KR20100079539A - Pegylated conjugated linoleic acid having antiobesity acitivity - Google Patents

Abstract

PURPOSE: A PEGylated conjugated linoeic acid(CLA) is provided to enhance solubility of CLA an d to improve biocompatibility. CONSTITUTION: A pharmaceutical composition having high anti-obesity activities contains phegylated linoleic acid(PCLA). The pharmaceutical composition further contains carrier, excipient, and diluent. A food composition contains linoleic acid as an active ingredient. The composition is used in the form of tablet, capsule, granule, and pill.

Description

Pegylated conjugated linoleic acid having antiobesity acitivity

The present invention relates to pegylated conjugated linoleic acid (PCLA) with anti-obesity activity, and more particularly to novel pharmaceutical uses for the anti-obesity activity of pegylated conjugated linoleic acid.

Obesity is a major risk factor for a variety of diseases, including hypertension, hyperlipidemia and heart disease. Type 2 diabetes is also characterized by impaired glucose and lipid metabolism and is associated with obesity. It is generally believed that excessive consumption of dietary fats promotes obesity in animal models as well as humans. Increasing evidence suggests that some adipose tissue-derived molecules are involved in the pathophysiology of obesity-related insulin resistance and atherosclerosis. For this reason, many scientists stress the importance of better understanding these epidemics for the purpose of developing effective therapies.

Unfortunately, however, few treatments are available for the treatment of obesity. It has been shown that trans-10, cis-12 conjugated linoleic acid (CLA) or a mixture of trans-10, cis-12 and cis-19, trans-11 isomers reduced body fat in vitro and in vivo. CLA has become an experimental model, either in free form or as esterified in triglycerides, but can be improved in chemical or physical modifications of CLA to improve potency to enhance water solubility and increase clinical use.

It has been found that poly (ethylene glycol) (PEG) increases the water solubility of liposomes and reduces secondary aggregation. In addition, the Food and Drug Administration has approved the use of PEG for human vein, oral and skin. In fact, the benefits of PEGylation include improved in vivo circulation time, reduced antigenicity and immunogenicity, improved bioavailability and reduced toxicity. PEG is also ideal for pharmaceutical use because it can be removed through a combination of renal and hepatic pathways.

In general, the C57BL / 6J (ob / ob) model has been used for the investigation of human obesity and metabolic syndrome. When the low fat diet was increased in C57BL / 6J (ob / ob) mice, the mice exhibited normal blood glucose at low and normal insulin levels and blood pressure (euglycemic). However, high fat (HF) dietary supply increased central hyperlipidemia, hyperinsulinemia, hyperglycemia and hypertension. These syndromes are thought to be associated with abnormalities in adrenergic regulation of adipocyte function, which in turn is associated with hyperinsulinemia. In addition, the development of insulin resistance, hyperglycemia and obesity in C57BL / 6J (ob / ob) mice is closely parallel with the progression of common forms of human disease.

The inventors have reported that PEGylated CLA (PCLA) has increased water solubility and promoted bioavailability with respect to the free fatty acid (FFA) form due to the biocompatible and hydrophilic nature of the conventional PEG. Based on our previous in vitro observations, we investigated whether PCLA induces or inhibits metabolic pathways related to lipid metabolism as efficiently as CLA in fat C57BL / 6J (ob / ob) mice fed HF diets. .

It is therefore an object of the present invention to provide novel anti-obesity active uses of PEGylated CLA (PCLA).

The above object of the present invention was achieved by identifying HF diet containing PEGylated CLA (PCLA) in C57BL / 6J (ob / ob) mice by inhibiting insulin resistance in metabolic pathways associated with lipid metabolism.

The present invention provides an anti-obesity active pharmaceutical composition containing PEGylated conjugated linoleic acid (PCLA) as an active ingredient.

The present invention also provides an anti-obesity active functional food composition containing pegylated conjugated linoleic acid (PCLA) as an active ingredient.

In the present invention, the term "active ingredient" refers to a substance or a group of substances (a medicinal agent whose pharmacologically active ingredient is not known, etc.) which is expected to express the efficacy or effect of the drug directly or indirectly by inherent pharmacological action. It means containing a main component).

Pharmaceutical compositions comprising the PCLA of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

As defined herein, "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functional" means It means ingestion for the purpose of obtaining useful effects on health use such as nutrient control or physiological action on structure and function.

In addition, it is possible to manufacture and process as a dietary supplement in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of anti-obesity activity of the present invention.

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The present inventors have reported that PEGylation of CLA increases the water solubility and bioavailability of CLA due to the biocompatibility and hydrophilic properties of PEG. In order to further study these in vito observations, the present invention is intended to test the hypothesis that PCLA acts as a mediator that can induce or inhibit certain metabolic pathways in HF diet-induced diabetic animals.

The present invention investigated the binding effect of dietary PCLA on induced pre-diabetic status and obesity characterized by insulin resistance in HF-fed C57BL / 6J (ob / ob) mice. Although many other researchers have reported lipoatrophia and hepatic steatosis in mice fed a CLA rich normal diet, in the present invention the CLA or PCLA rich HF diet is equally ob / ob despite the decrease in AF. It has been found that mice are protected from hepatic steatosis, which corresponds to conventional findings.

The results of the present invention showed that the final body weight and weight gain in HF-fed mice were significantly greater than in mice fed a normal diet. Previous studies support the fact that HF diets increase body weight and increase insulin resistance. Feeding HF-CLA or HF-PCA prevented the increase in body weight resulting from PEG addition or free HF diet. Due to similar feed uptake in HF (with or without PEG) -feeding and HF-CLA (or HF-PCLA) -feeding mice, respectively, throughout the entire test, fat loss in feces was digested and / or transported in CLA-treated mice. It could be expected through speed change. There was no apparent stool fat loss, suggesting that the absence of weight gain due to CLA promoted the use of fat in response to physical energy demands.

Plasma insulin levels and HOMA index were significantly lower in mice fed the HF-CLA diet than in mice fed the HF-diet alone, indicating that CLA was used to treat obesity in HF-fed C57BL / 6J (ob / ob) mice. It indicates that you are a good candidate. However, CLA used at 1.5% in conventional diets did not show any particular effect on many organs of Fisher rats, whereas AKR / J mice fed CLA showed insulin resistance due to a paradoxically increased circulating insulin level. Therefore, CLA treatment resulted in hepatic fat accumulation in AKR / J mice as well as normal C57BL / 6J mice. All these data suggest that the biological effects of CLA depend on the animal species and mouse strains used. Interestingly, Purusham et al. Demonstrated that insulin resistance induced by male C57BL / 6J mice was inhibited by leptin, which differed in the ability of CLA to increase hyperinsulinemia and hepatic steatosis in the presence of leptin. It has been shown to suggest the fact that it may depend on the basal level of factors such as leptin and adiponectin.

Therefore, further studies are needed to clarify the dietary effects of CLA or PCLA on HF-diet-induced obesity in various varieties in the absence of leptin.

In general, CLA is insoluble in FFA. Therefore, when provided as a dietary supplement, the CLAs must be physically or chemically modified to enhance their water solubility and be allowed for clinical use. In contrast, pegylated forms enhance the stability of CLA at pH, light and temperature compared to the free form. Indeed, we have previously reported that PEGylation of all transretinic acids increases the stability of RA under light exposure due to the formation of core shell-type polymer nanoparticles by PEGylation. PEG binding has also been shown to be a useful tool for increasing the water solubility of CLA.

The HF-CLA diet significantly reduced the cadaveric lipid content compared to the HF- diet. Park et al. Reported that CLA caused redistribution of body composition, which reduced body fat and increased lean body tissue, which supports this finding. TC plasma levels are also a major determinant of human vascular disease risk with or without prognostic signs of vascular disease. LDLC, which is usually the major atherogenic lipoprotein, constitutes up to 70% TC and contains a single apolipoprotein.

The present invention shows that lower TC levels correspond to lower LDLC in the HF-CLA diet group. In spite of the basal HF-diet, increasing FAS activity in abdominal disability of CAP-fed rats may suggest that it involves an increase in lipid synthesis, but the present invention also clearly reduced GPDH activity, which supports the major stage of fat formation. This was observed. In conclusion, fatty acid synthesis pathways may be weakened in adipose tissue with the discovery that CLA promotes lipodystrophy by increasing apoptosis.

Accumulation of lipids rich in unsaturated fatty acids in liver cells provides a sufficient base for lipid peroxidation in common oxidative stress conditions. Lipid peroxidation can start from electron transfer-mediated responses in peroxysomes, endoplasmic reticulum and more specifically in respiratory chain activity. MDA is a product of lipid peroxidation and is an indicator of cell oxidative stress. While CLA treatment significantly reduced MDA levels in liver homogenates, it was observed that they did not in the corresponding mitochondria, suggesting that mitochondrial antioxidant activity substantially confers their effects on the mitochondrial outer region.

This effect may result from a decrease in electron transfer by the respiratory chain and / or an increase in functional activity to weaken free radical levels. The former is not believed to be because the HF-CLA group is not significantly affected by more dietary fat through enhanced fatty acid oxidation in addition to stool loss. In contrast, the latter possibility was supported by an increase in GSH of liver homogenates through increased mitochondrial GPX activity. Indeed, GPX activity is believed to protect cells from free radicals and enhance the activity of respiratory facts.

PPARγ and C / EBPa are transcription factors that promote adipogenesis in NIH 3T3 fibroblasts and are involved in early stage adipocyte differentiation. Lipid accumulation and PPARγ expression increased simultaneously with HF feeding, which has been reported to meet the enhanced PPARγ and C / EBPa expression found for AF in HF fed-mouse. In contrast, according to other studies, the results of the present invention showed that CLA treatment inhibited PPARγ and C / EBPa expression, which could lead to a decrease in adipose tissue. Previous studies of the present inventors have reported on 3T3-L1 cells in which CLA caused adipocyte re-differentiation by downregulation of PPARγ2-induced adipogenesis, and CLA alleviates insulin resistance through a decrease in fat weight. It can correspond to in vivo experiments showing that it gives beneficial effects, including. Similarly, feeding CLA or PCLA decreased PPARγ and C / EBPa expression in AF in liver and adipose tissue of C57BL / 6J (ob / ob) mice, respectively, inhibiting TG accumulation.

According to the present invention, PEGylation of CLA increased the water solubility of CLA and thus improved its biocompatibility, and the main properties of CLA, especially those associated with the reduction of fat accumulation by inhibiting lipogen activity, are maintained in PEG-CLA. And, despite the HF-diet, there is a potent effect of strongly relieving insulin resistance in natural obese (ob / ob) mice.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to these examples.

Example

PEG (MW 2,000) was supplied by Aldrich (Milwukee, WI, USA). CLA mixtures (trans-10, cis-12, cis-9, trans-11 and other CLA isomers, 40/40/20 weight ratios respectively) were supplied by Lipogen (Gyeonggi, South Korea). The CLA isomer of the mixture was coupled to PEG using our conventional PEGylation method. PCLA was dissolved in d-chloroform and the composition of CLA in PCLA was calculated to be 65 mol% using 1 H NMR (Bruker, Avance 500).

All data are presented in comparison to the control (HF-fed animals) and expressed as mean (n = 10) ± S.D. All data were analyzed using the one-way analysis of variance (ANOVA) followed by the alleles t test for multiple comparisons. Western blot data were analyzed using the Student's t test. Statistical significance was indicated by P values below 0.05 and P values below 0.01.

Example 1 Evaluation of Oxidative Stability of PCLA

In this example, it was evaluated whether PCLA increased oxidative stability compared to CLA itself. As shown in FIG. 1, the decrease in the upper oxygen level by CLA occurred faster than in PCLA. In contrast, PCLA increased oxidative stability compared to CLA. In addition, binding to PEG also increased thermal stability. This suggests that the binding of CLA and PEG is a way to enhance oxidative stability.

Example 2 Experimental Design

The protocol of the present invention is accredited by Seoul National University's Animal Care and Use Committee (ACUC). Five-week-old male C57BL / 6J (ob / ob) mice purchased from Central Lab. Animal were exposed to temperature (20.22 ° C), humidity (55.60%) and illumination (9: 0021: 00 h). Breeding in a controlled room. The mice were fed a commercial AIN-93G diet (HanSam R & D, Gyeonggi, South Korea) over 7 days. After this purification process, the mice were randomly divided into five groups (n = 10), and over 6 weeks each had a normal diet (control), an HF diet according to initial composition, or an HF diet containing 1% PEG (HF-PEG), CLA Containing HF diet (HF-CLA), PCLA containing HF diet (HF-PCLA). Pay intake and body weight were recorded every three days.

Example 3 Blood Analysis

All mice were killed and blood was collected by orbital venipuncture. Blood was coagulated and centrifuged at 3000 rpm for 15 minutes and serum was stored at -20 ° C until analysis. Triglyceride (TG), total cholesterol (TC) and high density lipoprotein (HLD) cholesterol (HDLC) were enzymatically analyzed using a commercial kit (Asan Pharmaceutical, Seoul, South Korea). Low density lipoprotein (LDL) cholester (LDLC) was calculated from the TG, TC and HDLC concentrations using the Friedwald equation LDLC = TC-[(HDLC-TG) / 5]. HOMA parameters were used to estimate insulin resistance index from plasma insulin and glucose:

HOMA = plasma insulin (μU / ml) × plasma glucose (mM) /22.5; Here, the larger the HOMA value, the greater the level of insulin resistance.

Example 4: Tissue Analysis and Activity

Example 4-1: Liver Antioxidant Defense

Immediately after killing mice, mitochondrial fractions were prepared from the liver according to conventional methods. Diagnostic EIA kits (Bioxytech GPX-340, Bioxytech GR-340 and Bioxytech) obtained with glutathione (GSH), peroxidase (GPX), GSH reductase and GSH content from Oxy Research (Portland, OR, USA) Using GSH-400, respectively). The activity of superoxide dismutase (SOD) in liver homogenate and the activity of manganese SOD in mitochondrial fractions were measured using a Bioxytech SOD-525 kit. Malondialdehyde (MDA) was measured according to the method of Okhawa et al.

Example 4-2 Lipid Tissue Activity

Fatty acid synthase (FAS) and glycerol-3-phosphate dehydrogenase (GPDH) were measured with a commercial kit (Takara, Kyoto, Japan) using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA) It was. Enzyme activity is expressed in units of 1 nmol of oxidized NADH per minute. Protein concentration was measured by the Bradford method.

Example 4-3 Body Composition

Cryoextracted carcasses were homogenized in distilled water using Polytron (PRO300D PRO; Scientific). The homogeneous was dried to rated weight in an oven at 70 ° C., then ground and mixed thoroughly using a mortar and pestle. The final product was analyzed three times for lipid, protein and ash content. Lipids were analyzed by Soxhlet system HT 1043 extraction unit using 2: 1 chloroform-methane, proteins were analyzed by Perkin-Elmer Series II nitrogen analyzer 410, and ash was analyzed by CEM MAS 7000 microwave muffle furnace. The water content of the carcass was calculated by subtracting the dry carcass weight from the original weight of the viscera carcasses.

Example 4-4 Protein Expression

For Western blot analysis, 500 μg of AF sample was collected in elution buffer (20 mM Tris, 145 mM NaCl, 10% glycerol, 5 mM EDTA, 1% Triton-X, 0.5% Nonidet P-40, 100 μM phenylmethylsulfonyl fluoride). , 50 μM NaF, 1 mM sodium orthovanadate) in 1 mL. The eluate was centrifuged at 8850 g at 4 ° C. for 10 minutes. The upper liquid phase was removed from the lipid tissue sample and they were recentrifuged under the same conditions. Supernatants were collected and protein concentrations were measured by the Bradford method using bovine serum albumin as standard. 15 μg of protein was loaded in each lane. After sodium dodecyl sulphate polyacrylamide gel electrophoresis, the protein was transferred to a poly (vinylidene difluoride) (PVDF) membrane (Amersham Pharmacia Biotech., Buckinghamshire) using a Kem-En-Tec semidry blotter. , England). Proteins were transferred to PVDF as same while culturing the proteins to prevent freeze-thaw degradation of phosphoproteins. After transfer, the membrane was blocked overnight in TBS containing 5% degreasing dry milk and 0.1% Tween 20 (Biosesang, Seoul, Korea). Incubated with primary and secondary antibodies for 2 hours in TBS containing 5% nonfat dry milk and 0.1% Tween 20 (Biosesang, Seoul, Korea). After incubation with the antibody, the membrane was washed with TBS containing 0.1% Tween 20. The primary antibody used (2000: 1 primary antibody-TBS-T) was mouse monoclonal against PPARγ isoforms and mouse CCAAT / enhancer-binding protein alpha (C / EBPα). It was a mouse monoclonal peroxysome proliferator-activated receptor gamma (PPARγ) antibody that recognizes both antibodies. The secondary antibody used (3000: 1 secondary antibody-TBS-T) was a horseradish peroxidase-conjugated antimouse antibody (Santa Cruz Biotech, San Francisco, CA, USA). Enhanced chemiluminescence (ECL) (Intron Biotech., Seoul, South Korea) was used for detection. The signal intensity on PVDF after Western blot using various antibodies was measured by FluorChem densitomer equipped with Alpha-Ease-FC image processing and analysis software (Alpha Innotech). It measured using. After normalization with GAPDH protein, the density values for the desired protein zones are expressed as% of control. All figures showing quantitative fragmentation include data from five or more independent experiments.

Example 5 Evaluation of the Effect of Liquid PCLA Supplementation in Feeding on CLA Concentration

After oral administration of CLA and PCLA to SD male rats, respectively, the effect of PLCA supplementation on CLA concentration of visceral adipose tissue was evaluated as% of total fatty acids.

In addition, after oral administration of CLA and PCLA to SD male rats, the effect of PLCA supplementation on the concentration of CLA in plasma over time was evaluated as% of total fatty acids.

The following results were obtained through Examples 1 to 5 as described above.

1. Experimental design

When using a diet containing 0.252.0% CLA, body fat content and fat tissue weight in various experimental animals decreased dose-dependently. In addition, more than 0.5% CLA was required to significantly reduce body fat content, and in most studies, a 1% CLA diet was used to study the physiological activity of CLA. For this reason, the present invention used 1% CLA in the HF diet (see Table 1).

TABLE 1

Experimental Dietary Composition ¹

As also indicated above, the CLA in the PCLA mixture was estimated to be 65 mol% by 1 H NMR. All results obtained with or without PEG were used to limit this repetition to the overall available observations. In the tables and figures, HF represents PEG addition or no addition, and the same in HF-CLA, but such systematic repetition is omitted in the detailed description of the invention.

2. Weight and Dietary Intake

After 6 weeks, HF-fed mice were nearly 8.9g heavier than HF-CLA-fed mice (see Table 2). In contrast, the final body weight of the HF-CLA diet group was significantly decreased compared to the HF diet group.

TABLE 2

Body weight and feed in mice fed the experimental diet for 6 weeks

3. long-term weight

The HF diet did not affect the spleen, kidney or lung weight compared to the normal and HF-CLA diets (see Table 3). However, abdominal fat (AF) weight was significantly heavier in the HF group than in the other groups. In contrast, liver and AF weights of HF-CLA fed mice were significantly lighter than those of the normal and HF diet fed groups. In addition, Fig. 4 shows the histologically observed AF of PCLA-weighted mice.

TABLE 3

Long-term weight in mice fed the experimental diet for 6 weeks

4. Carcass Composition

The cadaveric lipid of HF-CLA-fed animals averaged 31.57 g compared to 25.32 g in HF-CLA-fed mice (see Table 4). The carcass protein, moisture and ash content of the HF-CLA diet group were hardly increased as compared to the HF diet group. In contrast, there was little difference in moisture and ash content between the HF-CLA and normal diet groups.

TABLE 4

Carcass composition in mice fed the experimental diet for 6 weeks

5. Determination of Plasma Insulin or Cholesterol

TC levels were significantly increased in mice fed the HF diet compared to mice fed the other diet, but plasma HDLC levels did not differ between the diet groups (see Table 5). In addition, the HF diet increased the plasma LDLC level by 2.2 times compared to the HF-CLA diet. In contrast, LDLC levels were significantly decreased in the HF-CLA diet group compared to the normal diet or the HF diet group. In addition, HF-CLA fed mice had lower TC concentrations when compared to normal diet or mice fed the HF diet. Plasma insulin levels of the HF diet group increased significantly, while HOMA levels tended to decrease in the HF-CLA-fed group, significantly different from the levels observed in HF-fed animals.

TABLE 5

Cholesterol Levels and HOMA Analysis of Mouse Plasma Feeding Experimental Diets for 6 Weeks

6. GPDH and FAS Activity Using HF Diet

FAS activity was significantly lower in the HF-fed group than in the HF-CLA-fed group (see Table 6). However, using HF-CLA, FAS activity was higher and GPDH activity was lower than that of normal or HF diet.

TABLE 6

GPDH and FAS Activities in Mouse Lipid Tissues Fed Experimental Diet for 6 Weeks

7. Liver MDA and Antioxidant Defense Levels

Compared to HF-fed animals, MDA levels decreased in liver homogenates from mice fed the HF-CLA diet (see FIG. 2). Moreover, HF-CLA-fed mice increased GSH levels per g liver, but did not increase GSH levels per mg mitochondrial protein. However, there was no significant difference in SOD activity in liver homogenates or mitochondrial fractions between diets. In contrast, GPX was higher in the mitochondrial fraction of HF-CLA-fed mice than in HF-fed mice, but GPX activity in liver homogenates was not different between dietary groups. Interestingly, GPX activity in mitochondrial fractions in HF-CLA-fed animals was significantly higher than in normal or HF-fed diets.

8. Adipocyte Marker Protein Expression

Adipocyte marker protein expression (see FIG. 3) was lower in HF-CLA-fed mice than in HF-fed mice.

9. Effect of PCLA Oral Administration on Growth Behavior

The effect of oral administration of PCLA on the growth behavior of experimental animals was evaluated and the results are shown in Table 7.

<Table 7>

Effect of Oral PCLA Administration on Growth Behavior

ab is the mean in the same row with significantly different superscripts (P, 0.05).

1) Number of animals in treatment group = 5

2) Number of animals in treatment group = 10

3) SEM: Standard Deviation

10. Effect of Liquid PCLA Supplementation on Feeding on CLA Concentration

After oral administration of CLA and PCLA to SD male rats, the effects of PLCA supplementation on CLA concentrations of visceral adipose tissue and the effects of PLCA supplementation on plasma CLA concentrations over time were evaluated. 6.

1 is a diagram showing the results of comparing the oxidation stability of PCLA and CLA.

2 is a diagram showing the effect of PCLA diet on MDA and antioxidant defense levels in liver homogenates and mitochondrial fractions.

Figure 3 shows the effect of PCLA diet on adipocyte marker protein expression.

4 is a diagram showing the results of histochemical observation of AF of PCLA-weighted mice.

5 is a diagram showing the effect of PLCA supplementation on CLA concentration of visceral adipose tissue in SD male rats.

Figure 6 shows the effect of PLCA supplementation on the concentration of CLA in plasma over time in SD male rats.

Claims (2)

An anti-obesity active pharmaceutical composition containing PEGylated conjugated linoleic acid (PCLA) as an active ingredient. An anti-obesity active functional food composition containing pegylated conjugated linoleic acid (PCLA) as an active ingredient.

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