Diseases induced by atherosclerosis are the first death cause in developed countries. In China, with the development of social economy and the aging of population, morbidity and mortality of cardio-cerebrovascular diseases have increased significantly in recent years. The origin and pathology of atherosclerosis are complex and have not been elucidated completely at present, but it is known that they are closely related to following factors: blood-fat abnormality, hypertension, diabetes, obesity, smoking, and so on. In these factors, the most important factor to induce the formation of atherosclerosis is blood-fat abnormality. Blood-fat abnormality mainly appears as the rise of LDL cholesterol level and descent of HDL cholesterol level.
Phospholipid transfer protein (PLTP) was called as fat transfer protein II previously, it is glucoprotein present in blood plasma and can mediate the net transfer and exchange of phospholipids between phospholipid vesicle and high-density lipoprotein (HDL) and between main lipoproteins. PLTP contains 476 amino acids residues distributing in all tissues. It exhibits high expression in placenta, pancreatic gland, fat tissue and lung, and low expression in liver, kidney and heart. Its biological synthesis is mainly completed in liver and fat tissue. There are two kinds of PLTP in blood plasma, one of which is high activity form (binding to apo E), and the other is low activity form (binding to apo A-I). The high activity form accounts for 46% in blood plasma (Tol, A. V. 2002; Janis M. T., Siggins S., Tahvanainen E., et al. J. Lipid. Res. 2004; 45(12):2303-2309). PLTP plays a very important role in the metabolism of lipoprotein.
PLTP has three principal actions in the metabolism of lipoproteins: the first action is phospholipid transfer activity, i.e., transferring phospholipids from surface remaining particles of chylomicron, very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) in lipolysis to HDL to increase HDL particles; the second action is remodeling HDL, i.e., regulating the particle size and subclass composition of HDL, mediating the fusing of two HDL3 particles, producing macrobeads of HDL2 and pre-β-HDL, moreover, phospholipid transfer is precondition to remodel HDL, pre-β-HDL is an effective acceptor of cholesterol during antiport of cholesterol; the third action is to regulate liver cells to secrete apolipoprotein B (apoB), to increase the content of VLDL in blood, PLTP deficiency leads to the reduction of VLDL secretion. Besides, PLTP participates in the transfer of vitamin E from lipoprotein to cell membrane, if its activity is reduced, it can increase the VE content in lipoproteins (VLDL and LDL) containing apo B, and antioxidation action of lipoproteins is improved; PLTP further has the activity to transfer lipopolysaccharide to enhance the response of organism to inflammation (Huuskonen J., Olkkonen V. M., Jauhiainen M., Ehnholm C. Atherosclerosis 2001, 155, 269-281; Albers J. J., Cheung M. C. Current Opinion in Lipidology 2004, 15, 255-260; Huuskonen J., Olkkonen V. M., Ehnholm C. et al. Biochemistry 2000, 39, 16092-16098).
PLTP and CETP both belong to fat transporting/lipopolysaccharide binding protein family. The protein family has four members, the other members are bactericidal permeability increasing protein (BPI) and lipopolysaccharide binding protein (LBP)). CETP was called as fat transfer protein I previously, which mediates the transfer of neutral fats such as cholesterol ester, etc. from HDL to LDL, and consequently reduces the particles of HDL. It has been found that human heritage deficiency of CEPT leads to significant rise of HDL level and moderate descent of LDL level, and thereby the research and development of CETP inhibitors have been initiated (Brown M. L., Inazu A., Hesler C. B. et al. Nature 1989, 342, 448-451). Clinic studies showed that CETP inhibitors can elevate HDL, and thus can be used for prevention and treatment of atherosclerosis. CETP inhibitors Torcetrapib (Brousseau M. E., Schaefer E. J., Wolfe M. L. et al. N. Engl. J. Med. 2004, 350, 1505-1515) and JTT-705 (de Grooth G. J., Kuivenhoven J. A., Stalenhoef A. F. et al. Circulation 2002, 105, 2159-2165) have been respectively in III and II phases of clinic trials. In III phase of clinic trials, Torcetrapib is used in combination with Atorvastatin for anti-atherosclerosis and treatment of blood fat abnormality.
In these four members, only X-ray diffraction crystal structure of human BPI has been reported. The crystal structure of BPI has shown that it is in boomerang shape, consists of two subunits, and takes on pseudosymmetric structure. In two concave surfaces of the boomerang, there are respectively two non-polar pockets, which each binds one lecithin molecule, mainly interacts with acyl chain of phospholipids, therefore it has been presumed that BPI binds the acyl chain of lipopolysaccharides. From the structure of BPI can be supposed the mechanism for the fat conveying protein family to convey fat, in which two non-polar pockets are its main functional structure (Beamer L. J., Carroll S. F., Eisenberg D. Science 1997, 276, 1861-1864). Huuskonen et al. have homologically modeled the structure of PLTP with BPI as template protein. Research on amino acid directed mutagenesis has shown that the N-terminal pocket of PLTP is very important to phospholipid transfer activity, and the C-terminal pocket thereof mainly functions to bind HDL (Huuskonen J., Wohlfahrt G, Jauhiainen M. et al. J. Lipid Res. 1999, 40, 1123-1130; Ponsin G., Qu S. J., Fan H. Z. et al. Biochemistry 2003, 42, 4444-4451).
Research results in epidemiology have shown that PLTP content in blood plasma of patients suffering from coronary artery diseases is remarkably higher than that of control group (25.5 vs 22.4 pmol/μL/h; P<0.0001). The probability of one-fifth of the patients in which the PLTP content in blood plasma is the highest suffering from coronary artery diseases is 1.9 times more than that of one-fifth of the patients in which the PLTP content in blood plasma is the lowest. Multivariable regression analysis has shown that after the correction of the factors such as age, blood plasma fat, smoking, diabetes, hypertension, etc. PLTP activity is an independent factor affecting the anticipation of coronary heart diseases. The rise of phospholipid transfer protein level in blood plasma is a danger factor of coronary artery diseases (Schlitt A., Bickel C., Thumma P. et al. Arterioscler Thromb. Vasc. Biol. 2003, 23(10), 1857-62). Blood plasma PLTP activity is elevated in the patients suffering from diabetes I stage and II stage, obesity and insulin tolerance symptoms (Tol, A. V 2002).
The PLTP activity level in blood plasma of transgenic mice in which human PLTP is over-expressed elevated by 2.5-4.5 times, so that blood plasma HDL cholesterol level is reduced by 30-40% as compared with that of wild type animals, and simultaneously the capability of formatting pro-β-HDL is enhanced (Van Haperen R., van Tol A. Vermeulen P. et al. Arterioscler Thromb Vasc Biol. 2000, 20, 1082-1088). Over-expression of PLTP in heterozygote mice with deficiency of LDL receptors causes PLTP activity to be increased by 1.3-2 times, thereby dose-dependently increasing the damage of atherosclerosis (Yang X. P., Yan D., Qiao C. et al. Arterioscler Thromb. Vasc. Boil. 2003, 23, 1601-1607).
The secretion of apolipoprotein B in PLTP gene knockout mice liver was reduced, which causes the secretion of VLDL to be decreased and anti-oxidation capability of lipoprotein to be enchanced, thereby the anti-inflammation action of HDL is improved, and the damage which suffer from coronary artery diseases is significantly decreased (Jiang X. C., Zhou H. W Curr. Opin. Lipidol. 2006, 17, 302-308; Jiang X. C., Qin S., Qiao C. et al. Nat. Med. 2001, 7, 847-852). Feeding mice with deficiency of PLTP with high fat food did not cause blood plasma lipoprotein abnormality, but interfered the transfer of all main blood plasma phospholipids from VLDL to HDL, thereby remarkably reduced HDL level (Jiang, X. C.; Bruce, C.; Mar, J. et al. J. Clin. Invest. 1999, 103, 907-914). The secretion of VLDL in the liver of mice in which PLTP is over-expressed is increased by 48%, this proves from opposite aspect that the crucial action of PLTP to the secretion of VLDL (Lie J., de Crom R., van G. T. et al. J. Lipid Res. 2002, 43(11), 1875-80). The causes for PLTP deficient state to enhance anti-oxidation capability of blood plasma lipoprotein are the increase of vitamin E accumulation and the increase of the bioavailability of vitamin E in lipoproteins (LDL and VLDL) inducing atherosclerosis (Jiang X. C., Tall A. R., QIn S. et al. J. Biol. Chem. 2002, 277(35), 31850-56). Schlitt et al. has reported that mice with deficiency of PLTP exhibit enhanced organism anti-inflammation capability (Schlitt A., Liu J., Yan D. et al. Biochim Biophys Acta. 2005, 1733(2-3), 187-91).
In summary, reducing blood plasma PLTP activity has anti-atherosclerosis effect, which at least has following three action mechanisms: the first one is to decrease the secretion of apolipoprotein B in liver so as to make the secretion level of VLDL reduced; the second one is to enhance the anti-oxidation action of LDL and mitigate the formation of atherosclerosis caused by oxidation of LDL; the third one is to enhance anti-inflammation capability of organism and mitigate the damage of atherosclerosis induced by inflammation. Therefore, PLTP inhibitor is target of novel pharmaceuticals for the prevention and treatment of atherosclerosis.
PLTP and CEPT are both members of fat conveying/lipopolysaccharide binding protein family. Researches have shown that both are not overlapped in physiological function, reduction of PLTP activity can decrease the secretion of VLDL, while reduction of CETP activity can elevate HDL cholesterol level, these two aspects are favorable for the prophylaxis and treatment of atherosclerosis. Consequently, Jiang et al. has advanced that PLTP and CETP dual inhibitor is a hopeful target for treating atherosclerosis (Jiang X. C., Tall A. R. PCT Int. Appl. WO 2002068450; Jiang X. C., Zhou H. W. Curr. Opin. Lipidol. 2006, 17, 302-308). N-{2-[2-(2,2-dimethyl-propanamido)-phenyldithio]-phenyl}-2,2-dimethyl-propanamide is the only one PLTP inhibitor that has been reported in literatures at present, and is PLTP and CETP dual inhibitor, the Ki values to inhibit both are 15 μM and 13 μM, respectively (WO 2002068450).
Researches have shown that PLTP selective inhibitor, PLTP and CETP dual inhibitor and CETP selective inhibitor can all be used for the treatment and/or prevention of various diseases associated with the increased plasma PLTP activity in a mammal (including human), such as atherosclerosis, peripheral vascular diseases, dyslipidemia, hyperlipemia, hypercholesterolemia, hypertriglyceridemia, familial hypercholesterolemia, cardiovascular diseases, angina pectoris, ischemia, heart ischemia, stroke, myocardial infarction, reperfusion injury, hypertension, diabetes with vascular complications, obesity or endotoxemia, etc.
Term “PLTP selective inhibitor” means a compound capable of inhibiting PLTP activity but having no inhibition action to CETP activity.
Term “PLTP and CETP dual inhibitor” means a compound capable of inhibiting both PLTP activity and CETP activity.
Term “CETP selective inhibitor” means a compound capable of inhibiting CETP activity but having no inhibition action to PLTP activity.