WO2019192112A1

WO2019192112A1 - Pharmaceutical composition for promotion of bioavailability of oral statins and applications thereof

Info

Publication number: WO2019192112A1
Application number: PCT/CN2018/098524
Authority: WO
Inventors: Oliver Yoa-Pu Hu; Yu-Ying Lai
Original assignee: Johnson Chemical Pharmaceutical Works Co., Ltd.
Priority date: 2018-04-02
Filing date: 2018-08-03
Publication date: 2019-10-10
Also published as: CN110339365A

Abstract

A pharmaceutical combination for promotion of bioavailability of oral statins comprises statins, pH modifiers, metabolic enzyme inhibitors.

Description

PHARMACEUTICAL COMPOSITION FOR PROMOTION OF BIOAVAILABILITY OF ORAL STATINS AND APPLICATIONS THEREOF

Technical Field

The present invention relates to a pharmaceutical composition for promotion of bioavailability of oral statins and applications thereof in order to reduce fluctuation of drug absorption increased between individuals as well as serious side effects of drugs and enhance drug safety for fewer lethal side effects.

Background Art

Hyperlipidemia is a problem to those who are slow metabolism, overeating, or family cardiovascular disease. In Europe and America, the incidence of obesity, coronary atherosclerosis and heart disease is higher than that of other countries because of high calorie intake. Over the past decade, cardiovascular diseases have also been one of the ten leading causes of death in the United State. Data on deaths caused by stroke or heart disease in Taiwan have also risen in recent years. Accordingly, lowering blood lipid is essential to health.

Statins (HMG-CoA reductase inhibitors) are common antilipemic agents inhibiting syntheses of mevalonate (a cholesterol precursor) which have become the rate-limiting step of cholesterol syntheses and been used in treating hypercholesterolemia and preventing cardiovascular diseases. Amid statins, atorvastatin (brand name

) synthesized by Pfizer in 1985 was the hot-selling prescription medicine statistically in 2003. Statins which are known as functions of reducing blood lipid and inhibiting vascular inflammation have become potent medicines which even replace aspirin well-known in the past for all illnesses.

With the metabolic pathways of phase I and phase II, the prototype drug of atorvastatin (pKa=4.46) enables metabolism of CYP3A4 for generation of active metabolites including 2-OH atorvastatin acid and 4-OH atorvastatin acid or is metabolized by CYP3A4, UGT1A1 and UGT1A3 for generation of atorvastatin lactone, 2-OH atorvastatin lactone and 4-OH atorvastatin lactone without pharmacological activities (FIG. 1) . The fact that HMG-CoA reductase is inhibited by 70%of total active metabolites implies the curative effect. On the other hand, the active metabolites such as 2-OH-atorvastatin acid and 4-OH-atorvastatin acid have proved in-vitro tests that the inhibitory effect on HMG-CoA reductase is equivalent to the prototype drug of atorvastatin. According to literatures disclosed, the metabolites of atorvastatin are categorized as pH-dependent compounds because of the non-enzymatic conversions of the metabolites between the acid form and the lactone form in a gastrointestinal tract environment. Currently, silybin of the known UGT enzyme inhibitor categorized as flavonoids has been commonly used as antioxidants, anticancer agents or even hepatoprotectants in the clinic. It is also reported that drug-drug interactions are enabled between silybin and metabolic enzymes of phase I as well as phase II drugs, silybin inhibits a series of UGT enzymes like UGT 1A6 and UGT 1A9 and particularly UGT 1A1 in-vitro tests.

How to reduce the conversion of atorvastatin into inactive atorvastatin lactone, 2-OH atorvastatin lactone and 4-OH atorvastatin lactone by adding UGT inhibitor to promote the effective concentration and bioavailability inside human body is a problem to be solved by the invention.

The common side effects of statins are associated with myotoxic, such as lethal rhabdomyolysis. In Japan, more than 20 people have been killed by statins due to the side effects of excessive concentration of drugs in the blood. The reasons can be summarized as: (1) combined oral administration of statins and cholesterol lowing drugs gemfibrozil clinically may increase the risk of the muscle toxicitybecause of drug-drug interactions, (2) the amount of drug absorption varies from person to person because of the differences among individuals, the drug metabolizing enzyme genotypes in the organism are divided into normal metabolic enzyme type such as extensive metabolizer, intermediate metabolizer, and extreme metabolic enzyme type such as ultrarapid metaolizer and poor metabolizer.

Based on the bioavailability of statins is low, wherein the absolute bioavailability of atorvastatin is only 14%, the effective concentration of drugs in vivo relies on administrations of more drugs which probably give rise to serious or even lethal side effects such as rhabdomyolysis attributed to different drug absorption among individuals. Accordingly, it is necessary to avoid excessive concentration of drugs in the blood so as to reduce side effects. If the bioavailability of statins can be increases from 14%to 98%, the difference in the amount of drug absorption among individuals is reduced from 7.14 times (14%～100%) to 1.02 times (98%～100%) . In virtue of high bioavailability of the organisms, the probability of serious or even lethal side effects such as rhabdomyolysis will be greatly reduced.

Although it has been reported previously that the metabolites of atorvastatin are also pH dependent compounds, there is still no successful study on improving the bioavailability of atorvastatin by pH regulation.

US 20040138290 discloses a pharmaceutical form comprising atorvastatin calcium as an active ingredient and a pH adjusting substance. It is characterized in that atorvastatin calcium comprises micronized amorphous atorvastatin calcium and aqueous media dissolved in 900ml aqueous solutions (pH=3) . For the pH value of aqueous media inside a pharmaceutical formulation equal to or greater than pKa+1 of atorvastatin calcium, bioavailability of atorvastatin was promoted when the pharmaceutical formulation with basic or buffering agents was dissolved in aqueous solutions easily or speedily. However, there was no embodiment for promotion of bioavailability disclosed in US20040138290 even if the pharmaceutical formulation easily dissolved at pKa+ 1 (equal to pH=5.5) was favorable to bioavailability of atorvastatin.

US 20080138429 discloses a particles composition, the particles comprise active substances which are HMG-CoA reductase inhibitors and coatings wherein the coatings comprise: (a) film-former selected from a group consisting of polyvinyl alcohol (PVA) and sodium carboxymethylcellulose (NaCMC) , hydroxyethyl cellulose (HEC) and combinations thereof; (b) at least a pharmaceutical excipient selected from the group consisting of a buffering agent, an alkalizing agent and a surface active agent wherein the alkalizing agent and the coating are added for stability of a drug.

US 20110165239 discloses a pharmaceutical composition comprises atorvastatin and an alkalizing additive selected from the group consisting of L-arginine and sodium bicarbonate wherein the atorvastatin is atorvastatin free acid or alkalizing agents selected from pharmaceutically acceptable salts, the molar ratio of the alkalizing additive to atorvastatin is from 1: 1 to 6: 1, and the alkalizing additive contributes to stability of atorvastatin.

Although the aforesaid prior arts used pH modifier (alkaline agent) as a drug stabilizer or to increase the solubility of the drug, they were not disclosed the pH modifiers used for increasing the bioavailability of atorvastatin. Moreover, the range of the pH modifier content in the above disclosures is unlimited or unspecified. However, as the oral drug passes through the acid stomach and then to the alkaline intestinal tract, the drug needs a certain amount of pH modifier to regulate the pH level of the stomach and intestinal tract to an appropriate pH environment that can improve the bioavailability of atorvastatin. Therefore, too much or too little the pH modifier cannot achieve the purpose of the invention.

Modified release drugs can change the time of release in the organism. At present, no one has disclosed what specific pH value can improve the bioavailability. Because of the higher total dosage of the modified release formulation, the bioavailability of statins such as atorvastatin is low, and the difference among individuals is large, which leads to higher absorption of some people or stronger reactivity to the drug. Therefore, it is necessary to promote the bioavailability, reduce the individual difference after the use of drugs, so that the degree of inconsistency in blood concentration reduced, and the safety and curative effect of the average medication increased, and the compliance of patients taking medicine was increased, thereby reducing the chance that atorvastatin as a pH modifier can cause severe or even lethal side effects such as rhabdomyolysis.

Therefore, providing a new statin formulation with higher bioavailability to reduce the inconsistency of blood concentration caused by individual patient differences after medication is essential to avoid serious or even lethal side effects such as rhabdomyolysis. Further, the use of the modified-release formulation enables the effective ingredients of statins to reach the intestinal tract safety through the destruction of gastric acids and the pH modifiers are used for adjusting the pH level of the intestinal tract to improve the stability and absorptivity of drugs. Furthermore, it can control the time of release in organism and reduce the failure of treatment because the patient forgot to take medicine. The development of a new statin formulation with the above functions has become an important topic to be solved by the present invention.

Summary of Invention

The present invention is to provide a pharmaceutical composition for promotion of bioavailability of oral statins, which comprises statins and substances to promote the bioavailability, wherein the substances for promotion of the bioavailability contain: (a) pH modifiers and a modified-release formulation and/or (b) metabolic enzyme inhibitors; the pH modifiers are used for adjusting the pH level of the intestinal tract and the modified-release formulation enables the effective ingredients of statins to reach the intestinal tract safety through the destruction of gastric acids.

To this end, the pH modifiers can adjust the pH value of the intestinal tract to 4.9～8.5.

To this end, the pH modifiers can adjust the pH value of the intestinal tract to 5.8～7.8.

To this end, the pH modifiers contain at least one of sodium carbonate, sodium bicarbonate, sodium hydroxide, boric acid, potassium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, glycine, ammonia, ammonium lactate, ammonium bicarbonate, ammonium hydroxide, ammonium phosphate dibasic, monoethanolamine, diethanolamine, triethanolamine, trihydroxymethylaminomethane, ethylenediamine, N-methyl glucamide, 6N-methyl glucamine, meglucamine, L-lysine and 2-amino-2- (hydroxymethyl) -1, 3-propanediol or a combination thereof.

To this end, the weight of the pH modifiers is 200～11800 mg.

To this end, the pH modifiers account for 20～90% (w/w) of the pharmaceutical composition.

To this end, the statins are selected from the group consisting of atorvastatin, pravastatin, simvastatin, lovastatin, mevastatin, compactin, fluvastatin, cerivastatin, rosuvastatin and pitavastatin.

To this end, the UGT1A3 inhibitors contain at least one of sodium lauryl sulfate (SLS) , acesulfame potassium, Aerosil 200, apigenin, baicalin, benzyl alcohol, benzyl benzoate, butylated hydroxyanisole (BHA) , Brij 58, Brij 76, cherry, citric acid, croscarmellose sodium, dextrates, EDTA 2 Na, glycerin, glycyrrhizin, hydroxy propyl cellulose, hydroxypropyl methylcellulose, kollidon VA64, lactose, lactose monohydrate, lactose S. G, lemon oil, locust, low-substituted hydroxypropylcellulose, maltodextrin, mannitol, methyl paraben, menthol, Myri 52, methyl cellulose, NF hydrate, β-naphthoflavone, PEG 400, PEG 2000, PEG 4000, Pluronic F68, Pluronic F127, polyoxyl 40 hydrogenated castor oil (RH 40) , propyl paraben, PVP K90F, saccharin, sodium cyclamate, sodium starch glycolate, sodium benzolate, sorbic acid, sorbitol solution, Span 60, Span 80, sucralose, starch acetate, trans-aconitic acid, triethyl citrate, trisodium citrate, Tween 20 (TW20) , Tween 40 (TW40) , Tween 80 (TW80) , ursolic acid, sodium dihydrogen phosphate, dicalcium phosphate dihydrate, dextrates (NF hydrate) and cholic acid or a combination thereof. To this end, the UGT1A1 inhibitors contain at least one of sodium lauryl sulfate (SLS) , acesulfame potassium, Aerosil 200, apigenin, baicalin, benzyl alcohol, benzyl benzoate, butylated hydroxyanisole (BHA) , Brij 58, Brij 76, cherry, citric acid, croscarmellose sodium, dextrates, EDTA 2 Na, glycerin, glycyrrhizin, hydroxy propyl cellulose, hydroxypropyl methylcellulose, kollidon VA64, lactose, lactose monohydrate, lactose S. G, lemon oil, locust, low-substituted hydroxypropylcellulose, maltodextrin, mannitol, methyl paraben, menthol, Myri 52, methyl cellulose, NF hydrate, β-naphthoflavone, PEG 400, PEG 2000, PEG 4000, Pluronic F68, Pluronic F127, polyoxyl 40 hydrogenated castor oil (RH 40) , propyl paraben, PVP K90F, saccharin, sodium cyclamate, sodium starch glycolate, sodium benzolate, sorbic acid, sorbitol solution, Span 60, Span 80, sucralose, starch acetate, trans-aconitic acid, triethyl citrate, trisodium citrate, Tween 20 (TW20) , Tween 40 (TW40) , Tween 80 (TW80) , ursolic acid, sodium dihydrogen phosphate, dicalcium phosphate dihydrate, dextrates (NF hydrate) and cholic acid or a combination thereof.

To this end, the modified-release formulation further features an extended release effect.

To this end, the pharmaceutical composition can be tablets, granules, capsules, powders or other pharmaceutically acceptable formulations.

To this end, the pharmaceutical composition can be further comprises excipients.

To this end, the excipients can be used as diluents, fillers, binders, disintegrants, lubricants or other pharmaceutically acceptable excipients.

The present disclosure is to provide a use of a pharmaceutical composition for promotion of the bioavailability of oral statins wherein the pharmaceutical composition comprises (a) pH modifiers and a modified-release formulation and/or (b) metabolic enzyme inhibitors, wherein the modified-release formulation enables the effective ingredients of statins to reach the intestinal tract safety through the destruction of gastric acids and the pH modifiers are used for adjusting the pH level of the intestinal tract to improve the stability and absorptivity of drugs.

To this end, the UGT1A3 inhibitors contain at least one of sdium lauryl sulfate (SLS) , acesulfame potassium, Aerosil 200, apigenin, baicalin, benzyl alcohol, benzyl benzoate, butylated hydroxyanisole (BHA) , Brij 58, Brij 76, cherry, citric acid, croscarmellose sodium, dextrates, EDTA 2 Na, glycerin, glycyrrhizin, hydroxy propyl cellulose, hydroxypropyl methylcellulose, kollidon VA64, lactose, lactose monohydrate, lactose S. G, lemon oil, locust, low-substituted hydroxypropylcellulose, maltodextrin, mannitol, methyl paraben, menthol, Myri 52, methyl cellulose, NF hydrate, β-naphthoflavone, PEG 400, PEG 2000, PEG 4000, Pluronic F68, Pluronic F127, polyoxyl 40 hydrogenated castor oil (RH 40) , propyl paraben, PVP K90F, saccharin, sodium cyclamate, sodium starch glycolate, sodium benzolate, sorbic acid, sorbitol solution, Span 60, Span 80, sucralose, starch acetate, trans-aconitic acid, triethyl citrate, trisodium citrate, Tween 20 (TW20) , Tween 40 (TW40) , Tween 80 (TW80) , ursolic acid, sodium dihydrogen phosphate, dicalcium phosphate dihydrate, dextrates (NF hydrate) and cholic acid or a combination thereof. To this end, the UGT1A1 inhibitors contain at least one of sodium lauryl sulfate (SLS) , acesulfame potassium, aerosil 200, apigenin, baicalin, benzyl alcohol, benzyl Benzoate, butylated hydroxyanisole (BHA) , Brij 58, Brij 76, cherry, citric acid, croscarmellose sodium, dextrates, EDTA 2 Na, glycerin, glycyrrhizin, hydroxy propyl cellulose, hydroxypropyl methylcellulose, kollidon VA64, lactose, lactose monohydrate, lactose S. G, lemon oil, locust, low-substituted hydroxypropylcellulose, maltodextrin, mannitol, methyl paraben, menthol, Myri 52, methyl cellulose, NF hydrate, β-naphthoflavone, PEG 400, PEG 2000, PEG 4000, Pluronic F68, Pluronic F127, polyoxyl 40 hydrogenated castor oil (RH 40) , propyl paraben, PVP K90F, saccharin, sodium cyclamate, sodium starch glycolate, sodium benzolate, sorbic acid, sorbitol solution, Span 60, Span 80, sucralose, starch acetate, trans-aconitic acid, triethyl citrate, trisodium citrate, Tween 20 (TW20) , Tween 40 (TW40) , Tween 80 (TW80) , ursolic acid, sodium dihydrogen phosphate, dicalcium phosphate dihydrate, dextrates (NF hydrate) and cholic acid or a combination thereof. To this end, the statins are selected from the group consisting of atorvastatin, pravastatin, simvastatin, lovastatin, mevastatin, compactin, fluvastatin, cerivastatin, rosuvastatin and pitavastatin.

In summary, the new statin formulations in the present disclosure have the following advantages:

(1) The pH level in the intestinal tract is adjusted by using a certain quantity of pH modifiers for promotion of the bioavailability of atorvastatin amid statins.

(2) Use of the metabolic enzyme inhibitors inhibit atorvastatin and their active metabolites including 2-OH atorvastatin acid and 4-OH atorvastatin acid which are metabolized into no pharmacological activity atorvastatin lactone, 2-OH atorvastatin lactone and 4-OH atorvastatin lactone for promotion of the bioavailability.

(3) In order to release the drug in the most appropriate pH value environment, the drug is coated with the modified-release formulation to make the drug dissolve in the intestinal tract with the appropriate pH value rather than in gastric fluid, so that the drug can be carried with sufficient alkaline agents to change the pH value in the intestinal tract to the appropriate pH value for further promotion of the bioavailability.

(4) The promotion of the bioavailability of drugs reduces the probability of serious or even lethal side effects such as rhabdomyolysis caused by individual absorption differences, thereby increasing the safety of the drugs and further ensuring the safety of the modified-release formulation with high dosage.

Brief Description of Drawings

FIG. 1 illustrates metabolic pathway of atorvastatin in organisms.

FIG. 2A illustrates the in-vitro permeation efficiency of atorvastatin in the intestinal wall of rats at different time points under different pH conditions.

FIG. 2B illustrates the in-vitro permeation efficiency of atorvastatin in the intestinal wall of rats in fifth hours under different pH conditions.

FIG. 3A illustrates time-varying concentrations of a pharmaceutical composition (AT+2AT+4AT) formed by a sum of atorvastatin (AT) and two active metabolites (2AT+4AT) in blood, using SD Rat oral

(water, n=8) , atorvastatin (pH 7.4, n=11, AT) and an experimental group of atorvastatin+HUEXC41 (pH 7.4, n=6) , respectively.

FIG. 3B illustrates time-varying concentrations of an active metabolite (2AT) in blood, using SD Rat oral

(water, n=8) , atorvastatin (pH 7.4, n=11) and an experimental group of atorvastatin+HUEXC41 (pH 7.4, n=6) , respectively.

FIG. 4A illustrates time-varying concentrations of a pharmaceutical composition (AT+2AT+4AT) formed by a sum of atorvastatin (AT) and two active metabolites (2AT+4AT) in blood, using SD Rat oral

(water, n=8) , atorvastatin (pH 7.4, n=11, AT) and an experimental group of atorvastatin+HUEXC85 (pH 7.4, n=6) , respectively.

FIG. 4B illustrates time-varying concentrations of an active metabolite (2AT) in bloods, using SD Rat oral

(water, n=8) , atorvastatin (pH 7.4, n=11) and an experimental group of atorvastatin+HUEXC85 (pH 7.4, n=6) , respectively.

FIG. 5A illustrates time-varying concentrations of a pharmaceutical composition (AT+2AT+4AT) formed by a sum of atorvastatin (AT) and two active metabolites (2AT+4AT) in blood, using SD Rat oral

(water, n=8) , atorvastatin (water, n=8, AT) and an experimental group of atorvastatin (pH 7.4, n=11) , respectively.

FIG. 5B illustrates time-varying concentrations of an active metabolite (2AT) in bloods, using SD Rat oral

(water, n=8) , atorvastatin (water, n=8) and an experimental group of atorvastatin (pH 7.4, n=11) , respectively.

Description of Embodiments

A pharmaceutical composition for promotion of bioavailability of oral statins and applications thereof are explained in but not limited to the following embodiments.

A new compound for promotion of bioavailability of statins is explained in but not limited to embodiments of atorvastatin.

Experimental facilities and method for the in-vitro permeation test

The vertical diffusion cell used in the in-vitro permeation test was a columnar glass vessel with an upper part and a lower part, each of which was separated from the other: the upper part was a columnar double-deck glass diffusion cell as a donor in which permeable drugs were stored; the lower part was a columnar glass tube as a receptor which had a contact surface at the bottom to collect the transdermal absorption of the permeable substances content. A permeable barrier which was designed for a permeation test and a Sprague

rat’s duodenum membrane was fixed between upper and lower glass parts. The outer glass was mainly used as the circulating water flow filled by a water bath to control the temperature at 37 ℃. When the upper and lower parts were tightly integrated, the sectional area inside was 0.785 cm ² (actual permeation area) . A total of 1 ml atorvastatin solution prepared with 0.5 mg/ml phosphate buffer with different pH values was filled into the interior space of the upper part, while 6 ml phosphate buffer (pH 7.4) was filled into the interior space of the lower part in which a magnetic stir bar was placed at the bottom to be stirred at a speed of 600 rpm on a multipoint mixer. 0.5 ml liquid drug was extracted at 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours and 5 hours respectively, then the pH 7.4 of 0.5 ml phosphate buffer solution (pH value in the simulated blood) was supplemented to the receptor for a total volume kept unchanged. The accumulative permeated liquid drug of the receptor was analyzed with a high-efficiency liquid chromatography followed by the Student’s t-test to analyze whether there are statistical differences among different groups with respect to the control group.

Reagents and methods for in-vitro screening

Preparation of microsomal enzymes

As the resource for preparations of microsomal enzymes, 250～300g male rats fasting for more than 16 hours were sacrificed for removals of livers which were cleaned in icy KCl (1.15%) and the weights were recorded. The shredded livers were added to the icy KCl (the weight ratio of liver to KCl=1: 4) and then placed in a homogenizer. The homogenized liver fluids were placed in high-speed centrifugal tubes (each about 15ml) for centrifugation at 4℃ and 9000rpm (gravitational acceleration=12, 500g) in the next 20 minutes. After centrifugation, supernatants were removed and the remaining liver fluids were then placed in an ultra-high-speed centrifuge for extra centrifugation at 4℃ and 40000rpm (gravitational acceleration=100,000g) in the next two hours. After the centrifugation was completed, the supernatants were discarded and the pellets deposited in the tubes were scraped and then the KH ₂PO ₄ buffers (pH=7.4) with the weight of liver were added to the pellets to form a mixture. The mixture was then homogenized and separately loaded into each of the 2ml centrifugal tubes which were kept in a refrigerator at -80℃.

Measurement of the protein content

The prepared hepatic microsomal enzymes used the well-known and credible Lowry method to quantify the concentration of protein content. The sensitivity of this quantitative method is moderate and the detection limit is about 0.05～0.5mg/ml.

In-vitro metabolism test

The model drugs were midazolam, estradiol and chenodeoxycholic acid, respectively, corresponding to enzymes of CYP3A4, UGT1A1 and UGT 1A3, respectively. The test group included “positive control group” , “negative control group” and “experimental group” . In each sample of each group, the sample size was three (n=3) and the base materials added were KH ₂PO ₄ (53mM; pH=7.4) , MgCl ₂ (5mM) , NADPH and UDPGA. The concentrations of the model drugs of midazolam, estradiol and chenodeoxycholic acid were 1.65μg/ml, 0.24mM and 0.24mM, respectively; the concentration of inhibitors of ketoconazole and silybin were 0.03mM and 0.075mM, respectively. The concentration of hepatic microsomal enzymes used to activate the reactions in Tables 8 and 9 was 0.5mg/ml. After each sample was added to the reagent, the oscillator was oscillating for 10 seconds and then placed in a 37℃ water bath for 1 hour.

The base materials added in samples of the experimental group were identical to those added in samples of control groups except inhibitors which were replaced by excipients of drugs to be screened. The concentrations of added excipients ranged from 33.3 to 16.7 and finally to 3.33mg/ml; the reactant concentrations of added excipients ranged from 0.033 to 0.0167 and finally to 0.003μg/ml. After each sample was added to the reagent, the oscillator was oscillating for 10 seconds and then placed in a 37℃ water bath for 1 hour.

After 1-hour of water bath, 1ml icy acetonitrile labeled “1’-OH-midazolam-D4” was added into test samples for stopping reactions. The total volume of 1.5ml test samples were transferred to a 2ml tube and centrifuged for 15000 rpm for 10 minutes. After centrifugation, the 0.2ml supernatants were removed from test samples for analyses. The data for production rate, %control and %inhibition were derived from analysis results substituted into formulas. The in-vitro screening tests were divided into 13 batches with statistically significant difference in each batch of test samples between the control group and the positive control group (P < 0.05) .

Creation of calibration curves for the in-vitro metabolism test

A total of three metabolites were analyzed in this experiment, so the three detection lines were prepared. The 1'OH-Midazolam (CYP 3A4) concentrations ranged from low to high respectively are 16.7, 33, 83, 167, 330, 830 and 1670 ng/ml, and the QC points were 50, 750 and 1500 ng/ml. Similarly, the B-Estradiol 3- (B-D-Glucuronide) *Sodium and CDCA 24-Acyl-β-D-glucuronide (UGT 1A1 &UGT 1A3) concentrations ranged from low to high respectively were 16.7, 33, 83, 167, 330, 830 and 1670 ng/ml, and the QC points were 50, 750 and 1500 ng/ml.

Formulas:

I. Production rate V (pmol/min/mg) = (produced metabolites (ng/mL) /molecular weight) ×0.0005 L/60 min/0.5 mg protein ×1000000

II. %control = (measured metabolites concentration /control mean) ×100

III. %inhibition = 100|%control

IV. Estimation of the concentration for in-vitro screenings:

Oral dosage given to a 60kg health adult (mg) /3000ml (volume of liver and intestines) ×100%= dosage for in-vitro screenings (mg)

Preparations of rats’liver microsomal enzymes

Measurement of the protein content

Preparation of animal test samples

SD rats are used for the Pharmacokinetic experiment. Bloods were collected from rats to which oral drugs were administered at 0min, 10min, 20min, 40min, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 12hr and 24 hr, respectively. The metabolites were extracted from test samples with HLB cartridges for solid phase extractions on ice.

A total of six kinds of metabolites were analyzed in the present disclosure: namely atorvastatin acid (AT) , 2-OH-atorvastatin acid (2AT) , 4-OH-atorvastatin acid (4AT) , atorvastatin lactone (AT-lactone) , 2-OH-atorvastatin lactone (2AT-lactone) and 4-OH-atorvastatin lactone (4AT-lactone) .

The concentration ranges in standard calibration curves of atorvastatin acid and 2-OH-atorvastatin acid from low to high were 0.25, 0.5, 1, 5, 10, 20, 50 and 100 ng/ml, and the concentrations ranges of QC were 0.75, 45 and 90 ng/ml. The concentrations ranges in standard calibration curve of atorvastatin lactone, 2-OH-atorvastatin lactone and 4-OH-atorvastatin lactone from low to high were 0.25, 0.5, 1, 2, 5, 10, 15 and 25 ng/ml, and the concentrations ranges of QC were 0.75, 9 and 20 ng/ml.

Procedures for preparation of animal test samples with HLB cartridges for solid phase extractions:

Embodiment 1: Effects of pH levels on in-vitro permeation of atorvastatin

For a better atorvastatin formulation, the drug permeation efficiency under different pH environmental conditions was evaluated, so Franz-cells was used for in-vitro permeation test. FIG. 2A illustrates the results of the in-vitro permeation test in which atorvastatin through Franz-cells and the rats duodenum membranes. The concentration of atorvastatin permeated at every point of time showed that the difference of permeation caused by the pH value increased significantly after 2 hours. The results show that atorvastatin has a good permeation efficiency for the duodenum membrane of SD Rat in pH=5.8 ^**, pH=6.3 ^*, pH=6.8 ^**, pH=7.4 ^***, pH=7.8 ^*, pH=8.0 ^***and pH=8.5**, and has the best permeation effect in pH=7.4 at all test periods. According to the results of FIG. 2B, the permeation concentration of atorvastatin at fifth hours reached the highest in pH7.4 at fifth hours reached the highest in pH=7.4, and there was a significant difference compared with the results of

gastrointestinal tract in pH=4.5 ( ^*P<0.05; ^**P<0.01; ^***P<0.001) . Because atorvastatin (pKa=4.46) is an acidic drug (BCS system class II) with physicochemical properties of low solubility and high permeatability. Therefore, from the results of the in-vitro permeatation test throguh duodenum membrane in rats, it is known that when the pH value of the ambient is larger than the pKa value of the drug itself, the permeability of atorvastatin will increase progressively, until the ambient pH value up to 7.4 reaches the highest point of permeatation concentration. In animal tests also proved that the lipophilic drug atorvastatin can improve the degree of dissociation and make the metabolites of atorvastatin permeate the membrane of the gastrointestinal tract (mucosa) , thereby achieving the effect of increasing the concentration of drug in the blood.

Embodiment 2: Effects of pure ingredients in common excipients or Chinese medicinal ushers on activity of metabolic enzymes of atorvastatin

The protein content of hepatic microsomal enzymes purified from the livers of 250～300g Sprague Dawley rat was measured based on the Lowry method and compared with the BSA (bovine serum albumin) standard which had the standard calibration curve with concentrations of 5, 10, 20, 30 and 40mg/ml after measurement of absorbance by a UV spectrometer (OD 550) . The measured mean concentration of hepatic microsomal enzymes was about 10mg/ml. The data in Tables 1～4 illustrate the effects of common excipients on the activities of major screening enzymes UGT1A3, UGT1A1 and CYP3A4, and the screening concentrations of excipients from high to low are 33.3, 16.7 and 3.33mg/ml respectively. The ranking of inhibitors affecting on the metabolic degree of microsome is mainly UGT1A3 which is the dominant enzyme involved in the acidification reaction of glucosaldehyde of atorvastatin active metabolites, followed by UGT1A1 with Silybin (0.036μg/ml) as the positive control group of UGT1A1/UGT1A3, and with Ketoconazole (0.016μg/ml) as the positive control group of CYP3A4 ( ^*p<0.05, ^**p<0.01, ^***p<0.001) . The invention needs to find safety compounds that can inhibit UGT1A1 and UGT1A3 but do not inhibit CYP3A4, so as to promote the bioavailability of statins.

Table 1 Effects of common excipients on activities of UGT1A3, UGT1A1 and CYP3A4

a. Silybin is used as UGT1A1/UGT1A3 positive control at concentration of 0.036μg/ml

b. Ketoconazole is used as CYP3A4 positive control at concentration of 0.016μg/ml

Statistic method: 2-way t-test. *p<0.05, **p<0.01, ***p<0.001 compared with control group.

Table 2 Effects of pure ingredients in common excipients or Chinese medicinal ushers on activities of UGT1A3, UGT1A1 and CYP3A4

Table 3 Effects of pure ingredients in common excipients or Chinese medicinal ushers on activities of UGT1A3, UGT1A1 and CYP3A4

Table 4 Effects of pure ingredients in common excipients or Chinese medicinal ushers on activities of UGT1A3, UGT1A1 and CYP3A4

Table 5 Effects of common excipients on activities of UGT1A3, UGT1A1 and CYP3A4

Statistic method: 2-way t-test. *p<0.05, **p<0.01, ***p<0.001 compared with control group

Table 6 Effects of common excipients on activities of UGT1A3, UGT1A1 and CYP3A4

Table 7 Effects of common excipients on activities of UGT1A3, UGT1A1 and CYP3A4

Table 8 Effects of common excipients on activities of UGT1A3, UGT1A1 and CYP3A4

Table 9 Effects of common excipients on activities of UGT1A3, UGT1A1 and CYP3A4

Table 10 Effects of common excipients on activities of UGT1A3, UGT1A1 and CYP3A4

Table 11 Effects of common excipients on activities of UGT1A3, UGT1A1 and CYP3A4

Table 12 Effects of common excipients on activities of UGT1A3, UGT1A1 and CYP3A4

Table 13 Ranking for effects of common excipients on the activity of UGT1A1

a. 0.033 μg/ml, b. 0.0167 μg/ml, c. 0.003 μg/ml

Statistic method: 2-way t-test *P＜0.05, **P＜0.01, ***P＜0.001 compared with control group.

Table 13 Ranking for effects of common excipients on the activity of UGT1A1 (continued)

a. 0.033μg/ml, b. 0.0167μg/ml, c. 0.003μg/ml

Table 14 Ranking for effects of common excipients on the activity of UGT1A3

a. 0.033 μg/ml, b. 0.0167 μg/ml, c. 0.003 μg/ml

Table 14 Ranking for effects of common excipients on the activity of UGT1A3 (continued)

a. 0.033μg/ml, b. 0.0167μg/ml, c. 0.003μg/ml

Table 13 shows the ranking for effects of common excipients on the activity of UGT1A1, wherein the top five inhibitors are HUEXC40 (sodium lauryl sulfate, SLS) , HUEXC07 (Brij 76) , HUEXC14 (Span 80) , HUEXC77 (polyoxyl 40 hydrogenated castor oil, RH 40) and HUEXC85 (trans-aconitic acid) respectively. However, the strongest inhibitory effect is HUEXC40 (sodium lauryl sulfate, SLS) , with a inhibition of 84.4%when the concentration was 0.01667μg/ml, followed by HUEXC07 (Brij 76, the second best inhibitor) with a inhibition of 52.1%when the concentration was 0.03334μg/ml. Table 14 shows that the top four UGT1A3 inhibitors in-vitro screening are HUEXC40 (sodium lauryl sulfate, SLS) , HUEXC32 (propyl paraben) , HUEXC01 (Tween 20, TW20) and HUEXC33 (methyl paraben) respectively, wherein all of the inhibition of UGT1A3 were about 50%when these inhibitors concentrations were 0.01667μg/ml. It can be seen from in-vitro screening inhibitors that the most effective types of inhibitory effects were surfactant. However, surfactant in animal tests may get the opposite effect, which may be caused by surfactant-induced diarrheas in experimental animals, resulting in poor absorption of oral drug.

Embodiment 3: Effects of pH levels and common excipients on bioavailability of atorvastatin

(water, n=8) , atorvastatin (pH 7.4, n=11, AT) and an experimental group of atorvastatin+HUEXC41 (pH 7.4, n=6) , respectively. FIG. 3B illustrates time-varying concentrations of an active metabolite (2AT) in blood, using SD Rat oral

(water, n=8) , atorvastatin (pH 7.4, n=11) and an experimental group of atorvastatin+HUEXC41 (pH 7.4, n=6) , respectively. The results of Table 15 show that the experimental group (atorvastatin (1mg/kg) + inhibitor HUEXC41 (10mg/kg at pH 7.4) are compared with the control group

(in water) , wherein AUC _∞, AUC _t and C _max are increased by 3.79 times (P<0.001) , 3.56 times (P<0.001) and 3.38 times (P=0.001) , respectively.

Table 15 Pharmacokinetic parameters and statistic analyses for animals oral administration of both the atorvastatin and excipients (HUEXC41)

Statistic method: one-way ANOVA test

*P<0.05, **P<0.01, ***P<0.001

(water, n=8) , atorvastatin (pH 7.4, n=11, AT) and an experimental group of atorvastatin+HUEXC85 (pH 7.4, n=6) , respectively. FIG. 4B illustrates time-varying concentrations of an active metabolite (2AT) in bloods, using SD Rat oral

(water, n=8) , atorvastatin (pH 7.4, n=11) and an experimental group of atorvastatin+HUEXC85 (pH 7.4, n=6) , respectively. The results of Table 16 show that the experimental group (atorvastatin + inhibitor HUEXC85 (6mg/kg, pH 7.4) are compared with the control group

(in water) , wherein the AUC _∞, AUC _t and C _max are increased by 3.8 times (P<0.001) , 3.74 times (P<0.001) and 3.21 times (P=0.001) , respectively. Compared with

(in water) , a pharmaceutical composition (AT+2AT+4AT) formed by a combination of an animal oral atorvastatin (AT) and two other active metabolites (2AT+4AT) as inhibitors presented T _max= 1～1.94 hr, C _max=0.11～0.12μmol/L and AUC _∞=0.66hr*μmol/L, and the individual active metabolite of the atorvastatin (AT) presented T _max=0.61～0.69 hr, C _max=6.23～11.43 ng/ml and AUC _∞=23.97～28.92 hr*ng/ml, and 2 OH-atorvastatin (2AT) presented T _max=1～2 hr, C _max=67.17～70.55 ng/ml and AUC _∞=383.62～406.06 ng/ml. It can be seen that the improvement of atorvastatin drug formulation and the combination of inhibitors can increase the concentration of active metabolites in the blood and prolong the duration of action of the drugs.

Table 16 Pharmacokinetic parameters and statistic analyses for animals oral administration of both the atorvastatin and excipients (HUEXC85)

Statistic method: one-way ANOVA test

*P<0.05, **P<0.01, ***P<0.001

By improving the formula of atorvastatin with appropriate enzyme inhibitors and pH levels, the invention promotes the solubility and absorptivity of drugs in the gastrointestinal tract, making it better to permeate into the blood circulation and prolong the retention time of the drugs inside the body. Compared with the animal test results of the original

the invention showed that the formulations for adding UGT1A1/UGT1A3 inhibitors enable the AUC _∞ and C _max of the atorvastatin to be increased by 3.79～3.8 times and 3.21～3.38 times, respectively, thereby significantly increasing the bioavailability of the atorvastatin.

Embodiment 4: Effects of pH levels on bioavailability of atorvastatin

Rats oral

(in water) or oral atorvastatin (1mg/kg) dissolved in pure water and buffer solution (pH 7.4) , wherein the experimental groups were

(in water) , atorvastatin (in water) and atorvastatin (in pH 7.4 buffer solution) , respectively. FIG. 5A illustrates time-varying concentrations of a pharmaceutical composition (AT+2AT+4AT) formed by a sum of atorvastatin (AT) and two active metabolites (2AT+4AT) in bloods, using SD Rat oral

(water, n=8) , atorvastatin (water, n=8, AT) and an experimental group of atorvastatin (pH 7.4, n=11) , respectively. FIG. 5B illustrates time-varying concentrations of an active metabolite (2AT) in blood, using SD Rat oral

(water, n=8) , atorvastatin (water, n=8) and an experimental group of atorvastatin (pH 7.4, n=11) , respectively. It can be seen from Table 17 that the pharmacokinetic result of

(in water) is similar to atorvastatin (in water) , while the comparison between the experimental group atorvastatin (in pH 7.4 buffer) and

(in water) indicates that AUC _∞ is increased by 3.39 times (P<0.001) , AUCt is increased by 3.02 times (P<0.001) and Cmax is increased by 1.77 times (P=0.002) . After oral

dissolved in pure water was administered by animals, the sum of atorvastatin and two active metabolites (AT/2AT/4AT) presented T _max= 0.58 hr, C _max=0.03μmol/L and AUC _∞=0.17hr*μmol/L. Other test data were T _max=0.54 hr, C _max=11.99 ng/ml and AUC _∞=30.91 hr*ng/ml for the individual active metabolite of atorvastatin acid (AT) and T _max=1 hr, C _max=16.07 ng/ml and AUC _∞=91.04 ng/ml for the individual active metabolite of 2 OH-atorvastatin acid (2AT) .

Table 17 Pharmacokinetic parameters and statistic analyses for animals oral administration of atorvastatin after adjusting pH level in intestinal tract

Statistic meyhod: one-way ANOVA test

*P<0.05, **P<0.01, ***P<0.001

Embodiment 5: Feasibility of adjusting pH level in intestinal tract by pH modifiers

From the above embodiments confirm that both permeability and bioavailability of atorvastatin at pH=7.4 were the best. Therefore, this embodiment further explores the feasibility of adding pH modifier to adjust the intestinal pH value in pharmaceutical composition.

150 ml of quasi artificial intestinal fluid (pH 4.5, according to the United States Pharmacopoeia USP 39 formula) was added to 80 mg atorvastatin powder (the daily dose of atorvastatin is about 10～80mg. ) After measuring the pH value, the required volume of buffer solutions was determined, in order to maintain the pH value of gastrointestinal tract at 7.4. As shown in Table 18, a variety of alkaline buffers were used to adjust the pH value of the quasi artificial intestinal fluid from pH 4.5 to pH 7.4 and estimate the dosages of the required buffer and all kinds of salts. The buffer Na2CO3/NaHCO3 (pH 8.6～pH 10.1) , NaHCO3/NaOH, alkaline borate (boric acid/KCL/NaOH) (pH 10) only need to use 236～826mg buffer salts to successfully adjust the quasi artificial intestinal fluid from pH 4.5 to 7.4, and the dosage of the buffer salt used is less than 1 gram. Therefore, adding pH modifier to pharmaceutical composition is feasible for adjusting the intestinal pH value. Moreover, it is found that the higher the dosage of the original pH, the less the dosage needed to adjust the quasi artificial intestinal fluid from pH 4.5 to pH 7.4.

Table 18 Dosages of alkaline buffer agents to keep intestinal tract at pH 7.4 simulated by 150 ml quasi intestinal fluid of pH 4.5

The above results confirm that the pharmaceutical compositions of the statins of the invention have the following prominent effects.

1) Using special dosage of pH modifiers and enteric membrane encapsulated drugs can make the dissolution of oral drugs only after entering the intestine, and to adjust the pH value of the intestinal tract.

2) By combining the use of inhibitors with metabolic enzymes, the possibility of reducing the atorvastatin and its active metabolites, such as 2-OH atorvastatin acid and 4-OH atorvastatin acid being metabolized into atorvastatin lactone and 2-OH atorvastatin lactone and 4-OH atorvastatin lactone without pharmacological activity can be achieved, thereby increasing the bioavailability of atorvastatin amid statins, reducing the serious or even lethal side effects caused by the individual absorption difference, such as rhabdomyolysis, and increasing the drug safety. Therefore, the pharmaceutical composition of the statin can be made as a modified-release formulation.

While the preferred embodiments of the invention have been set forth for the purpose of disclosure, modifications of the disclosed embodiments of the invention as well as other embodiments thereof may occur to those skilled in the art. Accordingly, the appended claims are intended to cover all embodiments which do not depart from the spirit and scope of the invention.

Claims

A pharmaceutical composition for promotion of bioavailability of oral statins, comprising: statins and substances which promote the bioavailability and contain

a) pH modifiers and a modified-release formulation and/or

b) metabolic enzyme inhibitors;

wherein the pH modifiers are used for adjusting a pH level of an intestinal tract and the modified-release formulation enables effective ingredients of statins to reach the intestinal tract safety through a stomach.
The pharmaceutical composition according to claim 1, wherein the pH level of the intestinal tract is ranged from 4.9 to 8.5.
The pharmaceutical composition according to claim 1, wherein the pH level of the intestinal tract is ranged from 5.8 to 7.8.
The pharmaceutical composition according to 1, wherein the pH modifiers contain at least one of sodium carbonate, sodium bicarbonate, sodium hydroxide, boric acid, potassium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, glycine, ammonia, ammonium lactate, ammonium bicarbonate, ammonium hydroxide, ammonium phosphate dibasic, monoethanolamine, diethanolamine, triethanolamine, trihydroxymethylaminomethane, ethylenediamine, N-methyl glucamide, 6N-methyl glucamine, meglucamine, L-lysine and 2-amino-2- (hydroxymethyl) -1, 3-propanediol or a combination thereof.
The pharmaceutical composition according to claim 1, wherein a weight of the pH modifiers is ranged from 200 mg to 11800 mg.
The pharmaceutical composition according to claim 1, wherein a weight ratio of the pH modifiers to the pharmaceutical combination is ranged from 20%to 90%.
The pharmaceutical composition according to claim 1, wherein the statins are selected from the group consisting of atorvastatin, pravastatin, simvastatin, lovastatin, mevastatin, compactin, fluvastatin, cerivastatin, rosuvastatin and pitavastatin.
The pharmaceutical composition according to claim 1, wherein the metabolic enzyme inhibitors comprise at least one of sodium lauryl sulfate (SLS) , acesulfame potassium, Aerosil 200, apigenin, baicalin, benzyl alcohol, benzyl benzoate, butylated hydroxyanisole (BHA) , Brij 58, Brij 76, cherry, citric acid, croscarmellose sodium, dextrates, EDTA 2 Na, glycerin, glycyrrhizin, hydroxy propyl cellulose, hydroxypropyl methylcellulose, kollidon VA64, lactose, lactose monohydrate, lactose S.G, lemon oil, locust, low-substituted hydroxypropylcellulose, maltodextrin, mannitol, methyl paraben, menthol, Myri 52, methyl cellulose, NF hydrate, β-naphthoflavone, PEG 400, PEG 2000, PEG 4000, Pluronic F68, Pluronic F127, polyoxyl 40 hydrogenated castor oil (RH 40) , propyl paraben, PVP K90F, saccharin, sodium cyclamate, sodium starch glycolate, sodium benzolate, sorbic acid, sorbitol solution, Span 60, Span 80, sucralose, starch acetate, trans-aconitic acid, triethyl citrate, trisodium citrate, Tween 20 (TW20) , Tween 40 (TW40) , Tween 80 (TW80) , ursolic acid, sodium dihydrogen phosphate, dicalcium phosphate dihydrate, dextrates (NF hydrate) and cholic acid or a combination thereof.
The pharmaceutical composition according to claim 1, wherein the modified-release formulation further features an extended release effect.
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition can be tablets, granules, capsules, powders or other pharmaceutically acceptable formulations.
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises excipients.
The pharmaceutical composition according to claim 11, wherein the excipients can be diluents, fillers, binders, disintegrants, lubricants or other pharmaceutically acceptable excipients.
Use of a pharmaceutical composition for promotion of bioavailability of statins, comprising:

a) pH modifiers and a modified-release formulation, and/or

b) metabolic enzyme inhibitors;

wherein the modified-release formulation enables the effective ingredients of statins to reach an intestinal tract safety through the destruction of gastric acids and the pH modifiers are used for adjusting a pH level of the intestinal tract to improve the stability and absorptivity of drugs.
The use of the pharmaceutical composition according to claim 13 wherein the pH modifiers contain at least one of sodium carbonate, sodium bicarbonate, sodium hydroxide, boric acid, potassium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, glycine, ammonia, ammonium lactate, ammonium bicarbonate, ammonium hydroxide, ammonium phosphate dibasic, monoethanolamine, diethanolamine, triethanolamine, trihydroxymethylaminomethane, ethylenediamine, N-methyl glucamide, 6N-methyl glucamine, meglucamine, L-lysine and 2-amino-2- (hydroxymethyl) -1, 3-propanediol or a combination thereof.
The use of the pharmaceutical composition according to claim 13, wherein the metabolic enzyme inhibitors contain at least one of sodium lauryl sulfate (SLS) , acesulfame potassium, Aerosil 200, apigenin, baicalin, benzyl alcohol, benzyl benzoate, butylated hydroxyanisole (BHA) , Brij 58, Brij 76, cherry, citric acid, croscarmellose sodium, dextrates, EDTA 2 Na, glycerin, glycyrrhizin, hydroxy propyl cellulose, hydroxypropyl methylcellulose, kollidon VA64, lactose, lactose monohydrate, lactose S.G, lemon oil, locust, low-substituted hydroxypropylcellulose, maltodextrin, mannitol, methyl paraben, menthol, Myri 52, methyl cellulose, NF hydrate, β-naphthoflavone, PEG 400, PEG 2000, PEG 4000, Pluronic F68, Pluronic F127, polyoxyl 40 hydrogenated castor oil (RH 40) , propyl paraben, PVP K90F, saccharin, sodium cyclamate, sodium starch glycolate, sodium benzolate, sorbic acid, sorbitol solution, Span 60, Span 80, sucralose, starch acetate, trans-aconitic acid, triethyl citrate, trisodium citrate, Tween 20 (TW20) , Tween 40 (TW40) , Tween 80 (TW80) , ursolic acid, sodium dihydrogen phosphate, dicalcium phosphate dihydrate, dextrates (NF hydrate) and cholic acid or a combination thereof.
The use of the pharmaceutical composition according to claim 13, wherein the statins are selected from the group consisting of atorvastatin, pravastatin, simvastatin, lovastatin, mevastatin, compactin, fluvastatin, cerivastatin, rosuvastatin and pitavastatin.
The use of the pharmaceutical composition according to claim 13, wherein the modified-release formulation further features an extended release effect.