GB2523527A - Screen compounds for the modulation of proprotein convertase subtilisin/kexin type 9(PCSK9) - Google Patents

Abstract

A cell-based, functional assay incorporating a recombinant gain of function PCSK9 protein comprising mutants D374Y and S127R to identify inhibitors or modulators of PCSK9. The assay is carried out in HepG2 cells and test compounds dissolved 200 fold in DMSO. Also claimed is an inhibitor of PCSK9 together with a statin for the treatment of atherosclerosis, coronary heart disease and type 2 diabetes. The inhibitor may be colchicine.

Description

SCREEN COMPOUNDS FOR THE MODULATION OF PROPROTEIN

CONVERTASE SUBTI LISIN/KEXIN TYPE 9(PCSK9)

FIELD OF THE INVENTION

This invention relates to methods for screening of the PCSK9 modulators. Such method and pharmaceutical composition are useful for the treatment of atherosclerosis, coronary heart disease and type-2 diabetes diseases. In particular, this invention relates to method and composition of colchicine compound, a prescribing drug for treatment of acute gout flares and familial Mediterranean fever. Our disclosure shows that coichicine inhibits PCSK9-mediated degradation of LDLR. This is a novel finding and giving an ancient remedy a new usage for atherosclerosis, cardiovascular disease and type-2 diabetes treatment.

BACKGROUND OF THE INVENTION

Cardiovascular Disease (CYD) is among the main causes of premature death in the world.

Plasma LDL cholestrol is a major determinant of CVD. Recently, stains, a class of drug of HMG-CoA rcductasc inhibitors, havc bccn provcd to bc a grcat succcss in trcatmcnt of ccardiovascular diseases in many people. However, some people may require 2 or 3 differcnt drugs to obtain cholcstcrol lcvcls of <lOOmgtdL, but carry a high risk of sidc efffects which includes type 2 diabetes. So there is a need to develop new cholesterol-lowering therapies. Given that high cholesterol related obesity is a taime bomb awaiting the next generation, this need is pressing, to protect current and future generations. Recent genetic studies have shown, one of the key genes, PCSK9, plays a critical role in cholesterol metabolism by controlling the level of low-density lipoprotein rcccptor(LDLR)[1,2]. Rcccnt population gcnctic studics have shown that the none-sense mutation of PCSK9 were associated with a 28 percent reduction in mean LDLc and an 88 percent reduction in the risk of a CDV in african-American, indicating PCSK9 is a gentic validated target for the reduction of LDLc in human population[3] PCSK9 is currently a "hot drug targef' for all m*jor pharmaceutical companies. Alnylam Pharmaceuticals Inc is developing antisense oligoes of PCSK9. isis pharamaceuticals Inc and Bristol-Myers Squibb Co. are using gene silencing approach to blocks the pmdcution of the PCSK9. Amgen Inc. Merck and Regeneron Pharmarceutical have all developed the antibodies against PCSK9. In recent 2012 American Heart Association meetin& at least five phase II clinicla trails of patients with AMC 145 antibodics(Amgcn) or SAR236553/RE0E727 antibody(Regeneron Pharmarceutical) indicate that anti-PCSK9 antibodies reduced LDL in patients with hypercholesterolemia[4]. Phase 3 results from these antibody therapy are eagerly waited.

SUMMARY OF TILE INVENTION

PCSK9 plays a critical role in cholesterol metabolism by conimiling the level of low-density lipopmtein receptor(LDLR). Recent population genetic studies have shown that PCSK9 is a gentic validated target for the reduction of LDLc. Several anti-PCSK9 antibodies are currently undergoing Phase II trails in patients with hypercholesterolemia with promising results. However, developing novel compounds that inhibit PCSK9 function or block it's binding to LDLR is pharmaceutically preferred over both antibody and siRNA routes. We have recently developed a unique, cell-based, functional assay incorporating recombinant PCSK9 protein for high-throughput screening of human liver cell HepG2. A pilot screen of the NINDS compound library has been successful in identifyng several potential compounds in duplicate screening. One of the lead compounds, eolchicine, has been validated with Western blot assays for PCSK9-mediated LDLR degradation in dose-reponsive assay. Colchicine is well known and FDA proved prescribing drug for the treatment of acute gout flares and familial Mediterranean fever.

Our finding, coincidcnt with previous observation of eolchicinc lowing cholesterol in clinical setting highlights the novel use of this drug for cardiovascular disease treatment This is the first successful assay of its kind developed for screening for PCSK9 inhibitor,

representing a landmark in the field.

According to the present invention there is thus provided a method for high-throughput screening a substance, which inhibits or prevents degradation of LDL receptor by PCSIK9 and its gain of function mutant forms', such as D374Y-PCSK9.

* Tn one embodiment, the invention provides an novel screening methods and DM50 dilution for such screening methods.

* In another embodiment, the invention provides cell lines, which express human PCSK9 and PCSK9, gain of function mutations, such as D374Y and S127R.

* In another embodiment, the invention provides methods for carrying out high-throughput assay with cell culture condition and dilution.

1. In another embodiment, a method for identifying a substance, which may be used in the treatment of atherosclerosis, coronary heart disease and/or diabetes, which method comprises determine whether a test substance is an inhibitor or modulators of pcsk9.

* In another embodiment, the invention provides methods for identi'ing colchicine as a substance to inhibit PCSK9-mediated degradation of LDLR.

Brief description of the Drawin2s

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as is commonly understood by one of the skill in the art to which this invention belongs. All publications and patents referred to herein are incorporated by reference, Figure 1 illusn-ates cell-based PCSK9-inhibition function assays with recomninant D374Y-PCSK9 protein. FlepG2 cells were incubated with the vehicle (PBS) and different dilutions of DM50 (0.5 tl or ipi DM50 in 100 p. medium) in D374Y-PCSK9 treated HepG2 cells.

LDL uptake was quantified on a Fusion fluorescence plate reader. The average fluorescence reading ± SD are shown. (* PcO.05 vs. corresponding vesicle only, n = 3 per group).

Figure 2 illustrates experimental design of cell-based high-throughput assay for identifying PCSk9 inhibitors. HepG2 cells were seeded in 2x105 /ml in 96-wells plate. Vehicle (PBS) or D374Y-PCSK9 protein (13.4 nM) was added in the culture medium with pooling 5 compounds into each well (500 nM final conc.) in a NINDS library. The positive pools in duplication were broken down to each individual compound. Each individual positive compound was then subject to second screen assay by westem blot.

Figure 3 illustrates representing readings of the fluorescence LDL uptake in the high-throughput assay. The positive poois (total 9) were broken down to each individual compound and 6 positives have been detected in a duplication assay to have significant higher LDL uptake than control weHs (*p<O.O5 vs. corresponding vesicle only, n = 6).

Figure 4 illustrates screening analysis of PCSK9-mediated LDLR degradation by western blot. A. 6 positive compounds (compound A-F) were added to the culture medium individually with D374Y-PCSK9 to HcpG2 cells. Reduction of cndogcnous mature LDLR (M) in HepG2 cells was analyzed by westem blot. Compound B (Fig. 4, lane 4, Table 1, colchicine) has been detected to inhibit D374Y-mediated LDLR degradation, showing increased LDLR after treatment. Equal loadings were confirmed using anti tubulin antibody. B. Dose response of the compound B (colchicine) on inhibiting D374Y-mediated LDLR degradation in the westem blot analysis. The ratio of LDLRitubulin in untreated sample (Fig.4, lane 1) was assigned a value of 100%. The ratio of LDLR/tubulin in D374Y-PCSK9 treated sample (13.4nM, 16h, Fig.4, lane 2) has a value of 25%. Four different concentrations of colchicine were used to calculate the ICSO of inhibition of PCSK9-D374Yby colchicine (n=3). The 1C50 was 0.131 p.M.

FigS. The proposed model for the action of chochicine in PCSK9-mediated LDLR degradation.

Table I shows the list of the potential positive hits in high-throughput assay.

DETAILED DESCRIPTION OF THE INVENTION

Present invention relates to establish a novel cell-based high-throughput program to identify PCSKY modulators.

We have set up a cell-based screening method using fluorescent-labeled LDL uptake in HepG2 cells as end point assay. We treated human liver HepG2 cells with a gain-of-function' mutant PCSK9-D374Y protein at 1 ig/mI concentration (13.4 NM). As shown in Fig. 1A, the fluorescence -labelled LDL uptake was significantly decreased in the PCSK9-D374Y-treated samples than that of untreated samples. This fluorescence intensity difference can be easily detected by the fluorescence plate reader (Fig.IB, 1st two columns).

Because all compounds in our screening assay were dissolved in DM50, we have further tested the DMSO effects on the PCSK9-mediated LDL uptake. Although there was tendency of DM50 treatment reducing both fluorescence reading of PCSK9-treated and untreated samples, at 200 fold (0.5 tl in 100!1l) dilution, the treatment group and untreated group was statistically significant (P<0.05)(Fig.IB), therefore, we have carried out the screening program at this dilution. The Z' factor of the assay was determined to he 0.52, suggestion this fluorescence-cell based assay would perform well in the large-scale screening process.

Initially, a pool of S compounds (1mM of each) was added into 100 p1 of medium in each well in 96-well plate format, resulting each compound concentration at 500 nM (Fig.2). In the positive control wells, PCSK9-D374Y protein (13.4 nM final concentration) was added.

The negative control will be medium only. After I 6h, the medium was replaced with DMEM without scram. After 24h, the medium was replaced with fresh medium containing tg/ml Bodipy FL LDL without serum. The cells were incubated for 4 h at 37°C. The cells were washed with PBS and LDL uptake was quantified on the Fusion fluorescence plate reader. We have successfully screening the NINDS compound library by pooling 5 compounds into each well (500 nM final conc.), screening by duplication. Hits were defined as molecule that gave significantly increased LDL fluorescence (p c 0.05). Each screen will be duplicated once. Only the hit polls on both duplicates were selected and breakdown into each individual compound. (Fig.2). As shown on Fig.3, the wells that contain D374Y-PCSK9 protein has significant lower fluorescence-labeled LDL uptake as LDLR being degraded in comparison with the control wells (untreatcd)(Fig.3.1 -2 column N3). Then positive pools (total 9) were broken down to each individual compound and 6 positive hits have been detected in a duplication assay (Fig.3, Table 1).

We next used the western blot assay to access the 6 potential hits identified by FITS assay.

HepG2 cells were treated with lpiml PCSK9-D374Y protein (13.4nM) together with each of the 6 compounds. After 16h in serum-free DMEM medium incubation, the whole cell extracts were isolated and subjected to western blot analysis with the anti-human LDLR antibody. As shown on Fig.4A, there was significant increase of LDLR protein in one of the compounds, compound B (colchicinc) treatment (Fig.4, lane 4). In fact the level of LDLR in colchicine treated cells was similar to the untreated group (Fig.4A, lane 1). We further observed does-dependent increase of the LDLR protein content (Fig.4B). The half maximum inhibition of colehicine to PCSK9-D374Y degradation of LDLR protein was calculated as 0.131 tM(Fig.4B).

In fact, looking for past literatures we found there are two previous reports showing cholesterol lowing effects of colchicine. In 1967, Faloon et al reported that in six obese patients receiving constant low-calories diet, being treated with colehicine, serum cholesterol fell by 40 to 80 mgtlOOml in 6 days in four of the five patients [5]. Again, in the 1970, Rubulis et al reported the two hypercholesterol patients have been shown significant fall in serum cholesterol in 5 days treatment of colchicinc 2.5mg daily[61.

Despite these reports, the metabolic effects of coichicine on cholesterol regulation have not been explored. Our high-throughput result has pointed for the first time that eolchieine interaction with PCSK9 could be its key function in mediation of cholesterol regulation.

Colchicine is destructive in PCSK9/LDLR tight interaction in early endosome, partly may be due to directly biding to PCSK9 protein(Fig. 5) or alternatively, because such interaction need to be in lower PH>6.O. At the acidic pH of endosomes, the affinity of PCSK9 binds to LDLR increases approximately 150-fold[7], while chochicine is known to increases tissue pH, by inhibiting the oxidation of glucose. Colehieine &so inhibits microtubule polymerization by binding to tubulin. The details of colchicine!PCSK9 action remain to be determined.

EXAMPLE 1

Cell culture, Protein purification and western blot assays HcpG2 cells were obtained from European collection of cell culture (Wiltshire, UK). Cells were grown in DMEM containing 25mM glucose and 10% fetal calf serum, as described[8]. A stable expressed FLAG-tagged wild-type PCSK9 cell lines and a gain-of-function' mutant PCSK9-D374Y cell line were developed as previously described[9]. The cells were grown in DMEM medium with 10% FBS. The FLAG-tagged PCSK9 protein was isolated using the FLAG-immunoprecipitation kit (Sigma) following by elution with the 3xFLAG peptide (final concentration lSOngiml of 3xFLAG peptide). PCSK9-wild type and a gain-of-function' mutant PCSK9-D374Y protein purification were described previously. the protein purity was determined by SDS-PAGE and visualized by Coomassie Blue stain with over 90% purity. Protein concentration was measured using Bio-Rad Protein Assay kit, Cat: 500-0006, Bio-Rad. UK). The antibodies used were a rabbit antibody directed against amino acids 184-196 of human LDLR (Research Diagnostics Inc.) and a rabbit antibody against PCSK9 (Cayman). The methods of whole cell extract and western blots were carried out as described[8]. Western blot densitometry was carried out using the Vision works LS software (UVP, Cambridge, UK). All data were analysed by GB-Stat V5.4.4 program (written by Dr. Philip Friedman, Howard University) using student t-Test (two-tailed). As shown in Fig. 1A, the fluorescence -labelled LDL uptake was significantly decreased in the PCSK9-D374Y-treated samples than that of untreated samples. This fluorescence intensity difference can be easily detected by the fluorescence plate reader (Fig.1B, 1 two columns). Because all compounds in our screening assay were dissolved in DM50, we have further tested the DM50 effects on the PCSK9-mediated LDL uptake. Although there was tendency of DM50 treatment reducing both fluorescence reading of PCSK9-treated and untreated samples, at 200 fold (0.5 tl in R1) dilution, the treatment group and unneated group was statistically significant (Pc0.05)(Fig.IB), therefore, we have carried out the screening program at this dilution.

The Z' factor ol the assay was determined to he 0.52, suggestion this fluorescence-cell based assay would perfonn well in the irgescale screening process. Initially, a pool of S compounds (1mM of each) was added into 100 tl of medium in each well in 96-well plate format, resulting each compound concentration at 200 NM (Fig.2).

Example 2.

High-throughput screening of cell-based LDL uptake assay of NINDS library The National Institute of Neurological Disorders and Stroke (NINDS) compound library, which is a collection of known drugs and pharmacologically active compounds originally collated by the National Institute of Neurological Disorders and Stroke (US). The compound set is 1041 compounds (http://iccb.rned.harvardedu/screeninq/compound hbraries/nndshtm), a collection of known drugs and pharmacologically active compounds, consisting mainly of FDA-approved drugs [10,111, The NINDS library was kindly supplied to us by Dr Nicole Mathon and Dr Catherine Kettleborough from the Medical Research Council Technology, UK(MRCT) All the compounds in the NINDS were dissolved in DMSO at 1mM concentration, therefore, the sensitivity of the cell-based assay were determined in different dilutions of DMSO on the standard cell culture condition.

High-throughput screening was carried out in high throughput robotics facility in the university of Sheffield [12]. Human liver HepG2 cells were seeded in 96 well tissue plates at concentration 2x1 o cells/mi. After 24h, the medium was replaced with DMEM medium without FBS and purified PCSK9-D374Y (1ig/ml PCSK9I3.4 nM) plus pooling 5 compounds. The original compound aliquot was dissolved in 100% DMSO at the concentration of 1mM. In each well, 5 compounds (Ipi of each) will be added into 100 tl of medium, resulting each compound concentration at 200nM. In the positive control wells, only PCSK9-D374Y protein was added. The negative control was vehicle (DMSO) plus medium only. Vehicle (0.2% DMSO) was used in all assays. After 16h incubation, the medium was replaced with fresh medium containing 101g/ml Bopipy FL LDL (Invitrogen, UK) without FBS. The cells were incubated for 4 h at 37°C. The cells were washed with PBS and LDL uptake was quantified on the Fusion fluorescence plate reader (Packard BioScience, Beaconsfield, UK) with the excitation filter 485nm and emission filter 530nm.). As shown on Fig.3, the wells that contain D374Y-PCSK9 protein has significant lower fluorescence-labeled LDL uptake as LDLR being degraded in comparison with the control wells (untreated)(Fig.3.1-2 column N3). Then positive pools (total 9) were broken down to each individual compound and 6 positive hits have been detected in a duplication assay (Fig.3, Table 1).

The high-throughput analysis was calculatcd as Z' factor. The Z factor was determined according to Zhang et at [!3tusing the fbI lowing equation: I 3(SDncg -f SDpc:)/(Meanncg Mean00.

Z' values >0.5 have acccptablc characteristics for high-throughput screening (HTS).

Example 3.

Secondary screening analysis of PCSK9-mediated LDLR degradation by western blot.

HepG2 cells were seeded in 6 well tissue plates at concentration of 2x105 cells/ml. After 24h, the medium was replaced with DMEM medium without FBS and added the purified D374Y protein (1 tg/ml). In the control experiments, without PCSK9 protein was added. In compound function assay, the compound was added in the same time as purified PCSK9.

After 1 6h, cells was washed twice in PBS and harvested for western blot analysis as previous described. 6 positive compounds ( Fig. 4A and Table I compound A-F) were added to the culture medium individually with D374Y-PCSK9 to HepG2 cells. Reduction of endogenous mature LDLR (M) in HepG2 cells was analyzed by western blot.

Compound B (Fig. 4, lane 4, Table 1, colchieine) has been detected to inhibit D374Y-mediated LDLR degradation, showing increased LDLR after treatment. Equal loadings were confirmed using anti tubulin antibody. B. Dose response of the compound B (colehicine) on inhibiting D374Y-mediated LDLR degradation in the western blot analysis.

The ratio of LDLRItubulin in untreated sample (Fig.4, lane 1) was assigned a value of 100%. The ratio of LDLRitubulin in D374Y-PCSK9 treated sample (13.4nM, 16h, Fig.4, ane 2) has a value of 25%. Four different concentrations of eokhicine were used to calculate the ICSO of inhibition of PCSK9-D374Y by colchicine (n3). The ICSO was 0.131 RM.

References [fl NI. Abifadel, M. Varret, j.P. Rabes, D. Allard, K. Ouguerram, M. Devillers, C. Cruaud, S. Benjannet, L. Wiclcham, D. Erlich, A. Derre, L. Villeger, M. Farnier, I. Beucler, F. Bruckert, J. Chambaz, B. Chanu, J.M. Lecerf, G. Luc, P. Moulin, J. Weissenbach, A. Prat, M. Krempf, C. Junien, N.G. Seidah, C. Boileau, Mutations in PCSK9 cause autosomal dominant hypercholesterolemia, Nat Genet 34 (2003] 154-156.

[21 J.D. Horton, J.C. Cohen, H.H. Hobbs, Molecular biology of PCSK9: its role in LDL metabolism, Trends Biochem Sci 32 (2007) 71-77.

[31 J.C. Cohen, E. Boerwinkle, TB. Mosley, Ir., flU. Hobbs, Sequence variations in PCSK9, low LDL, and protection against coronary heart disease, N EngI j Med 354 (2006) 1264-1272.

[41 A. King, Lipids: Antibodies against PCSK9-a new era of therapy, Nat Rev Cardiol [2012).

[51 W. Faloon, D, Webb, Race, 1. homas, Cholesterol lowering effect of colchicine, Ann Intern Med 66 (1967) 1058-1058.

[61 A. Rubulis, M. Rubert, W.W. Faloon, Cholesterol lowering, fecal bile acid, and sterol changes during neomycin and colchicine, Am J Clin Nutr 23 (1970) 1251-1259.

[71 D. Cunningham, D.E. Danley, K.F. Geoghegan, MC. Griffor, J.L. Hawkins, T.A.

Subashi, A.H. Varghese, M.J. Ammirati, J.S. CuIp, L.R. Hoth, MN. Mansour, K.M.

McGrath, A.P. Seddon, S. Shenolikar, 1<4. Stutzman-Engwall, L.C. Warren, D. Xia, X. Qiu, Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia, Nat Struct Mol Biol 14 (2007) 413-419.

[81 W. Xu, L. Liu, 1G. Chades, S. Moncada, Nitric oxide induces coupling of mitochondrial signalling with the endoplasmic reticulum stress response, Nat Cell Biol 6 (2004) 1129-1134.

[9] W. Xu, L. Liu, D. Hornby, c-IAP1 Binds and Processes PCSK9 Protein: Linking the c-IAP1 in a TNF-alpha Pathway to PCSK9-Mediated LDLR Degradation Pathway, Molecules 17 (2012) 12086-12101.

[10] C.T. Aiken, A.J. Tobin, ES. Schweitzer, A cell-based screen for drugs to treat Huntington's disease, Neurobiol Dis 16 (2004) 546-555.

[11] W. Wang, W. Duan, S. Igarashi, H. Morita, M. Nakamura, C.A. Ross, Compounds blocking mutant huntingtin toxicity identified using a Huntington's disease neuronal cell model, Neurobiol Dis 20 (2005) 500-508.

[121 D.H. Wyllie, K.C. Sogaard, K. Holland, X. Yaobo, M. Bregu, A.V. Hill, E. Kiss-Toth, Identification of 34 novel proinflammatory proteins in a genome-wide macrophage functional screen, PLoS One 7 (2012] e42388.

[13] j.H. Zhang, T.D. Chung, K.R. Oklenburg, A Simple Statistica' Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, J Biomol Screen 4 (1999] 67-73.

Claims (19)

1. Claims 1. A method for identifying a substance which is an inhibitor or modulators of Proprotein convertase subtilisinlkexin type 9(pcsk9).
2. 2. A method according to claim 1, comprising determining whether thc test substance is an inhibitor o f PCSK9 activity.
3. 3. A method according to claim 1, comprising determining whether the test substance is an inhibitor o f PCSK9 activity by using colchicine as positive control.
4. 4. A method according to calimi or 2 or 3, wherein determining whether a test substance is an inhibitors of PCSK9 comprises: (i) providing a cell-based screening method.(ii) Providing fluoreseence-LDL uptake method (iii) Providing a fluorescence microplate-rcadcr reading method (iv) DMSO dilution in 200 fold in the screening process (vProviding a cell overexpressing human PCSK9 and its gain of thnction' mutations of D374Y and S127R (vi) Purification methods of human PCSK9 and its gain of function' mutations of D374Y and S127R proteins.(vii)High-throughput screening methods, including seeding HepG2 cells in 2xl06/ml concentration in serum free DMEM medium.(viii) Incubation time with the test substance in certain time, together with PCSK9 or its gain of function' mutations of D374Y and S127R proteins.4. An inhibitor according to claim 1, method and pharmaceutical composition useful for the treatment of atherosclerosis comprising: (a) an effective amount of colchicine or physiologically functional derivative thereof and/or colchicine or physiologically functional derivative thereof, and (b) an effective, conjoint amount of another material statins, alone or in an acceptable pharmaceutical vehicle.
5. 5. An inhibitor according to claim 1, method and pharmaceutical composition useful for the treatment of coronary heart disease comprising: (a) an effective amount of colchicine or physiologically functional derivative thereof and/or colchicine or physiologically functional derivative thereof, and (b) an effective, conjoint amount of another material statins, alone or in an acceptable pharmaceutical vehicle.
6. 6. An inhibitor according to claim 1, method and pharmaceutical composition useful for the treatment of type 2 diabetes comprising: (a) an effective amount of colchicine or physiologically functional derivative thereof and/or colehicine or physiologically functional derivative thereof, and (b) an effective, conjoint amount of another material statins, alone or in an acceptable pharmaceutical vehicle.
7. 7. The composition of claim 4 or 5 or 6, presented for administration in doses comprising 0.6mg/day to 3.9 mg/day of colchicine or one half one third or one quarter of said amount.
8. 8. The composition of claim 4 or 5, or 6 presented for administration in 4days to 2ldays or more days with monitor the total cholesterol level, which could fell by 40-SOmg/lOOml in 6 days.
9. 9. A pharmaceutical composition comprising the colchicine derivative of claim 5, claim 6, claim 7, and a pharmaceutically acceptable carrier.
10. 10. A mcthod for treatment of atherosclerosis in a mammal, comprising administcring to the mammal a therapeutically effective amount of a composition comprising the colchicine derivative of claim 5, claim 6 or claim 7.
11. 11. The method of claim 9, wherein the mammal is a human.
12. 12. The method of claim 9, wherein the therapeutically effective amount of the composition comprising the colchicine or its derivative is present in a tablet or a pill.
13. 13. However, these are known adverse side effects to colchicine therapy, which need to be monitored by the health professionals to look for the symptoms such as abdominal pain with cramps, diarrhea, nausea, and vominting from the patient results of diagnostic tests indicative of the adverse side effect, and stop using it in the effected patients.Amendments to the claims have been filed as follows.Claims 1. A method for identifying a substance, which is an inhibitor or modulator of Proprotein convertase subtilisin/kexin type 9(pcsk9).2. A method according to claim 1, comprising determining whether the test substance is an inhibitor o fPCSK9 activity.3. A method according to claim 1, comprising determining whether the test substance is an inhibitor o fPCSK9 activity by using colchicine as a positive control.4. A method according to calimi or 2 or 3, wherein determining whether a test substance is an' inhibitors of PCSK9 comprises: (i) Providing a cell-based screening method.(ii) DMSO dilution in 200 fold in the screening process (iii) Providing a cell overexpressing human PCSK9 and its gain of function' mutations of D374Y and S127R (iv) Purification methods of human PCSIC9 and its gain of function' mutations of D374Y and S127R proteins.(v)l-ligh-throughput screening methods, including seeding l-lepG2 cells in 2x105/ml concentration in serum free DMEM medium.(vi) Incubation time with the test substance in certain time, together with PCSK9 or its gain of function' mutations of D374Y and S127R proteins.5. An inhibitor according to claim I, method and pharmaceutical composition useful for the treatment of atherosclerosis comprising: (a) an effective amount of colchicine or physiologically functional derivative thereof and/or colchicine or physiologically * : fithetional derivative thereof, and (b) an effective, conjoint amount of another material statins, alone or in an acceptable pharmaceutical vehicle. c *C: 6. An inhibitor according to claim I, method and pharmaceutical composition useful for the r treatment of coronary heart disease comprising: (a) an effective amount of colchicine or physiologically functional derivative thereof and/or colchicine or physiologically functional derivative thereof, and (b) an effective, conjoint amount of another material statins, alone or in an acceptable pharmaceutical vehicle.7. An inhibitor according to claim 1, method and pharmaceutical composition useful for the treatment of dyslipidaemia comprising: (a) an effective amount of colchicine or physiologically functional derivative thereof and/or colchicine or physiologically functional derivative thereof, and (b) an effective, conjoint amount of another material statins, alone or in an acceptable pharmaceutical vehicle.8. An inhibitor according to claim 1, method and pharmaceutical composition useful for the treatment of type 2 diabetes comprising: (a) an effective amount of coichicine or physiologically functional derivative thereof and/or colchicine or physiologically functional derivative thereof; and (b) an effective, conjoint amount of another material statins, alone or in an acceptable pharmaceutical vehicle.9. The composition of claim 4 or 5 or 6, presented for administration in doses comprising 0.6mg/day to 3.9 mg/day of colchicine or one half one third or one quarter of said amount.10. The composition of claim 4 or 5, or 6 presented for administration in 4days to 2idays or more days with monitor the total cholester& level, which could fell by 40-80mg/I OOml in 6 days.II. A pharmaceutical composition comprising the coichicine derivative of claim 5, claim 6, claim 7, and a pharmaceutically acceptable carrier.12. A method for treatment of atherosclerosis in a mammal, comprising administering to the st's mammal a therapeutically effective amount of a composition comprising the 555øt* . . . * * colchicine derivative of claimS, claim 6 or claim 7.IS.....* 13. The method of claim 10, wherein the mammal is a human.S'S.'. * S*
14. 14. The method of* claim 10, wherein the therapeutically effective amount of the * composition comprising the colchicine or its derivative is present in a tablet or a belleS pill.
15. 15. A pharmaceutical composition comprising the inhibitors of microtubule-associated proteins derivative of claim 5, claim 6, claim 7, and a pharmaceutically acceptable carrier.
16. 16. The method for treatment of atherosclerosis in a mammal, comprising administering to the mammal a therapeutically effective amount of a composition comprising the inhibitors of Microtubule-associated proteins of claim 5, claim 6 or claim 7.
17. 17. The method of claim 14, wherein the mammal is a human.
18. 18. The method of claim 9, wherein the therapeutically effective amount of the composition comprising the he inhibitors of microtubule-associated proteins are present in a tablet orapill.
19. 19. However, these are known adverse side effects to colchicine therapy, which need to be monitored by the health professionals to look for the symptoms such as abdominal pain with cramps, diarrheal, nausea, and vomiting from the patient results of diagnostic tests in&cative of the adverse side effect, and stop using it in the effected patients. * .*..**. * *C * C *. * * S * * S.C* ** *.S * *