WO2015191179A1 - Ttp inhibitiors of low density lipprotein secretion for the management of diseases associated with elevated blood ldl levels - Google Patents

Abstract

The present invention provides for the novel method for the inhibition of microsomal triglyceride transfer protein (MTP) and/or apolipoprotein B (Apo B) secretion having Formulas I-XI in lowering serum lipids and in the prevention and treatment of related disease. Regulation of the VLDL/LDL pathway as a therapeutic means to reduce hyperlipidemia is supported from studies in liver cells and in an animal model. Accordingly, the inhibition of regulation described herein is identified as a new therapeutic target for treatment of cardiovascular disease or diseases associated with cardiovascular disease such as hyperlipidemia. Treatment of cardiovascular disease and associated conditions or diseases with the novel TTP inhibitors of the invention reduces MTP protein associated lipid production without side effects that occur with other methods of MTP protein associated lipid inhibition. We describe small molecules (disubstituted tetrahydro-tetrazolo(l,5a)pyrimidine (TTP) and a method of their use to inhibit the secretion of ApoB and LDLs from liver derived cells in tissue culture and in animal models. We further describe how to use these small molecules in the management of diseases associated with elevations of circulating LDLs.

Description

TTP INHIBITORS OF LOW DENSITY LIPPROTEIN SECRETION FOR THE MANAGEMENT OF DISEASES ASSOCIATED WITH ELEVATED BLOOD LDL LEVELS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C.§ 119(e) to U.S. Provisional Application Serial No. 61/987,748, which was filed on May 02, 2014. The disclosure of which is

incorporated herein by reference.

BACKGROUND OF THE INVENTION We describe small molecules (disubstituted tetrahydro-tetrazolo(l,5a)pyrimidine (TTP) and a method of their use to inhibit the secretion of ApoB and LDLs from liver derived cells in tissue culture and in animal models. We further describe how to use these small molecules in the management of diseases associated with elevations of circulating LDLs.

Excess plasma lipid accumulation is one of the most common causes of disease in Western societies because they enhance risks for various cardiovascular and metabolic disorders such as hyperlipidemia, atherosclerosis, heart disease, and metabolic syndromes. In plasma, lipids are transported on lipoproteins that provide endogenously produced and dietary lipids to tissues. Plasma lipid levels are controlled by lipoprotein assembly and their catabolism. Hence, reducing lipoprotein production is a useful approach to prevent or treat various cardiovascular and metabolic disorders.

Lipoproteins, such as Low Density Lipoprotein (LDL) and Very Low Density

Lipoprotein (VLDL), are synthesized by the liver using a structural protein, apolipoprotein B (Apo B) with the assistance of microsomal triglyceride transfer protein (MTP). MTP catalyzes the transport of triglyceride, cholesteryl ester, and phospholipids and has been implicated as a mediator in the assembly of Apo B-containing lipoproteins.

Accordingly, compounds which inhibit MTP and/or otherwise inhibit ApoB secretion are useful in the treatment of hyperlipidemia, atherosclerosis, heart disease and other conditions related thereto. Such compounds are also useful in the treatment of other diseases or conditions in which, by inhibiting MTP and/or Apo B secretion, serum cholesterol and triglyceride levels may be reduced, such conditions may include hypercholesterolemia, hypertriglyceridemia, pancreatitis, and obesity (Wetterau et al., Science, 258, 999-1001, (1992), Wetterau et al., Biochem Biophys Acta, 875, 610-617 (1986), European patent application publication Nos. 0 584 446 A2, and 0 643 057 Al). Examples of MTP inhibitors may be found in U.S. 5,712,279; 5,741,804; 5,968,950; and 6,121,283; PCT International Patent Application publications WO 96/40640, WO 97/43257, WO 98/27979, WO 99/33800 and WO 00/05201; and EP 584,446 B and EP 643,057 A.

Prior inhibitors of MTP reduced low density lipoprotein (LDL) in individuals with hypercholesterolemia. However, side effects such as elevated serum transaminase and hepatic fat accumulation, made it unlikely for large scale clinical use in the treatment of

hypercholesterolemia. Combinations using MTP inhibitors and other cholesterol or triglyceride drugs have been previously disclosed (U.S. Pat. Nos. 6,066,653 and 5,883,109) but suffer the same drawbacks as described for MTP inhibitors used alone. Thus, there is a need to develop beneficial methods for the reduction MTP activity.

SUMMARY OF THE INVENTION

The present invention provides a means to prevent LDL assembly and secretion by inhibiting the MTP protein using disubstituted tetrahydro-tetrazolo(l,5a)pyrimidine (TTP) compounds. One embodiment of the present invention incorporates the use of TTP compounds identified by ELISA in a high-throughput screen from a library for compounds that inhibit surface antigen of the hepatitis B virus (HBsAg) secretion. While these compounds inhibit HBsAG secretion they also serve as a selective inhibitor of major LDL proteins such as

Apoliprotein B, but not HDL Apolipoprotein Al. TTP treatment results in the loss of recognition by conformation-specific HBsAg antibody, suggesting a change in conformation may be the same mechanism associated with inhibition of LDL. No compound-induced cytotoxicity was observed.

Another embodiment incorporates compounds associated with 7-(2-chloro-6-fluoro- phenyl)-5-(4-chloro-phenyl)-4,5,6,7-tetrahydro-tetrazolo(l,5a)pyrimidine compounds (see Formulas I through VI) (US2009/0088397 Ciconati et al.). Another embodiment incorporates compounds associated with 7-(2-Chloro-6- fluorophenyl)-5-(4-chlorophenyl)-4,7-dihydro-[l,2,4]triazolo[l,5-a]pyrimidine compounds (see Formulas VII through XI) (US8,921,381 Xu et al.).

The present invention describes embodiments incorporating a pharmaceutical composition comprising an effective amount of a compound of formulas (I-XII) and a pharmaceutically acceptable carrier are disclosed for methods for reducing the level of Low Density Lipoprotein (LDL) and or Very Low Density Lipoprotein (VLDL) in individuals with hypercholesterolemia. DETAILED DESCRIPTION OF THE FIGURES

Figure 1: Treatment of HepG2 and HepG.2.215 cells with 10.0 micromolar tetrazolo- and triazolo-pyrimidine inhibitors of HBsAg secretion causes reduction in secretion of

Apolipoprotein B and HBsAg in Hep2.2.15, of Apolipoprotein B in HepG2, but not of

Apolipoprotein Al in either cell line. ApoB was detected with ApoB ELISA kit (Cat. No.

108807) and ApoAl was detected with ApoAl ELISA kit (Cat. No. 108804).

Figure 2: Treatment with Triazolo-pyrimidine test compound (PBHVB-2-15) of UPA/SCID mice with humanized livers ("PXB mouse", produced by PhoenixBio Ltd, Japan) causes reduction in serum levels of Apolipoprotein B of Apolipoprotein Al in either cell line

Figure 3: Treatment of HepG.2.215 cells with tetrazolo- and triazolo-pyrimidine inhibitors (10.0 micromolar PBHBV-001, 10.0 micromolar PBHBV-2-15, and 8, 4 and 2 micromolar HBF-0259) of HBsAg secretion did not induce endoplasmic reticulum stress and unfolded protein response (UPR) in HBsAg producing cells, as measured by induction of GRP78 expression. Tunicamycin and Brefeldin A treatment for 24 hrs were positive controls for UPR induction. Top figure, GRP78 mRNA levels were measured over several days by quantitative reverse-transcription PCR; Bottom figure, GRP78 protein levels were determined by western blotting after 6 days of treatment as shown

Figure 4: Pharmacokinetic parameters in UPA/SCID mice with humanized

livers ("PXB mouse", produced by PhoenixBio Ltd, Japan). Figure 5: Triazolo-pyrimidine test compound (PBHBV-2-15) does not cause body weight change during 14 days of twice-daily (BID) dosing, and is well-tolerated in UPA/SCID mice with humanized livers ("PXB mouse", produced by PhoenixBio Ltd, Japan).

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method to prevent LDL assembly and secretion by inhibiting the MTP protein using TTP compounds.

One embodiment of the present invention incorporates an effective amount of a compound selected from Formulas I, II, III and mixtures thereof;

wherein

Ri-R6 are independently phenyl or Het, wherein each phenyl or Het is optionally substituted with at least one substituent independently selected from the group consisting of (C₁_7)alkyl, (C₂_

₆)alkenyl, (C₂_6)alkynyl, (C₂_₇)alkanoyl, (C₂_₇)atkanoyloxy, (C₃_₁₂)cycloalkyl, (C_1-7)acyl, aryl, halo, OR_a, trifluoromethoxy, trifluoromethyl, NO_2> NR_aRb, cyano, CONR_aRb, CO₂R_a, SO_mR_a, S(O)_mNR_aR_b, P(=O)(OR_a)(R_a), and Het, wherein (C_1-7)alkyl or (C₃_₁₂)cycloalkyl are each independently optionally substituted with from 1 to 5 aryl, Het, OR_a> halo, NO₂, NR_aR_b, cyano, CONR_aR_b, CO₂R SO_mR_a, S(O)_mNR_aR_b, or P(=0)( OR_a)(R_a);

Xi-X₆ are independently a bond or a saturated or unsaturated alkylene group;

R_a and R_b are each independently H, (C₁_₇)alkyl, (C₃_₁₂)cycloalkyl, (C₂_₇)alkanoyl, (C₂_₇)alkanoyloxy, or aryl, or R_a and R_b together with a nitrogen to which they are attached form a Het;

m is 0, 1, or 2;

n is 0, 1, 2, 3, or 4;

or a derivative of said compound selected from the group consisting of N-oxide derivatives, prodrug derivatives, protected derivatives, isomers, and mixtures of isomers of said compound; or a pharmaceutically acceptable salt or solvate of said compound or said derivative.

Another embodiment provides for an effective amount of a compound selected from Formulas IV, V, VI and mixtures thereof:

wherein

R₇ and Rg are independently phenyl or Het, wherein each phenyl or Het is optionally substituted with at least one substituent independently selected from the group consisting of (C₁_₇)alkyl, (C_2-

₆)alkenyl, (C₂-₆)alkynyl, (C₂_₇)alkanoyl, (C₂_₇)atkanoyloxy, (C₃_₁₂)cycloalkyl, (C_1-7)acyl, aryl, halo, OR_a, trifluoromethoxy, trifluoromethyl, N0_2> NR_aR_b, cyano, CONR_aR_b, C0₂R_a, SO_mR_a,

S(0)_mNR_aR_b, P(=0)(OR_a)(R_a), and Het, wherein (C_1-7)alkyl or (C₃_₁₂)cycloalkyl are each independently optionally substituted with from 1 to 5 aryl, Het, OR_a> halo, N0₂, NR_aR_b, cyano, CONR_aR_b, C0₂R SO_mR_a, S(0)_mNR_aR_b, or P(=0)( OR_a)(R_a);

X₇ and X₈ are independently a bond or a saturated or unsaturated alkylene group;

R_a and R_b are each independently H, (C₁_₇)alkyl, (C₃_₁₂)cycloalkyl, (C₂_₇)alkanoyl, (C₂_₇)alkanoyloxy, or aryl, or R_a and R_b together with a nitrogen to which they are attached form a Het;

m is 0, 1, or 2;

n is 0, 1, 2, 3, or 4;

or a derivative of said compound selec ted from the group consisting of N-oxide derivatives, prodrug derivatives, protected derivatives, isomers, and mixtures of isomers of said compound; or a pharmaceutically acceptable salt or solvate of said compound or said derivative;

provided that the compound is a compound of Formula VI when R_y is methyl, p-MeOC₆H₄ or p- BrOC₆H₄, Rg is unsubstituted phenyl, methyl, p-MeOC₆H₄, or p-BrOC₆H₄, and X₇ and Xg are each a single bond; and

the compound is a compound of Formula IV or VI when R_y is H, Rg is an unsubstituted phenyl, p-MeOC₆H₄ or p-BrOC₆H₄, and X₇ and X₈ are each a single bond.

One embodiment of the present invention is a pharmaceutical composition, wherein Ri and R₂ of Formula I are phenyl independently optionally substituted with at least one substitute independently selected from (C₁_₇)alkyl, halo, and OR_a.

A further embodiment (HBF-0259) of the invention is a compound wherein Xi and X₂ are each a bond and

Ri is and. R-2 is

Another embodiment of the present invention incorporates an effective amount of compound selected from Formulas VII, VIII, IX, X, XI, XII and mixtures thereof;

VMM IX

wherein

Ri- R₁₂ are independently phenyl or Het, wherein each phenyl or Het is optionally substituted with at least one substituent independently selected from the group consisting of (Ci_₇)alkyl, (C₂_ ₆)alkenyl, (C₂_6)alkynyl, (C₂_7)alkanoyl, (C₂_7)atkanoyloxy, (C3-i₂)cycloalkyl, (C_1-7)acyl, aryl, halo, OR_a, trifluoromethoxy, trifluoromethyl, N0_2> NR_aRb, cyano, CONR_aRb, SO_mR_a, S(0)_mNR_aR_b, P(=0)(OR_a)(R_a), and Het, wherein (Ci_₇)alkyl or (C₃-i₂)cycloalkyl are each independently optionally substituted with from 1 to 5 aryl, Het, OR_a> halo, N0₂, NR_aR_b, cyano, CONR_aR_b, C0₂R SO_mR_a, S(0)_mNR_aR_b, or P(=0)( OR_a)(R_a);

Xi-X₁₂ are independently a bond or a saturated or unsaturated alkylene group;

R_a and R_b are each independently H, (C₁_₇)alkyl, (C₃_₁₂)cycloalkyl, (C₂_₇)alkanoyl, (C₂_₇)alkanoyloxy, or aryl, or R_a and R_b together with a nitrogen to which they are attached form a Het;

m is 0, 1, or 2;

n is 0, 1, 2, 3, or 4;

or a derivative of said compound selected from the group consisting of N-oxide derivatives, prodrug derivatives, protected derivatives, isomers, and mixtures of isomers of said compound; or a pharmaceutically acceptable salt or solvate of said compound or said derivative.

Further embodiments of the invention are compounds of Formula XII

XII

wherein

X_1; X₂, Ri, R₂, R₇ are described as above;

R₁₃ is independently H, (C₁-₇)alkyl, (C₃_₁₂)cycloalkyl, (C₂_₇)alkanoyl, (C₂_₇)alkanoyloxy, or aryl;

Methods for treating elevated VLDL/LDL levels in blood in a patient include the administration of an effective amount of a compound(s) from the present invention, together with a pharmaceutically acceptable carrier, that will reduce the serum level of VLDL/LDL in a patient. In practice, a composition containing compounds from the present invention may be administered in any variety of suitable forms known in the art, for example, by inhalation, topically, parenterally, rectally, or orally.

A composition containing a compound of the present invention may be presented in forms permitting administration by the most suitable route. The invention also relates to administering compositions containing a compound of the present invention which is suitable for use as a medicament in a patient. These compositions may be prepared according to the customary methods, using one or more pharmaceutically acceptable adjuvants or excipients. The adjuvants comprise, inter alia, diluents, serial aqueous media and the various non-toxic organic solvents. The compositions may be presented in the form of oral dosage forms, or injectable solution, or suspensions.

The choice of vehicle and the compound in the vehicle are generally determined in accordance with the solubility and chemical properties of the product, the particular mode of administration and the provisions to be observed in the pharmaceutical practice. When aqueous suspensions are used they may contain emulsifying agents or agents which facilitate suspension. Diluents such as sucrose, ethanol, polyols such as polyethylene glycol, propylene glycol and glycerol, and chloroform or mixtures thereof may also be used. In addition, the compounds of the present invention may be incorporated into sustained-release preparations and formulations.

For parenteral administration, emulsions, suspensions or solutions of the compounds according to the invention in vegetable oil, for example sesame oil, groundnut oil or olive oil, or aqueous-organic solutions such as water and propylene glycol, injectable organic esters such as ethyl oleate, as well as sterile aqueous solutions of the pharmaceutically acceptable salts, are used. The injectable forms must be fluid to the extent that it can be easily syringed, and proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin. The solutions of the salts of the products according to the invention are especially useful for administration by

intramuscular or subcutaneous injection. Solutions of the compounds as a free base or

pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropyl-cellulose. Dispersion can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. The aqueous solutions, also comprising solutions of the salts in pure distilled water, may be used for intravenous administration with the proviso that their pH is suitably adjusted, that they are judiciously buffered and rendered isotonic with a sufficient quantity of glucose or sodium chloride and that they are sterilized by heating, irradiation, microfiltration, and/or by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

Sterile injectable solutions are prepared by incorporating the compounds in the required amount in the appropriate solvent with various amounts of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique, which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

Topical administration, gels (water or alcohol based), creams or ointments containing the compounds be used. The compounds may also be incorporated in a gel or matrix base for application in a patch, which would allow a controlled release of compound through transden-nal barrier.

For administration by inhalation, the compounds may be dissolved or suspended in a suitable carrier for use in a nebulizer or a suspension or solution aerosol, or may be absorbed or adsorbed onto a suitable solid carrier for use in a dry powder inhaler.

The percentage of compound in the compositions used in the present invention may be varied, it being necessary that it should constitute a proportion such that a suitable dosage shall be obtained. Obviously, several unit dosage forms may be administered at about the same time. A dose employed may be determined by a physician or qualified medical professional, and depends upon the desired therapeutic effect, the route of administration and the duration of the treatment, and the condition of the patient. In the adult, the doses are generally from about 0.001 to about 50, preferably about 0.001 to about 5, mg/kg body weight per day by inhalation, from about 0.01 to about 100, preferably 0.1 to 70, more especially 0.5 to 10, mg/kg body weight per day by oral administration, and from about 0.001 to about 10, preferably 0.01 to 10, mg/kg body weight per day by intravenous administration. In each particular case, the doses are determined in accordance with the factors distinctive to the patient to be treated, such as age, weight, general state of health and other characteristics, which can influence the efficacy of the compound according to the invention.

The compounds used in the invention may be administered as frequently as necessary in order to obtain the desired therapeutic effect. Some patients may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate. For other patients, it may be necessary to have long-term treatments at the rate of 1 to 4 doses per day, in accordance with the physiological requirements of each particular patient. Generally, the compounds may be administered 1 to 4 times per day. Of course, for other patients, it will be necessary to prescribe not more than one or two doses per day. Examples of TTP treatment

Figure 1 demonstrates the ability of TTP compounds to inhibit secretion of both LDL and the surface antigen of the hepatitis B virus (HBsAg). After treating HepG2 and HepG.2.2.15 cells with 10.0 micromolar tetrazolo- and triazolo-pyrimidine inhibitors, there was a reduction in the secretion of both Apo B and HBsAg in Hep2.2.15 cells, but not of Apo Al in HepG2 or HepG.2.2.15 cells. Figure 1, panel A shows Apo B and Apo Al ELISA for medium from HepG2 after treatment for 6 days where a significant reduction of Apo B compared with Apo Al was observed. Figure 1, panel B shows Apo B, Apo Al and HBsAg ELISA for medium from HepG2.2.15 cells after treatment for 3 days where a significant reduction of Apo B and HBsAg ELISA but not Apo Al was observed.

Accordingly, the TTP inhibitors of HBsAg secretion (approximately 1.0 micromolar) also inhibit the secretion of the major LDL protein Apo B, but not the HDL component Apo Al, suggesting that the mechanism for TTP inhibition is at least partially related to the LDL pathway in HBsAg secretion and LDL blood levels. HbsAg particles exhibit a lipoprotein-like structure in which a compact phospholipid monolayer surrounds a more hydrophobic and fluid inner core, likely composed of triglycerides, fatty acids and sterol esters. The HBsAg particle is similar to LDLs, differing only in the Apo B-100 location at the LDL surface with the S-proteins largely immersed in the lipid core. Also HbsAg particles contain a larger quantity of protein (approximately 60% in mass) as compared to LDLs (20-25% in mass).

Figure 2 demonstrated a reduction in plasma levels of Apo B in a mouse model. Figure 2, panel A show Apo B plasma levels measure over 14 days in mice given 25 mg/kg or 75 mg/kg compared to control. Using both dosing regimens, Apo B levels were significantly reduced by day 7. Figure 2, panel B confirms that Apo Al levels in mice does not change under the same conditions.

Figure 3, panel A demonstrates results from the treatment of HepG.2.2.15 cells with the tetrazolo- and triazolo-pyrimidine (10.0 micromolar PBHBV-001, 10.0 micromolar PBHBV-2-15, and 8, 4 and 2 micromolar HBF-0259) which inhibits HBsAg Secretion but did not induce endoplasmic reticulum stress or an unfolded protein response (UPR) in HBSAg producing cells, measured through the induction of GRP78 expression. Figure 3, panel B shows the induction of a UPR response with the treatment of Tunicamycin and Brefeldin A for 24 hours, demonstrating the ability of these same cells to elicit an unfolded protein response (UPR). Figure 3, panel B on the top gel GRP78 mRNA levels were measure over several days by quantitative reverse transcription PCR). Figure 3, panel B on the lower gel GRP78 protein levels were determined by western blotting after 6 days of treatment.

Pharmacokinetic summary from UPA/SCID mice, both by i.p. and p.o. administration, with humanized livers ("PXB mouse"; produced by PhoenixBio Ltd., Japan) is shown in Figure 4.

The Triazolo-pyrimidine compound (PBHBV-2-15) does not cause body weight change in mice during a 14 day dosing regimen of twice daily (BID) administration in UPA/SCID mice with humanized livers, see Figure 5.

The foregoing description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be rereadily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set for the in the claims. Such variations are not regarded as a departure from the spirit and script of the invention, and all such variations are intended to be included within the scope of the following claims.

Claims

1. A method for preventing or treating disorders associated with elevated VLDL or LDL serum levels in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of TTP.

2. The method of claim 1 wherein the disorder is selected from a group consisting of

hyperlipidemia, atherosclerosis, heart disease, metabolic syndromes, and combinations thereof.

3. The method of claim 1 wherein the therapeutically effective amount of a TTP compound contains a pharmaceutically acceptable salt, or a solvate or hydrate of said compound; and a pharmaceutically acceptable excipient, diluent, or carrier.

4. The method of claim 1 wherein the TTP compound is from a group consisting of a compound selected from Formulas I, II, III, IV, V, VI, and mixtures thereof.

5. The method of claim 4 wherein the TTP compound is HBF-0259.

6. The method of claim 1 wherein the TTP compound is from a group consisting of a compound selected from Formulas VII, VIII, IX, X, XI, XII, and mixtures thereof.

7. The method of claim 1 further comprising at least one additional pharmaceutical agent

selected from a lipid-lowering agent.

8. A method for inhibiting LDL assembly and secretion in a subject, comprising administering to said subject an effective amount of a TTP compound.

9. The use of a TTP compound in the manufacture of a medicament for treating a disease,

modulated by inhibition of MTP and/or apolipoprotein B secretion in animals.

10. The use of claim 9 wherein the disease is selected from a group consisting of hyperlipidemia, atherosclerosis, heart disease, metabolic syndromes, and combinations thereof.

11. The use of claim 9 wherein the TTP compound is from a group consisting of a compound selected from Formulas I, II, III, IV, V, VI, and mixtures thereof.

12. The use of claim 11 wherein the TTP compound is HBF-0259.

13. The use of claim 9 wherein the TTP compound is from a group consisting of a compound selected from Formulas VII, VIII, IX, X, XI, XII, and mixtures thereof