原虫ヒストンアセチルトランスフェラーゼインヒビター

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7732475B2 (en) 2005-07-14 2010-06-08 Takeda San Diego, Inc. Histone deacetylase inhibitors

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6068987A (en) * 1996-09-20 2000-05-30 Merck & Co., Inc. Histone deacetylase as target for antiprotozoal agents
US6110697A (en) * 1995-09-20 2000-08-29 Merck & Co., Inc. Histone deacetylase as target for antiprotozoal agents

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997011366A1 (fr) * 1995-09-20 1997-03-27 Merck & Co., Inc. Histone desacetylase comme cible pour des agents antiprotozoaires
AU4043897A (en) * 1996-07-23 1998-02-10 Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The P300/cbp-associated transcriptional co-factor p/caf and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6110697A (en) * 1995-09-20 2000-08-29 Merck & Co., Inc. Histone deacetylase as target for antiprotozoal agents
US6068987A (en) * 1996-09-20 2000-05-30 Merck & Co., Inc. Histone deacetylase as target for antiprotozoal agents

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1222308A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7732475B2 (en) 2005-07-14 2010-06-08 Takeda San Diego, Inc. Histone deacetylase inhibitors
US7741494B2 (en) 2005-07-14 2010-06-22 Takeda San Diego, Inc. Histone deacetylase inhibitors

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EP1222308A1 (fr) 2002-07-17
JP2003511054A (ja) 2003-03-25

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