FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
PROVISIONAL / COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION
HYPOLIPAEMIC PHARMACEUTICAL COMPOSITIONS
2.APPLICANT(S)
(a) NAME : CADILA PHARMACEUTICALS LTD.
(b) NATIONALITY : An INDIAN Company
(c) ADDRESS : "Cadila Corporate Campus", Sarkhej-Dholka Road, Bhat,
Ahmedabad - 38221 O.Gujarat, India,


3. PREAMBLE TO THE DESCRITION
PROVISIONAL
The following specification describes invention
COMPLETE

The following specification particularly doscribos the invention and the manner in which it is to bo performed
4. DESCRIPTION (Description shall start from next page)

FIELD OF INVENTION:
The present invention related to hypolipaemic pharmaceutical compositions comprising lipid lowering agents and BAR antagonist such as 3p-hydroxy pregna-5,16-dien-20-one (Herein known as 16 DP) Gugulsterones. 16 DP is also given a compound number
80/574.

BACKGROUND OF INVENTION:
Hyperlipidemia or an elevation in serum lipids is associated with an increase incidence of cardiovascular disease and atherosclerosis. Specific forms of hyperlipidemia include, for example, hypercholesteremia, familial dysbetalipoproteinemia, diabetic dyslipidemia, nephrotic dyslipidemia and familial combined hyperlipidemia. Hypercholesteremia is characterized by an elevation in serum low density lipoprotein-cholesterol and serum total cholesterol. Low density lipoprotein (LDL-cholesterol) transports cholesterol in the blood. Familial dysbetalipoproteinemia, also known as Type III hyperlipidemia, is characterized by an accumulation of very low density lipoprotein-cholesterol (VLDL-cholesterol) particles called p-VLDLs in the serum. Also associated with this condition, there is a replacement of normal apolipoprotein E3 with abnormal isoform apolipoprotein E2. Diabetic dyslipidemia is characterized by multiple lipoprotein abnormalities, such as an overproduction of VLDL-cholesterol, abnormal VLDL triglyceride lipolysis, reduced LDL-cholesterol receptor activity and, on occasion, Type III hyperlipidemia. Nephrotic dyslipidemia is difficult to treat and frequently includes hypercholesteremia and hypertriglyceridemia. Familial combined hyperlipidemia is characterized by multiple phenotypes of hyperlipidemia, i.e., Type Ha, lib, IV, V or hyperapobetal ipoproteinemia.
It is well known that the likelihood of cardiovascular disease can be decreased, if the serum lipids, and in particular LDL-cholesterol, can be reduced. It is also well known that the

progression of atherosclerosis can be retarded or the regression of atherosclerosis can be induced if serum lipids can be lowered. In such cases, individuals diagnosed with hyperlipidemia or hypercholesteremia should consider lipid-lowering therapy to retard the progression or induce the regression of atherosclerosis for purposes of reducing their risk of cardiovascular disease, and in particular coronary artery disease.
Cholesterol is transported in the blood by lipoprotein complexes, such as VLDL-cholesterol, LDL-cholesterol, and high density lipoprotein-cholesterol (HDL-cholesterol). LDL carries cholesterol in the blood to the subendothelial spaces of blood vessel walls. It is believed that peroxidation of LDL-cholesterol within the subendothelial space of blood vessel walls leads to atherosclerosis plaque formation. HDL-cholesterol, on the other hand, is believed to counter plaque formation and delay or prevent the onset of cardiovascular disease and atherosclerotic symptoms. Several subtypes of HDL-cholesterol, such as HDLj -cholesterol, HDL2 -cholesterol and HDL3 -cholesterol, have been identified to date.
The ideal Hypolipaemic agent should reduce LDL cholesterol and improve HDL cholesterol without significant side effects.
Several types of hypolipidemic agents have been developed to treat hyperlipidemia or hypercholesteremia or normolipidemics diagnosed with cardiovascular disease. Their effects on serum lipids is summarized in table below.
Effect of various lip id-modifying drugs:

Abbreviations: HDL-C, High-density lipoprotein cholesterol; LDL-C, Low-density lipoprotein cholesterol; TG, Triglycerides.
In general, these agents act (1) by reducing the production of the serum lipoproteins or lipids, or (2) by enhancing their removal from the serum or plasma. Drugs that lower the concentration of serum lipoproteins or lipids include inhibitors of HMG-CoA reductase, the rate controlling enzyme in the biosynthetic pathway of cholesterol. Examples of HMG-CoA

reductase inhibitors include mevastatin, U.S. Pat. No. 3,983,140, lovastatin also referred to as mevinolin, U.S. Pat. No. 4,231,938, pravastatin, U.S. Pat. Nos. 4,346,227 and 4,410,629, lactones of pravastatin, U.S. Pat. No. 4,448,979, velostatin, also referred to as synvinolin, simvastatin, U.S. Pat. Nos. 4,448,784 and 4,450,171, rivastatin, fluvastatin, atorvastatin and cerivastatin. For other examples of HMG-CoA reductase inhibitors, see U.S. Pat. Nos. 5,217,992; 5,196,440; 5,189,180; 5,166,364; 5,157,134; 5,110,940; 5,106,992; 5,099,035; 5,081,136; 5,049,696; 5,049,577; 5,025,017; 5,011,947; 5,010,105; 4,970,221; 4,940,800; 4,866,058; 4,686,237; 4,647,576; European Application Nos. 0142146A2 and 0221025A1; and PCT Application Nos. WO 86/03488 and WO 86/07054.
Statins are widely used for their hypofipaemic effects. TTie following tables provide information on hypolipaemic effects of various statins.
Mean Percent Change from Baseline with various dose of Simvastatin

Daily Dose TOTAL-C LDL-C HDL-C TG
5mg -19 -26 10 -12
10 mg -23 -30 12 -15
20 mg -28 -38 8 -19
40 mg -25 -29 13 -28
80 mg -31 -36 16 -33
Mean Percent Change from Baseline with various dose of Atorvastatin

Daily Dose TC LDL-C ApoB TG HDL-C
10 mg -29 -39 -32 -19 6
20 mg -33 -43 -35 -26 9
40 mg -37 -50 -42 -29 6
80 mg -45 -60 -50 -37 5

Mean Percent Change from Baseline with various dose of Rosuvastatin

Daily Dose Total-Cc LDL-CC ApoBc HDL-C TGa
10 mg -32 -46 -37 +7 -20
20 mg -37 -52 -43 +8 -26
40 mg -41 -57 -47 +8 -28
Other drugs which lower serum cholesterol include, for example, nicotinic acid, bile acid sequestrants, e.g., cholestyramine, colestipol DEAESephadex (Secholex.RTM. and Polidexide.RTM.), probucol and related compounds as disclosed in U.S. Pat. No. 3,674,836, lipostabil (Rhone-Poulanc), Eisai E505O (an N-substituted ethanolamine derivative), imantil (HOE-402) tetrahydrolipstatin (THL), isitigmastanylphosphorylcholine (SPC, Roche), aminocyclodextrin (Tanabe Seiyoku), Ajinomoto AJ-814 (azulene derivative), melinamide (Sumitomo), Sandoz 58-035, American Cyanimid CL-277,082 and CL-283,546 (disubstituted urea derivatives), ronitol (which has an alcohol which corresponds to nicotinic acid), neomycin, p-aminosalicylic acid, aspirin, quarternary amine poly(diaUyldimethylammonium chloride) and ionenes such as disclosed in U.S. Pat. No. 4,027,009, poly(diallylmethylamine) derivatives such as disclosed in U.S. Pat. No. 4,759,923, omega-3-fatty acids found in various fish oil supplements, fibric acid derivatives, e.g., gemfibrozil, clofibrate, bezafibrate, fenofibrate, ciprofibrate and clinofibrate, and other known serum cholesterol lowering agents such as those described in U.S. Pat. No. 5,200,424; European Patent Application No. 0065835A1, European Patent No. 164-698-A, G.B. Patent No. 1,586,152 and G.B. Patent Application No. 2162-179-A.
Nicotinic acid, also known as niacin, has been used for many years in the treatment of hyperlipidemia or hypercholesteremia. This compound has long been known to exhibit the beneficial effects of reducing total cholesterol, VLDL-cholesterol and VLDL-cholesterol remnants, LDL-cholesteroJ, triglycerides and apolipoprotein a, known as "Lp(a)," in the human body, while increasing desirable HDL-cholesterol.
16 DP (80/574) is a novel hypolipaemic compound. It is a subject matter of patent US6,579,862B1, EP1020191 and US6,875,758B2 Like gugulsterone it is a BAR antagonist(Molecular Endocrinology 2002; 16: 1590-97) . In human clinical trial

administration of 16 DP (80/574) 300 mg twice a day results in decrease in total cholesterol by 6.32%, LDL by 4.36%, Triglyceride by 14.87%. It increases HDL by about 10.13%.
Combination therapy of an HMG CoA reductase inhibitor with fibrates, bile acid resins and niacin have been studied with limited success.
The combination of fibrate and statin provide variable results and run the increased risk of side effects particularly myopathy.
Mean Percent Change from Baseline with atorvastatin and fenofibrate alone and in
combination

Atorvastatin 20mg Fenofibrate 200 mg Atorvastatin 20 mg + Fenofibrate 200mg
HDL +9% +16% +22%
TC -31% -16% -17%
Triglyceride -30% -41% -50%
LDL -40% -15% -46%
The combination of statin and niacin offers an alternative to the fibrate-statin therapy for patients with mixed hyperlipidemia as well as for patients with only elevated LDL-C. However, an increased chance of myopathy is again associated with this combination, and the vasodilatory effects of niacin are difficult to tolerate for many patients .
Mean Percent Change from Baseline with Simvastatin and Simvastatin + Niacin
(SIMCOR)

Daily Dose LDL-C Total-C HDL-C TGa ApoB
Simvastatin 20 mg -6.7% -4.5% 7.8% -15.3% -5.6%
SIMCOR 1000/20 j-11.9% -8.8% 20.7% -26.5% -13.2%
SIMCOR 2000/20 1-14.3% -11.1% 29.0% -38.0% -18.5%

Ezetimibe is a novel selective inhibitor of intestinal cholesterol absorption from dietary and biliary sources without affecting the absorption of fat soluble vitamins and triglycerides. Unlike other cholesterol-lowering compounds, ezetimibe does not inhibit cholesterol synthesis in the liver nor does it increase bile acid excretion. Ezetimibe acts at the brush border of the small intestine to inhibit the absorption of cholesterol leading to a reduction in the amount of cholesterol delivered to the liver. This in turn causes a reduction of hepatic cholesterol stores and an increase in cholesterol clearance from the blood which makes ezetimibe use an appropriate adjunct to HMG-Co A reductase inhibitors. Its combination with Simvastatin is associated with improved Hypolipaemic activity.
Mean Percent Change from Baseline with VYTORIN (Ezetimibe + Simvastatin)

Treatment (Daily Dose) N Total-C LDL-C ApoB HDL-C TGa Non-HDL-C
Ezetimibe lOmg 149 -13 -19 -15 +5 -11 -18
VYTORIN by dose 10/10 152 -31 -45 -35 +8 -23 -41
10/20 156 -36 -52 -41 +10 -24 -47
10/40 147 -39 -55 -44 +6 -23 -51
10/80 154 -43 -60 -49 +6 -31 -56
a For triglycerides, median % change from baseline
Torcetrapib is a CETP inhibitor that has been shown in monotherapy to result in a dose-dependent increase in HDL-C (15-70%), as well as a modest decrease in LDL-C (up to -20%) and inconsistent effect on triglyceride (TG) levels. When combined with atorvastatin , it improved the efficacy of atorvastatin, particularly for HDL elevation. It has been discontinued from development due to side effect profile in phase III studies.

References:
1. Diabetes Care 2002;25:1198-1202
2. Wolfe M. L., Vartanian S. F., Ross J. L., Bansavich L-L,Mohler E.R. Ill, Meagher E, et al. Safety and effectiveness of Niacin when added sequentially to a statin for treatment of dyslipidemia. Am. J. Cardiol. 87: 476-479 (2001).
3. Indian J. Chem., Sect. B 29B, 12 (1990), p. 1134-7.
4. Witzum, circulation, 80, 5 (1989), p. 1101-1114.
5. Illingworth, Drug, 36(SuppL3) (1988), p. 63-71.
6. The American Journal of Cardiology 90(10B) 44K-49K, 2002.
7. Hardman JG, Limberg JE, eds. Goodman and Gilman's The Pharmacologic Basis of Therapeutics. 9th ed. New York, NY: McGraw-Hill; 1996; pp 875-897.
8. Illingworth D.R. Management of hypercholesterolemia. Med. Clin. North Am. 84: 23-42(200).
9. Wolfe M. L., Vartanian S.F., Ross J. L., Bansavich L-L, Mohler E.R. Ill, Meagher E, et at. Safety and effectiveness of Niacin when added sequentially to a statin for treatment of dyslipidemia. Am. J. Cardiol. 87: 476-479 (2001).

10. Patti, G., Pasceri, V., et al. Atorvastatin pretreatment improves outcomes in patients with acute coronary syndromes undergoing early precutaneous coronary intervention results of the ARMYDA-ACS randomized trail J. Am. Coll. Cardiol. 49, 1272-8 (2007).
11. Freireich, E.J. et al. Quantitative comparison of toxicity of anti-cancer agenst inmouse, rat, dog, monkey and man. Cancer Chemother. Repts. 50, 219-244 (1966).
DETAIL DESCRIPTION OF FIGURES:
Fig. 1 : Plasma concentration profile of 16 DP (80/574) and metabolite Ml after oral
administration of 16 DP (80/574) (100 mg/kg) alone and in combination with
atorvastatin (10 mg/kg) Fig. 2 : Plasma concentration profile of 16 DP (80/574) (12.5 mg/kg) and metabolite Ml
after oral administration of 16 DP (80/574) alone and in combination with
atorvastatin (1.25 mg/kg) at reduced dose
SUMMARY OF INVENTION:
The main object of invention is to provide hypolipaemic pharmaceutical compositions comprising lipid lowering agents and BAR antagonist to achieve synergistic changes in lipid profile of mammals.

Yet another object is to improve efficacy of stains so that its side effect profile can be substantially reduced.
Yet another object of prevent invention is to provide a method for significant improvement in high density lipoprotein HDL.
Yet another object of invention is to alter HDL/TC total cholesterol ratio to improve its therapeutic benefit.
Yet another object of invention is to provide a pharmaceutical composition for effective management of dyslipidemia.
Yet another object of present invention is to provide better management of atherosclerosis disease / atheromatous plaque.
Yet another object if invention is to improve efficacy of each drug to reduce LDL cholesterol.
DETAILED DESCRIPTION OF THE INVENTION:
Statins, fibrates, Niacin, Ezetimibe, (16 DP (80/574)) etc are useful in management of dyslipidemia.
Statins are HMG CoA inhibitors while 16 DP (80/574) is BAR antagonist. Their effect on dyslipidemiaare as following,

16 DP
(80/574)
Atorvastatin
Total cholesterol
6.32%
23.03%
LDL
4.36%
31.49%
HDL
10.13%
5.29%
14.87%
TGA
23.46%
Surprisingly it is observed that lipid lowering drugs like atorvastatin and 16 DP (80/574) when co-administered; the lipid lowering effect of atorvastatin is significantly increased. The effect seen with 1/8 dose of atorvastatin gives better results than full dose of atorvastatin when combined with 16 DP (80/574). These effects are seen over wide range of ratio of atorvastatin and 16 DP (80/574). The effects also seen over wide range of dose administered. It is also surprisingly seen that when both drugs are co-administered bio-availability of both drugs are increased. Generally when two drugs are co-administered bioavailability of both drugs is not altered or bio-availability of one of the drug is altered. Following examples describes the invention without limiting scope-of the invention.
A. Anti-dyslipidemic effect in combination of statin and compound 16 DP (80/574) in high fat diet fed hamster model:

The effect of 16 DP (80/574) alone and upon co-administration with statin was evaluated in high fat diet fed hamster model. The anti-hyperlipidemic response of compound 16 DP (80/574) alone or in combination with atorvastatin was derived from their effect on important biochemical parameters like triglycerides (TG), total cholesterol (TC), high density lipoproteins (HDL).
Experimental methodology for evaluating lipid lowering effects:
Anti-hyperlipidemic activity of compound 16 DP (80/574) alone and upon co-administration with statin was performed in hyperlipidemic Male Golden Syrian hamsters weighing 110-120g.
Hyperlipidemia was produced by feeding high fat diet (HFD). These animals had a free access to the HFD and water ad libitum during the entire 11 day period of the experiment. The drugs for evaluation were given orally as an aqueous suspension from day 5 to day 11 (7 days) to the animals (Table 1). Control animals received only water and HFD during the period of experiment.
Body weight and diet intake of animals in each group was recorded daily in order to have a comparative account of food and water intake vis-a-vis body weight gain of the animals in all the three groups at the end of the experiment.
At the end of experiment i.e. on the 11 day, the blood of the animals was withdrawn in EDTA coated tubes. The blood samples were cooled to 4°C for 15 min. The cold plasma was separated by standard procedures. All the plasma samples were analyzed on the same day using commercially available diagnostic kits for lipid components e.g. triglycerides (TG), total cholesterol (TC), and high density lipoprotein (HDL) using Synchron CX-5 Clinical System, Beckmann Coulter Instrument.
Example 1: The effect of each drug and their low dose combination on serum lipids.
The animals were divided into three groups of eight animals in each:
Group-1: Control hyperlipidemic animals
Group-2:
a. Hyperlipidemic animals administered 16 DP (80/574) at a dose of 100 mg/kg
b. Hyperlipidemic animals administered atorvastatin at a dose of 10.0 mg/kg
Group-3:

a. Hyperlipidemic animals administered a combination of 12.5 mg/kg of 16 DP (80/574) and 1.25 mg/kg atorvastatin
Compound 16 DP (80/574) alone and in combination with atorvastatin was administered in the following manner as given in table-1.
Table-1: Dose and feeding schedule of water, HFD, 16 DP (80/574), atorvastatin discrete and
in combination

Male Golden Syrian Hamsters Group Compound/ Drug Dose Duration of administration of HFD Duration and day of administration
of drug Blood
sampling/ analysis
1 Control Plain water Day-1 toDay-
11 Day-5 to Day-11 Day-11
2a 16 DP
(80/574) 100 mg/kg

2c Atorvastatin 10 mg/kg

3b 16 DP
(80/574) Atorvastatin 12.5 mg/kg 1.25 mg/kg

Table-2 very clearly indicates that a combination of 1/8111 of the effective dose of 16 DP (80/574) i.e. 12.5 mg/kg and l/8th of the effective dose of atorvastatin i.e. 1.25 mg/kg was comparable to or more effective than 100 mg/kg of 16 DP (80/574) alone or 10 mg/kg of atorvastatin alone for its effect on total cholesterol and High density lipoprotein.
The most beneficial response of the combination was evident from a significant rise of 54% in HDL levels and a rise of 76% in HDL-Total Cholesterol ratio. Both are more than scopes of the effect seen with 100 mg / kg of 16 DP and 10 mg/kg of atorvastatin .
Triglyceride levels were also significantly reduced by 40% at such a low dose combination.
Table-2: Efficacy of 16 DP (80/574), atorvastatin discrete and in combination

HDL/TC
Drug
16 DP (80/574)
Atorvastatin
16 DP (80/574) Atorvastain
The effect on total cholestero

TG
TC
HDL
Dose
-60
-8
NC
+5
100 mg/kg
-63
-14
+15
+35
10 mg/kg
-17
-40
+54
+76
12.5 mg/kg 1.25 mg/kg
is more than that seen with each one takes separately.

Example 2: In an identical experiment the effect of low dose 16 DP (80/574) ,atorvastatin and their combination in ratio of 1:10 was evaluated.

The amount of each ingredient was kept identical i.e. 2.5 mg/kg for atorvastatin and 25 mg/kg for 16 DP (80/574).
The animals were divided into three groups of eight animals in each:
Group-1: Control hyperiipidemic animals
Group-2: a. Hyperiipidemic animals administered 16 DP (80/574) at a dose of 25 mg/kg
b. Hyperiipidemic animals administered atorvastatin at a dose of 2.5 mg/kg
Group-3: Hyperiipidemic animals administered a combination of25.0 mg/kg of 16 DP (80/574) and 2.5 mg/kg atorvastatin
Table 3 : Efficacy of lower doses of 16 DP (80/574), atorvastatin discrete and in combination
at a ratio 1:10

TC% HDL % J TG%
16 DP (80/574) 25 mg -16 NC -24
Atorvastatin 2.5 mg -28 NC -38
Atorvastatin 2.5 mg 16 DP (80/574) 25 mg -30 +82 -64
Tabel 3 indicates that both the drugs when co-administered provides significant higher effect them each drug given individually. The effect is most marked on HDL and Triglycerides.
Example 3 : In an identical experiment the effect of 16 DP (80/574) in combination with atorvastatin in ratio of 1:5, 1:10 and 1:20 was evaluated to study the effect of combination over a wider range of ratio.
The animals were divided into three groups of eight animals in each:
Group-1: Control hyperiipidemic animals
Group-2: Hyperiipidemic animals administered a combination 16 DP (80/574) and atorvastatin in ratios of 1:10,1:5 and 1:20.
The findings in Table 4 suggest that synergistic activity is seen at all three ratios. The effect on triglyceride was most marked at ratio of 1:20 while on total cholesterol was most marked at ratio of 1:5.
Table 4: Efficacy of combination at various ratios

TC% HDL % TG%
80/ and 574Atorvastatin 1.25 & 12.5 1:10 -13 +128 -23
Atorvastatin 1:5 -18 ,_ +127 -56

2.5 & 12.5
Atorvastatin and 16 DP
(80/574) 2.5 & 50 1:20 -14 +140 -66
B : Effect of combination of 16 DP (80/574) and atorvastatin on pharmacokinetic profile
of each other
I: Pharmacokinetic profile of compound 80- 574 on concomitant administration of
atorvastatin
Example 4 : Male SD rats (200 ± 20 g) were fasted overnight before dosing but allowed free access to water. Feed was provided ~2 hr post oral dose. 1st and 2nd Group received 16 DP (80/574) alone where as the 3rd and 4th group were administered 16 DP (80/574) and atorvastatin both (Table-5). The blood samples were collected at 0.25 0.5, 0.75, 1, 1.5, 2, 3, 4,5,6,8, 10, 12, 18 and 24 hr post dose. Not more than three blood samples were collected from each rat, two of which were by intra-cardiac (~0.5ml per time point) and one from vena cava in terminal phase. 16 DP (80/574) and metabolite M-l were analyzed by a validated LC-MS/MS method. All experimental procedures were carried out in accordance with the guidelines laid down by local ethics committee for experimental animal.
Comparison of pharmacokinetic parameters (Table-6 and 7) of 16 DP (80/574) at 100 mg/kg and 12.5 mg/kg alone and the combination of 16 DP (80/574) (12.5 mg/kg) and atorvastatin (1.25 mg/kg) revealed that atorvastatin significantly alter the pharmacokinetic of 16 DP (80/574) at a combination dose of 100 mg/kg 16 DP (80/574) and 10 mg/kg atorvastatin. Time to reach maximum plasma concentration (Tmax) was reduced by two fold indicating faster absorption. Co-administration of atorvastatin significantly increases AUCo-24 of 16 DP (80/574). Maximum plasma concentration (Cmax) did not increase significantly, which may be due to rapid and extensive peripheral distribution indicated by 2.5 fold increment in volume of distribution. Plasma elimination half life (tm) increased by 2.8 fold indicating prolonged and enhanced availability of drug in systemic circulation (Figure-1). AUCo-24 of metabolite M-l decreased significantly upon co-administration of atorvastatin with 16 DP (80/574) indicating decreased metabolism of 16 DP (80/574). Plasma-Concentration Time profile of 1/8th dose combination of 16 DP (80/574) (12.5 mg/kg) and atorvastatin (1.25 mg/kg) also reveals enhanced and prolonged systemic exposure of 16 DP (80/574) than 16 DP (80/574) alone at 12.5 mg/kg (Figure-2).
The above findings correlate very well with the therapeutically activity at l/8m dose combination of 16 DP (80/574) (12.5 mg/kg) and atorvastatin (1.25 mg/kg) (Figure-2). Thus

it can be concluded that atorvastatin significantly alter the pharmacokinetic of 16 DP (80/574) by decreasing its metabolism in male SD rats.
Table 5: Dosing schedule in male SD rats

Male SD Rats Group Drug Dose Dosing Schedule
I. 16 DP (80/574) 100 mg/kg Single, oral dose
2. 16 DP (80/574) 12.5 mg/kg

3. 16 DP (80/574) Atorvastatin 100 mg/kg
10 mg/kg

4. 16 DP (80/574) Atorvastatin 12.5 mg/kg 1.25 mg/kg

Table-6: Pharmacokinetic parameters of 16 DP (80/574) and metabolite Ml after oral administration of 16 DP (80/574) (100 mg/kg) alone and in combination with atorvastatin (10
mg/kg)

Parameters

16 DP (80/574) (100
mg/kg)

16 DP (80/574) (100
mg/kg) + atorvastatin (10
mg/kg)

Cmax-1 (ng/ml) 17.81 ±11.06** 14.47 ±7.97**
Cmax-2 (ng/ml) 12.64 ±7.78** 14.74 ±10.58**
Tmax-1 (h) 1.5 0.75
Tmax-2(h) 3 8
AUCo-24 (ng.h/ml) 111.8 ± 11.8* 196.86 ±20.17*
Vd(LAg) 70.84 181.8
CI (L.h/kg) 14 12.9
T,/2(h) 3.4 9.7
MRT(h) 9.6 10.8
Metabolite-Ml
1 (ng/ml) 26.13 ±13.54** 17.06 ±10.79**
Tmax-1 (h) 1.5 1.5
Cmax-2 (ng/ml) 8.94 ±6.67** 6.43 ±1.98**
Tmax-2 (h) 4 6
AUCo-24 (ng.h/ml) 66.03 ± 7.48* 46.9 ± 5.80*
MRT(h) 6.64 7.24
AUCp/AUMm 1.69 4.19
Mean ± SD, *Values are significantly different (p<0.5), **Values are not significantly different (p>0.5)

Table-7: Pharmacokinetic parameters of 16 DP (80/574) and metabolite Ml after oral administration of 16 DP (80/574) (12.5 mg/kg) alone and in combination with atorvastatin
(1.25 mg/kg)

mg/kg mg/kg)
2.10±0.83 0.5
16.75 ±0.814
Parameters Cmax-1 (ng/ml)
Tmax-1 (h)
AUCo.24 (ng.h/ml) Vd (L/kg) CI (L.h/kg) T1/2(h) MRT(h)
Metabolite-Ml Cmax-1 (ng/ml) Cmax-2 (ng/ml) Tmax-l(h)
Tmax-2 (h)
AUCo-24 (ng.h/ml)
MRT (h) *Mean±SD
B. II: Changes in pharmacokinetic of atorvastatin on concomitant administration of 16
DP (80/574)
Fasting wistar rats (8 weeks old , five in each group) were administered 16 DP (80/574) and
atorvastatin at a pre-determined dose in various experiments to study changes in
pharmacokinetic of atorvastatin with or without concomitant administration of 16 DP
(80/574).
The blood samples were taken at 0.5, 2.0 and 4 hrs following drug administration.
Example 5:
To evaluate the effect of 16 DP (80/574) on atorvastatin , the combination of atorvastatin and
16 DP (80/574) in the ratio of 1:10 was administered. The composition was administered to
provide 10 mg / kg of atorvastatin to each animal. The following table clearly demonstrates

that concomitant administration of 16 DP (80/574) increases significantly the blood levels of atorvastatin as well as its active metabolite o-atorvastain at all time points.

0.5 Hr | 2Hr r 4Hr
Atorvastatin levels
Atorvastatin + 16 DP
(80/574) 210.36 ±106.95 30.53 ±11.32 16.32 ±5.42
Atorvastatin
Atorvastatin + 16 DP
(80/574) Atorvastatin 154.90 ±28.50
O-Atorvas
515.44+233.91
396.29 ±138.62 14.67 ±10.03 atin levels
308.78 ±114.55
98.67 ±20.51 9,61 ±8.06
190.86 ±70.63 45.34 ±19.42
Example 6:
(i) Effect of various dose of combination:
To evaluate blood levels of atorvastatin at various dose of the pharmaceutical composition containing the combination of 16 DP (80/574) and atorvastatin in the ratio of 1:10 was administered at lmg/Kg, 2 mg/Kg, 5 mg/Kg of atorvastatin. The following table clearly demonstrates that the increase in the blood levels of atorvastatin with increasing dose of combination is non linear, i.e. 2 hrs the blood level of atorvastatin with 2 mg of atorvastatin are eight times lower than 5 mg of atorvastatin containing combination.

Amount of
atorvastatin in
combination Mean ± SD

0.5 Hr 2Hr 4Hr
1 mg/Kg 2.80±1.41 1.83±0.53 1.06 ±0.73
2 mg/Kg 5.01±1.62 4.08±1.96 1 1.91±1.12
5mg/kg 28.52±20,46 20.43±8.54 5.71±1.57
(ii) Effect of ratios of Atorvastatin to 16 DP (80/574) on blood levels of atorvastatin:
To evaluate the effect of various ratios Atorvastatin to 16 DP (80/574) on blood levels of atorvastatin, the combination of atorvastatin and 16 DP (80/574) was administered at dose of lOmg/Kg of atorvastatin. The following table clearly demonstrates that the increase in the blood levels of atorvastatin with increasing in ratio from 1:1 to 1:5 to 1:30 of combination. Again the increase was non-linear.

Ratio of Atorvastatin to 16 DP (80/574) Mean ± SD

0.5 Hr 2Hr 4Hr
1:5 1:1 59.90
±30.56
43.32
±27.25 34.93
±40.77
10.41
±3.98 7.74 ±3.39 4.52 ±0.85
1:30 126.12 ±108.97 25.49 ±11.16 8.06 ±3.53
C : Stable pahamaceutical composition of 16 DP (80/574) and lipid lowering agent. Example 7:
The following examples illustrates pharmaceutical composition containing 16 DP (80/574) and atorvastatin in different properties. The amount of atorvastatin is kept at 10 mg.

No. Product Batch No. Ratio
1 16 DP + Atorvastatin Calcium (50+10 mg) Capsules 05/030/027,05/030/031, 05/030/035 5:1
2 16 DP + Atorvastatin Calcium (100+10 mg) Capsules 05/030/028, 05/030/032, 05/030/036 10:1
3 16 DP + Atorvastatin Calcium (200+10 mg) Capsules 05/030/029, 05/030/033, 05/030/037 20:1
4 16 DP + Atorvastatin Calcium (300+10 mg) Capsules 05/030/030, 05/030/034, 05/030/038 30:1
Qualitative Formula:
The following example illustrates qualitative formula of blend for filling into capsule..

S.No. Ingredients
1 16-DP (16 DP (80/574))
2 Atorvastatin Calcium
3 MicroCrystalline Cellulose (Ran Q 102)
4 Cross Carmellose Sodium
5 Colloidal Silicone Dioxide
6 Magnesium Stearate
7 Purified Talc

Microcrystalline cellulose is used as a Diluent, cross carmellose sodium is used as disintegrant, colloidal silicone dioxide is used as glidant and purified talk as glidant.
Stability: The above pharmaceutical composition are found to be stable even at accelerated conditions for more than 6 months.
The following are some of the pharmaceutical compositions. Title: 16 DP + Atorvastatin Calcium (100+10 mg) Mfg. Date: 28/05/08 Formula:

S.No. Ingredients Qty per Unit
(mg)
1 16-DP 100.0
2 Atorvastatin Calcium 10.69
3 MicroCrystalline Cellulose (Ran Q 102) 48.31
4 Cross Carmellose Sodium 6.00
5 Colloidal Silicone Dioxide 1.00
6 Mg. stearate 1.00
7 Purified Talc 1.00
Title: 16 DP + Atorvastatin Calcium (200+10 mg)
Mfg. Date: 28/05/08
Formula:

S.No. Ingredients Qty per Unit (mg)
1 16-DP 200.0
2 Atorvastatin Calcium 10.69
3 MicroCrystalline Cellulose (Ran Q 102) 107.31
4 Cross Carmellose Sodium 10.80
5 Colloidal Silicone Dioxide 2.40
6 Mg. stearate 2.40
7 Purified Talc 2.40

Title: 16 DP + Atorvastatin Calcium (300+10 mg)
Mfg. Date: 28/05/08
Formula:

S.No. Ingredients Qty per Unit (mg)
I 16-DP 300.0
2 Atorvastatin Calcium 10.69
3 MicroCrystalline Cellulose (Ran Q 102) 164.31
4 Cross Carmellose Sodium 16.00
5 Colloidal Silicone Dioxide 3.00
6 Mg. stearate 3.00
7 Purified Talc 3.00
Title: 16 DP + Atorvastatin Calcium (50+10 mg)
Mfg. Date: 28/05/08
Formula:

S.No. Ingredients Qty per Unit
(mg)
1 16-DP 50.00
2 Atorvastatin Calcium 10.69
3 MicroCrystalline Cellulose (Ran Q 102) 18.81
4 Cross Carmellose Sodium 2.70
5 Colloidal Silicone Dioxide 0.60
6 Mg. stearate 0.60
7 Purified Talc 0.60