PAEONIFLORIN PREPARATIONS AND USES THEREOF FOR FAT REDUCTION

Abstract

Disclosed are methods and preparations useful for reducing fat at a targeted area(s) on a human. The preparations comprise as an active ingredient an adipolysis enhancing (i.e., fat-melting) amount of an active ingredient, paeoniflorin (PF). The preparations may be provided as an injectable preparation or as a topically applied preparation, such as in the form of a crème or lotion. In topical preparations, the active ingredient paeoniflorin may be contained within nanospheres, such as albumin nanospheres. The PF-containing preparations may also include a permeant, such as azone. The method may be accompanied by the application of ultrasound to the area being treated prior to, during or after, or prior to, during, and after application of the paeoniflorin preparation to an area of the body in which fat reduction is desired. By way of example, the methods and preparations are effective for reducing targeted fat deposits at various anatomical sites of the body, such as the midsection (“love handles”), jowls, hips, arms, thighs and buttocks area.

Claims

1. A method for enhancing fat loss in a human comprising: administering an adipolysis enhancing paeoniflorin preparation comprising paeoniflorinin and a liquid carrier to a human; enhancing fat loss in the human, wherein fat loss in the human provided the paeoniflorin preparation is enhanced compared to fat loss in a human not provided the paeoniflorin preparation.

2. The method of claim 1 wherein the paeoniflorin preparation is not an injectable preparation.

3. The method of claim 1 wherein the paeoniflorin preparation comprises a concentration of paeoniflorin of about 0.02 mg/0.5 ml to about 0.25 mg/0.5 ml.

4. The method of claim 1 wherein the paeoniflorin preparation comprises a concentration of paeoniflorin of about 0.25 mg/0.5 ml.

5. The method of claim 1 wherein the paeoniflorin preparation comprises a concentration of paeoniflorin of about 0.2 mg/ml to about 0.3 mg/ml.

6. The method of claim 1 wherein the paeoniflorin preparation comprises a concentration of paeoniflorin of about 0.5 mg/ml.

7. An adipolysis enhancing and subcutaneous fat reducing preparation comprising an adipolysis enhancing amount of paeoniflorin in a suitable carrier, wherein the adipolysis enhancing amount of paeoniflorin in the preparation is a concentration of paeoniflorin of about 0.02 mg/0.5 ml to about 0.25 mg/0.5 ml.

8. The adipolysis enhancing and subcutaneous fat reducing preparation of claim 7 wherein the carrier is water.

9. The adipolysis enhancing and subcutaneous fat reducing preparation of claim 7 further comprising a permeant.

10. The adipolysis enhancing and subcutaneous fat reducing preparation of claim 7 comprising a concentration of paeoniflorin of about 0.25 mg/0.5 ml.

11. The adipolysis enhancing and subcutaneous fat reducing preparation of claim 7, said preparation containing a concentration of paeoniflorin that increases cyclic AMP levels in adipocytes compared to cyclic AMP levels in adipocytes in the absence of the concentration of paeoniflorin.

12. The adipolysis enhancing and subcutaneous fat reducing preparation of claim 7 comprising a concentration of paeoniflorin of about 0.2 mg/ml to about 0.3 mg/ml.

13. The adipolysis enhancing and subcutaneous fat reducing preparation of claim 7 comprising a liquid preparation.

14. The adipolysis enhancing and subcutaneous fat reducing preparation of claim 7 comprising a concentration of paeoniflorin of about 1 umol/L to about 5 umol/L.

15. An adipolysis enhancing and subcutaneous fat reducing preparation comprising an adipolysis enhancing amount of paeoniflorin in a suitable carrier, wherein the adipolysis enhancing amount of paeoniflorin in the preparation is about 11 moles/Liter.

16. The adipolysis enhancing and subcutaneous fat reducing preparation of claim 15 comprising a liquid preparation.

17. The adipolysis enhancing and subcutaneous fat reducing preparation of claim 16 wherein the suitable carrier is water.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0046] FIG. 1 demonstrates PF increased the release of glycerol by 3T3-L1 adipocytes significantly by the 7.sup.th day after exposure.

[0047] FIG. 2 demonstrates intracellular cAMP tested by ELISA after incubation with PF in 3T3-L1 adipocytes.

[0048] FIG. 3A-3B demonstrate expression of HSL in 3T3-L1 adipocytes after incubation with PF. FIG. 3B, PF is shown to increase the expression of HSL in 3T3-L1 from the 3.sup.rd day of exposure, which follows the increase of cyclic AMP and gives rise to release in glycerol as a byproduct of fat metabolism.

[0049] FIG. 4 demonstrates pathway of adipolysis.

[0050] FIG. 5 demonstrates Expression of AR in 3T3-L1 adipocytes exposed to PF.

[0051] FIG. 6A-6B demonstrate mechanisms of lipolysis stimulation by 13-ARs. 6A=Basal State, 6B=Activated State.

[0052] FIG. 7 demonstrates an expression of Cpe in 3T3-L1 adipocytes exposed to PF.

[0053] FIG. 8 demonstrates an expression of Adiporl in 3T3-L1 adipocytes exposed to PF.

[0054] FIG. 9 demonstrates an expression of Pparg in 3T3-L1 adipocytes exposed to PF.

[0055] FIG. 10 demonstrates the clinical effect: Comparison of PF. Serum cholesterol, LDL, Cholesterol, serum triglycerides in blood of eight patients was reduced after PF treatment.

[0056] FIG. 11 demonstrates a needle at 90° angle to the long axis of the syringe.

[0057] FIG. 12 demonstrates glycerol release after incubation for 3 days with Mesotheraphy (M) and PF.

[0058] FIG. 13 demonstrates glycerol release after incubation for 7 days with Meso (M) and PF. PF increased the release of glycerol significantly by the 7.sup.th day after stimulation (p<0.01).

[0059] FIG. 14 illustrates intracellular cAMP levels measured by ELISA after incubation with Meso (M) and PF for 72 hours. In the PF group, intracellular cyclic AMP was significantly increased by PF in the first day of stimulation (p<0.01). In the M group, the level of cAMP did not show much difference compared to the control group.

[0060] FIG. 15 present results of ELISA demonstrating the intracellular cAMP after incubation of fat cells (adipocytes) with Meso (M) and FF for 24 hours. In the PF group, intracellular cyclic AMP was significantly increased by PF in the first day of stimulation (p<0.01). In the Meso group (M), the level of cAMP did not show much difference compared to the control group.

[0061] FIG. 16 presents the expression of HSL in 3T3-L1 adipocytes after incubation with Meso (M) and PF. PF increased the expression of HSL in 3T3-L1 adipocytes from the third day after stimulation.

DETAILED DESCRIPTION

[0062] The present invention, in a general and overall sense, provides a variety of preparations and methods for enhancing lipolysis and fat reduction and/or loss in vivo in a patient animal. The preparations and/or compositions include as an active ingredient PF. PF is a natural, purified bioactive glucoside in Paeoniac Radix (PR), the roots of Paeonia Pall. PF, a natural plant extract purified to over 99%, is provided here as a natural organic potent lipolysis drug. Its mode of action is through significantly enhancing the expression of several obesity-related genes such as Adrb2, Adrb3, Cpe, Adiporl and Pparg.

[0063] PF enhances the expression of β-adrenergic receptors 2 & 3. β-adrenergic receptor 2 is a major biolytic receptor in human fat cells. (β-adrenergic receptor 3 is important in regulating thermogenesis and lipolysis in brown adipose tissue through autonomic nervous system (ANS) activity. Its biologic intracellular pathway showed that activation of β-adrenergic receptors goes through the cAMP pathway which in turn significantly enhances the level of expression of Hormone-Sensitive Lipase (HSL), to break down tri-glycerides into glycerol and fatty acids.

[0064] According to the present compositions/methods, PF significantly enhances fat loss by enhancing a fat cell's ability to burn fat into its natural by-products. PF can be administered in any of a variety of ways that accomplishes contact of the preparation with fat cells or a tissue comprising at least some fat cells. By way of example, and not limitation, the composition may be provided to an animal, such as a human, by subcutaneous injections, or by applying the preparation as a crème and/or lotion. As a crème and/or lotion, the PF may be driven through the skin to the fat cells. This may be accomplished, for example, by using a weak electric current similar to that used by a physiotherapist, or by mixing PF with albumin nano spheres and driving the mixture through the skin to the fat cells using ultrasound.

[0065] To test the effectiveness of PF on targeted fat loss, a 3T3-L1 cell line was used. The 3T3-L1 cell line provides an art-accepted model for fat loss. Fat loss is identified utilizing an in vitro adipolysis (digestion of fats) measure in a Swiss 3T3 mouse cell line. 3T3-L1 cells propagated under normal conditions have a fibroblastic phenotype. However, when treated with a combination of dexamethasone, isobutylmethylxanthine (IBMX) and insulin, 3T3-L1 cells adopt a rounded phenotype and accumulate lipids intracellularly in the form of lipid droplets.

[0066] As detailed below, PF was found to significantly increase the number of β-adrenergic receptors (β 1, 2 and 3 subtypes) responsible for the break down of fat into its natural byproducts. The exposure of 3T3-L1 fat cells to PF causes a significant increase in the level of expression of intracellular cyclic adenosine monophosphate (cAMP) on days 1 & 3 (P<0.01). This is followed by a significant increase in the level of expression of Hormone-Sensitive Lipase (HSL). HSL is a multifactorial tissue lipase that plays a critical role in fat metabolism. Such a significant increase in HSL expression is followed by a significant increase in glycerol release on day 7 of exposure to PF in vitro. In vivo, lowered levels of plasma triglycerides provide a clinical indicator of fat breakdown in a patient.

[0067] The present preparations are demonstrated to possess clinical effectiveness in patients, and to provide effective site-specific lipolysis (fat breakdown) such as that stored in the midsection region (“love handles”), stomach, jowls, hips and thighs. Blood analysis and a comparison of before and after treatment with PF revealed that blood sugar levels decreased upon treatment, and low-density-lipoprotein (LDL) levels showed a tendency towards reduction, compared to pretreatment blood sugar level measures and LDL levels.

[0068] Accordingly, the present preparations, formulations, methods and techniques provide among other things, the following advantages, characteristics and features:

[0069] 1. A purified, natural and effective product (PF) that dissolves fat stored subcutaneously in the adipose tissue upon direct contact.

[0070] 2. A predictable mode of PF's action. PF activates the expression of β adrenergic receptors as well as other fat metabolism genes.

[0071] 3. A fat-reducing product that is provided in a suitable carrier to deliver PF across the skin to the fat layers.

[0072] 4. A method that employs electric current and a charged carrier to effectively deliver the preparation with the active agent, PF, to the fat cells and into the fat layers in target areas.

[0073] 5. A method that delivers PF to fat cells through the use of albumin nanospheres driven through the skin by ultrasound.

[0074] In those embodiments where the preparation is a preparation suitable for injection, the preparation may be described as comprising a physiologically compatible carrier solution, such as saline and/or sterile water. In other embodiments, the injectable preparation will further include phosphatidylcholine, or any other of a variety of similar phospholipids and combinations of phospholipids.

Example 1: PF Promotes Adipolysis

[0075] The present example demonstrates the utility of the present formulations and/or preparations for promoting adipolysis, or fat break down.

[0076] The 3T3-L1 cell line is an accepted model by those of skill in the art for natural fat loss. This cell line is a substrain of Swiss 3T3 mouse cell line 3T3-L1. This cell line propagated under normal conditions has a fibroblastic phenotype. However, when treated with a combination of dexamethasone, isobutylmethylxanthine (a non-specific inhibitor of phosphodiesterases) (IBMX) and insulin, 3T3 cells adopt a rounded phenotype and accumulate lipids intracellularly in the form of fat droplets.

[0077] Differentiated 3T3-L1 adipocytes were cultured in media containing 1 umol/L PF as test group, and media without PF as control. As a result of lipolysis activity, glycerol will be generated as a result of triglyceride breakdown and released into the extracellular space. Glycerol content from 3T3-L1 adipocyte cell culture media may therefore provide an indicator of adipolysis. It was found that PF increased the release of glycerol significantly by the 7.sup.th day after exposure (p>0.01) (FIG. 1), compared to glycerol release from 3T3-L1 adipocytes in the absence of the same amount of PF.

[0078] It was found that PF increased adipolysis within one week after stimulation was initiated. The PF preparations are thus demonstrated to induce adipolysis indirectly by causing changes in gene expression that lead to triglyceride breakdown. Further studies were carried out to define the mechanism of action of PF.

Example 2: PF Increased cAMP

[0079] The present example, among other things, demonstrates the ability of the invention to provide effective fat loss through PF action on cyclic AMP levels. 3T3-L1 adipocytes were cultured in medium containing 11 mol/L PF as test group and medium only as control. Cells were lysed by adding 0.1 N HCL, and intracellular cyclic AMP was measured by ELISA. It was found that intracellular cyclic AMP was significantly increased by PF in the first day of exposure (P<0.001), and the third day as well (P<0.01) (FIG. 2).

[0080] The major pathway leading to lipolysis involves activation of cAMP-dependent Protein kinase A (PKA), which in turn activates other substrates such as HSL and perilipin. The agonists of β-adrenergic receptor bind to the F3-adrenergic receptor, which activates the G-protein, Gs. The activation of Gs stimulates adenylate cyclase (AC) to produce cyclic AMP. Protein kinase A (PKA) is activated by cAMP to phosphorylate the lipid droplet surface protein, perilipin (PL). Hormone-sensitive lipase (HSL) docks onto phosphorylated PL and breaks down triglyceride into glycerol and free fatty acid. Glycerol is released into the extracellular space through aquaporin adipose (AQPad). The release of glycerol was found to be increased significantly by the 7th day after PF stimulation (P<0.001), and that intracellular cyclic AMP was significantly increased by PF in the first day of stimulation (P<0.001), and the third day as well (P<0.01). The change and time-relationship of cyclic AMP and glycerol release demonstrates that PF induced adipolysis functions through the pathway of “second messenger” (cyclic AMP).

Example 3: Hormone Sensitive Lipase (HSL)

[0081] The present example demonstrates an increase in adipolysis in vivo as demonstrated by an increase in detectable levels of hormone sensitive lipase (HSL).

[0082] 3T3-L1 adipocytes were cultured in medium containing 1 mol/L PF as a test group and medium only as the control group. Total RNA was extracted and reverse transcribed to cDNA employing conventional methods known to those of skill in the art.

[0083] Primers used for polymerase chain reaction (PCR) amplification of mouse

[0084] HSL primers were selected based on published sequence (NM_010719) (expected PCR fragment: 409 bp):

TABLE-US-00001 forward primer,  5′-GCTGGTGCAGAGAGACAC-3′; reverse primer,  5′-GAAAGCAGCGCGCACGCG-3′

[0085] For semi-quantitative analysis, the amplification cycles were chosen within the linear range (HSL: 24 cycles with denaturation at 58° C., GAPDH: 21 cycles with denaturation at 58° C.). It was found that the PF increased the expression of HSL in 3T3-L1 cells from the third day after exposure (FIG. 3).

[0086] Hormone-sensitive lapse (HSL) is a multifunctional tissue lipase that plays a critical role in the process of fat metabolism. The enzyme has broad specificity, catalyzing the hydrolysis of tri-, di-, and monoacylglycerols, as well as cholesterol esters. HSL is thought to catalyze the major rate-limiting step in lipolysis. The lipase is acutely activated by cAMP-dependent phosphorylation, which also leads to its redistribution from the cytoplasm to the lipid droplet. Regulation of adipocyte HSL is the primary means by which lipolytic agents, such as catecholamines, stimulate the release of free fatty acids and thus control circulating levels. In this study, PF was found to increase the expression of HSL in 3T3-L1 from the third day of exposure, which follows the increase of cyclic AMP and gives rise to release of glycerol as a byproduct of fat metabolism (FIG. 4) in vitro.

Example 4: Nanosphere Formulation with PF

[0087] The present example is provided to demonstrate, by way of example only, one of the dermal preparations of PF. In particular, a nanosphere formulation with PF is presented.

[0088] Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) for the topical application to the skin are made of lipids such as glycerol behenate (Compritol® 888 ATO), glycerol palmitostearate (Precirol® ATO 5), or the wax, cetyl palmitate. For NLC at room temperature, liquid lipids such as medium chain triglycerides (Miglylol® 812) are added. Alternatively, oleic acid belonging to the frequently used penetration enhancers in semisolid vehicles applied to the skin, may be added to enhance drug uptake further (Lombard Borgia, Regehly et al. 2005). Mean particle size ranges of the nanospheres was from 50 to 1000 nm. Nano dispersions contained 5 to 40% lipid. The higher concentrated preparations are of semisolid appearance, and are cosmetically acceptable as they are. Depending on the mode and concentration of the lipid, 0.5 to 5% surfactant may be added for physical stabilization of the particles. For dermal use, these are Poloxamer 188, Polysorbate 80, lecitihin, Tyloxapol®, TegoCare® 450 (polyglycerol methylglucose distearate), Miranaol®Ultra C32 (sodium cocoamphoacetate) or saccharose fatty acid ester.

[0089] To facilitate dermal application, fluid dispersions which are obtained when the lipid content is low (<10%) can be incorporated into a crème or gel base which does not induce dissolution or aggregation of SLN particles. Photon correlation spectroscopy and differential scanning calorimetry results have not changed over a storage period of 6 months (Jenning, Thunemann et al. (2000) and Wissing and Muller (2001), copied from Scafer-Korting, Mehnert et al. (2007)).

Example 5: Microarray Analysis of 111 Obesity-Related Genes

[0090] The present example demonstrates that one of the clinical indicators of the fat-burning and obesity fighting activity of the present formulations and/or preparations includes an increase in the expression levels of obesity-related genes.

[0091] In order to explore a more detailed mode of action of PF on adipolysis, microarray analysis was carried out on a panel of obesity-related genes. Expression levels from this gene panel are shown to be increased by PF, compared to expression levels of these genes in the absence of PF.

[0092] 3T3-L1 adipocytes were cultured in medium containing 1 umol/L PF as test group and medium only as control. Total RNA was extracted and Oligo GEArray was tested using Superarray OMM-17. The GEArray includes 111 obesity-related genes that are directly involved in the regulation of energy intake and expenditure. The genes included orexigenic peptides, hormones, and receptors, anorectic peptides, hormones, receptors, and central and peripheral signaling molecules involved in energy expenditure. The increase changes or decrease changes of more than 1.5 fold are regarded meaningful according to the diagnosis discipline, as described in the insert literature of a metabolic disease/obesity gene panel product purchased from Superarray Bioscience Corporation. (Frederick, Md.).

[0093] In the following microarray analysis, the red color shows the expression of obesity-related genes increased by more than 1.5 fold, and the blue color shows the expression of the obesity-related genes decreased by more than 1 5 fold, i.e., the number less than 0 66 shows reduction of more than 1.5 fold. In the graphs, the data above the upper line and the data below the lower line show a significant change of gene expression.

Adrenergic Receptor (AR)

[0094] The adrenergic system plays a major role in the regulation of lipolysis in white adipose tissue, which is the major site of energy storage. Catecholamines are able to stimulate lipolysis by the activation of adipocyte 8-adrenergic receptors (131-, B2-, B3-AR). At the same time, catecholamines can also increase lipid storage through alb-AR. Since B and a2b-AR coexist on the same fat cell, the ratio of functional alb- and B-AR present in adipose tissue may deteimine whether fat storage or release is activated by catecholamines (Soloveva et al., 1997). The present data of microarray analysis after exposure to PF demonstrated a significant increase in the expression of Adrb1, Adrb2 and Adrb3, and no effect on Adra2b (Table 1, FIG. 5). Adrb1 was activated earlier followed by Adbr3 then Adbr2. Adrb2 and Adrb3 were increased by 7.4 fold and 5.65 fold, respectively, at 7 days post exposure to PF. Since the expression of Adra2b almost did not change during the whole study, while there was a significant increase in 13 receptor levels, that the ratio of 13-adrenergic receptors to a2b-adrenergic receptors is shown to be increased by PF. In other words, lipolysis exceeds lipogenesis when exposed to PF.

[0095] Table 1. Ratio of expression signals of AR in 3T3-L1 adipocytes exposed to PF compared to control

TABLE-US-00002 TABLE 1 Expression of AR in 3T3-L1 adipocytes exposed to PF Control PF-3 days PF-6 days PF-7 days β1-adrenergic 1 3.39 0.8 0.58 receptor (Adrbl) β1-adrenergic 1 0.74 1.96 7.40 receptor (Adrb2) β1-adrenergic 1 1.56 3.18 5.65 receptor (Adrb3) β1-adrenergic 1 0.92 1.44 1.27 receptor (Adra2b)

[0096] The three β-adrenergic receptor subtypes β1-, β2-, β-AR) are members of a large family of G protein-coupled receptors, which function through the production of cAMP and the activation of HSL (Soloveva et al., 1997). In the basal state (FIG. 6A), nonphosphorylated HSL is in the cytosol probably bound to some cytosolic acceptors such as lipotransin, and nonphosphorylated perilipin (PL) is tightly bound to the lipid droplet. HSLs do not have free access to the droplet. When catecholamines interact with the β-ARs (FIG. 6B), β-ARs alternatively couple to G-protein, Gs. The activation of Gs stimulates adenylate cyclase (AC) to produce cyclic AMP. Protein kinase A (PKA) is activated by cAMP to phosphorylate the lipid droplet surface protein, perilipin (PL). Hormone-sensitive lipase (HSL) docks onto phosphorylated PL and breaks down triglyceride into glycerol and free fatty acid. Glycerol is released into the extracellular space through aquaporin adipose (AQPad) (FIG. 4).

[0097] The results of β-ARs gene expression and that of the change of cAMP and HSL (shown in part IV) mirror the pathway above.

Carboxypeptidase E (Cpe)

[0098] Carboxypeptidase E (Cpe) is a key enzyme involved in the biosynthesis of peptide hormones and neurotransmitters, including insulin. Cpe plays a vital role in fat metabolism. The mutations in the gene of Cpe result in “fat mutation” (Naggert et al., 1995). “Fat mutation” represents the first demonstration of an obesity-diabetes syndrome elicited by a genetic defect in a prohormone processing pathway (Naggert et al., 1995). The fat mutation mouse does not express Cpe and presents as obese and hyperglycemic. The present data of microarray analysis for PF demonstrated a significant increase in the level of expression of Cpe on day 7 of exposure of 3T3-L1 adipocytes to PF. The expression of Cpe was increased by 2.67 fold compared to the control (Table 2, FIG. 7).

TABLE-US-00003 TABLE 2 Ratio of expression signals of Cpe in 3T3-L21 adipocytes exposed to PF compared to control Control PF-3 days PF-6 days PF-7 days Carboxypeptidase E (Cpe) 1 0.79 1.04 2.67

[0099] Adiponectin has been shown to increase insulin sensitivity and decrease plasma glucose by increasing tissue fat oxidation. AdipoR1 serves as receptor for globular adiponectin and mediates increased AMP-activated protein kinase, glucose uptake and fatty-acid oxidation by adiponectin (Yamauchi et al, 2003). The present data of microarray analysis revealed that the expression of Adiporl was increased by 1.86 fold on day 7 of exposure of 3T3-L 1 adipocytes to PF (Table 3, FIG. 8). Since it is proved that obesity-linked down-regulation of adiponectin is a mechanism whereby obesity could cause insulin resistance and diabetes, adiponectin receptor agonists and adiponectin sensitizers are suggested to serve as versatile treatment strategies for obesity-linked diseases such as diabetes and metabolic syndrome (Kadowaki and Yamauchi, 2005).

TABLE-US-00004 TABLE 3 Ratio of expression signals of Adiporl in 3T3-L1 adipocytes exposed to PF compared to control Control PF-3 days PF-6 days PF-7 days Adiponectin receptor 1 1 0.68 1.21 1.86 (Adiporl)

Peroxisome Proliferators Activated Receptor Gamma (Pparg)

[0100] Pparg encoded PPAR-gamma, is a regulator of adipocyte differentiation. PPAR-gamma has been implicated in the pathology of numerous diseases including obesity, diabetes, atherosclerosis and cancer. In other words, its reduction will inhibit the differentiation of new adipocytes. It was shown by microarray that the level of expression of Pparg was reduced by 1.54 fold on day 7 of exposure of 3T3-L1 adipocytes to PF (Table 4, FIG. 9). In the microarray analysis, the blue color shows gene expression decreased by more than 1 5 fold, i.e. the number less than 0.66 shows reduction of more than 1.5 fold. In the graph, the data below the lower line shows significant reduction of the gene expression.

TABLE-US-00005 TABLE 4 Ratio of expression signals or Pparg in 3T3-L I adipocytes exposed to PF compared to control Control PF-3 days PF-6 days PF-7 days Peroxisome proliferators 1 0.91 1.09 0.64 activated receptor gamma (Pparg)

Example 6

Comparison of the Effect of PF and Meso (M) on Fat Cells

[0101] The present example demonstrates that the effect of PF and mesotheraphy (M) on fat cells is distinct from one another. The present example particularly demonstrates that the mode of action of PF on lipolysis is distinct from the action of mesotheraphy. PF breaks down fat by activating fat cells to express more β-adrenergic receptors responsible for fat metabolism. Breaking down fat by PF is accompanied by an increase in cAMP. The increase in cAMP in turn increases the level of expression of HSL, which breaks down fat and causes a significant increase in glycerol released as a result of breaking down triglycerides into glycerol and fatty acids (See FIG. 12—Glycerol release after incubation for 3 days with Meso (M) and PF). Mesotheraphy, on the other hand showed no effect on most of the measured parameters, except on slightly enhancing the HSL as a result of the presence of aminophyline, which is known to act on β-adrenergic receptors.

[0102] There is evidenced a significant release of hormone sensitive lipase (HSL) when fat cells are exposed to PF. There is not a significant release of HSL upon Mesotherapy. The present application includes data in the attached figures that establishes that the use of PF results in a release of glycerol from fat cells, that a resulting increase in cAMP occurs and that an increase in hormone sensitive lipase (HSL) occurs. These events did not occur with mesotheraphy, and/or did not occur to the extent evidenced with PF, in comparison to control cultures (no drug added) or with mesotheraphy.

[0103] There is evidenced a significant increase in the expression of Adrb1, Adrb2 and Adrb3, and no influence on Adra2b, with exposure to PF. Adrb 1 was activated earlier followed by Adbr3, then Adbr2. Adrb2 and Adrb3 were increased by 7.4 fold and 5.65 fold, respectively, at 7 days post exposure to PF. Since the expression of Adra2b almost did not change, while there was significant increase in the 13 receptor levels, the ratio of 13-adrenergic receptors to a2badrenergic receptors is increased by PF. Lipolysis therefore exceeds lipogenesis upon exposure to PF. The data presented herein compares the effects of PF as contrasted to the effects of mesotheraphy and control treatments on cultures of 3T3 cells. This data demonstrates that activation of the intracellular pathway can only be triggered or induced by the activation of the beta adrenergic receptors, and that activation of the beta adrenergic receptors was only achieved with PF exposure, and there was no activation of beta adrenergic receptors with other treatments.

Example 7

In Vivo Clinical Data

[0104] The present example demonstrates that the present formulations of PF as part of an injectable preparation effectively provide for targeted fat reduction in a human.

[0105] Eight patients were treated with PF. The levels of three indices such as serum cholesterol, LDL Cholesterol, serum triglycerides in blood were tested before and after treatment. Serum cholesterol, LDL Cholesterol, serum triglycerides in blood for the eight patients were reduced after PF treatment (Table 5, FIG. 10). For serum cholesterol, the average level for these cases was higher than the high limit of normal range, and the average level fell into normal range after treatment, which showed significant change statistically (P<0.01). For LDL Cholesterol, serum triglycerides, although the change of pre-treatment and post-treatment does not show statistical change, the extent of decrease is meaningful at clinical aspect.

[0106] In human, PF has been found to dissolve 1 cm of fat per 1-1.5 sessions in the stomach and thigh areas while about 1 cm per session was observed in the upper arms areas. In almost all the patients, the total cholesterol blood levels were reduced significantly with the PF treatment.

[0107] The average level of cholesterol in these cases was higher than the high limit of normal range, and the average level fell into normal range after treatment, which showed statistically significant change (P<0.01).

TABLE-US-00006 TABLE 5 Clinical effect comparison of PF Serum Serum Serum Serum Serum Serum Cholesterol Cholesterol Cholesterol Cholesterol Cholesterol Cholesterol (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) Case Pre-tx NR: Post-tx Pre-tx NR: Post-tx Pre-tx NR: Post-tx No. 50-200 NR: 50-200 60-150 NR: 60-150 35-160 NR: 35-160 1 216 171 123 113 62 73 2 173 135 109 75 88 60 3 207 151 142 92 72 59 4 233 222 93 146 116 67 5 169 168 72 108 54 71 6 269 237 208.5 176.5 83 110 7 180 150 117 98 70 55 8 176 148 118 93 90 85 Ave 202.88 172.75 122.81 112.69 79.38 72.50 SD 35.37 37.05 40.41 33.07 19.38 17.89 P 0.002 0.452 0.462

Example 8

In Vivo Fat Reduction

[0108] Examples of actual treatment results are presented below and the periods of treatment. These patients were selected because they represented the results of treatment of different areas in the body (stomach, arms, and thighs). [0109] 1) Patient 1: Period of Treatment=6 weeks/11 sessions [0110] Measurements at the stomach area: Size before start: 90 cm [0111] Size at End of Treatment: 83 cm [0112] 2) Patient 2: Period of Treatment=5 weeks/9 sessions [0113] Measurements (at the stomach [0114] area) Size before Start: 96 cm [0115] Size at End of Tx: 90 cm [0116] 3) Patient 3: Period of Treatment=1 month/8 sessions In Both Arms: 8 Sessions in total, (i.e. 4 sessions per side) [0117] Areas treated: both upper arms [0118] Size Before Start: 40 cm [0119] Size at End: 36.5 cm

Complete Protocol for the Procedure

Injectable Preparations

Preparation of Solution

[0120] 1 ml of PF (1) at a concentration of 1 mol is mixed with 4 ml injectable water and 5 ml (phosphatidyl choline (PPC)) prepared from soya bean extract and 5 ml 2% lidocain. A total of 15 ml solution. This solution is mixed together in a 20 ml syringe and the needle is changed to a small insulin needle Gauge 30×½ as shown therein before the actual injections are given.

Mode of Action of the Mixture

[0121] PF is the active ingredient in the mixture. It acts upon contact with the fat cells in the adipose tissues. Phosphatidyl choline is a mild detergent that will bind to fat and thereby bring PF in contact with fat cells for an extended period of time (between 8-12 hours). The lidocain in the mixture increases vascularization into the injected site thus bring in more blood vessels to clear the dissolved fat byproducts.

[0122] A topical anesthetic is usually applied to the site prior to injections by 5-10 minutes.

[0123] The solution is then given subcutaneously directly into the fat areas at a dose of 0.5 ml per site totaling 20 injections. These injections are spread out to cover a big surface area, e.g. could be spread out through a whole midsection (“love handle”) in case of a larger patient, or if the patient were an average size it could be spread out to cover right and left love handles in one session. For reference, please refer to the patients injected in the thigh and buttocks area, for explanation. This patient was injected with 30 smaller injections of 0.2 ml per site, and they were spread throughout the whole outer and inner thigh and buttocks areas on the right side.

[0124] Each of these 20 smaller injections comprises a session. On average, a noticeable difference was seen within 4-5 sessions. The initial calculations measured 3 cm of fat loss in 5 sessions. The patient lost over 5 pounds of fat with 10 sessions.

Number of Sessions Required by the Patients

[0125] Each patient is different, depending on their size and the desired amount of fat to be lost. Each 10 sessions resulted in about 7 cm of fat loss. Active patients and those who followed instructions showed above average fat loss. Some patients needed to sculpture small areas and therefore discontinued treatment when the desired result was achieved. The addition of 2 sessions, together with an aerobic instructor, was also observed to result in a weight loss of over 3 pounds in 2 weeks.

Frequency of Injections

[0126] One patient evidenced more than 5 inches of fat loss, received 10 sessions over about 6 weeks. After the desired results were achieved, the patient can receive one session every one to two months for maintenance.

Alternative Mode of Application

[0127] With morbidly obese patients, such as the patient results reported above, the dose of the active ingredient was doubled to 2 ml of 1 mmol in 3 ml of injectable water and mixed with the rest of the solution as mentioned above. A booster injection was used where smaller injections were not appropriate, and the content of one syringe was concentrated into one or two areas of the fat and large needles were used in a retrograde manner of injection. For example, as the needle is pulled out, the injectable solution is released. The needle is rotated in a circular manner to cover a large surface area. It is important to note that the needle is bent to a 90° angle to the long axis of the syringe as shown in FIG. 11.

[0128] About 3 ml is injected in this manner, and then the needle is withdrawn, but not totally out of the initial injection site, and re-inserted into another plane at about 30° in a counter clock-wise manner. This process is repeated about 5 times until the content is all injected. For example, employing a circle injection site area to be treated as a targeted fat area, with its center as the initial site of insertion of the needle, then the needle is rotated around this circle in the manner described above.

[0129] Concentration ranges of PF included in injectable preparations; doses of PF provided to a patient at each treatment (injection) episode; specific steps that were used in preparing the injectable preparations.

[0130] 0.5 mg of PF was dissolved in 5 ml of injectable water rendering a concentration of 0.1 mg/ml. This was then mixed with 5 ml of Phosphatidyl Choline in a 20 ml syringe. The large gauge needle was then replaced with a smaller gauge (Gauge 27-30, in some embodiments, a 30 gauge×½) insulin needle and the 10 ml was injected into 20 different sites of a given area. 0.5 ml of the solution was injected into the stomach, with about 1 cm separating each injection site.

[0131] Concentration of PF included into the topical preparation: 0.2 mg to 0.3 mg of PF were dissolved in 1 ml of azone liquid.

[0132] This mixture was then massaged into the target area. Azone has been extensively studied using a range of permeates. The composition of the azone is C.sub.1811.sub.35NO. It is colorless, slightly yellowish or transparent in color, and is oil based. Heavy metal content in this compound is less than 0.001%. The azone used in the present studies was purchased from Nan Jing Long Tan chemical company, China. Azone has been shown to enhance permeability through the skin by disrupting the organized lipid structure in the intercellular region of the stratum corneum. This process leads to increased lipid fluidity and enhanced drug diffusion. This is the reason azone was chosen to be mixed with PF.

[0133] A low current of 2.5 amp is then passed through pads placed on the skin of the desired treatment area which then drives the PF through the skin into the adipose tissues, thus allowing the PF to come in contact with the fat cells.

Protocol Used in Creating Albumin Nanospheres

[0134] Many different preparations of nanospheres will be used in the preparations of the present crèmes and topical preparations. Any number of different nanosphere formulations known to those of skill in the art may be used in the practice of the present invention. By way of example, such include any known delivery in the area of nanospheres.

Patient Preparation

[0135] For Regular Injections: After sterilization of the surface area with 74% ethanol, topical application of lignocain ointment or lidocain gel was applied for 5 minutes to numb the skin where the injections are to take place.

[0136] For Booster Injection: The patient is prepared using 1 ml of 2% lidocain to anaesthetize the site of entry of the large needle. This way, the procedure is almost painless. 3-5 minutes later, the booster injection is given slowly over a 3 minute period.

[0137] The patient should ingest a meal before the session, or is provided a chocolate bar or a cookie before a session.

[0138] Patients are forewarned that bruising may occur due to the injections. Thus, some bruising in a patient may be expected. For example, bruising was observed in a patient injected in the thighs. The bruising, usually disappears within 1 week. In addition, redness and tenderness at the injection site of the booster, is very common. Redness lasts for a couple of hours and the tenderness lasts for a couple of days. Bruising, is encountered more with the booster than the regular injections, but also disappears within a week or so. Another observation is fatigue as a result of treatment in some patients. Two patients suffered from fatigue, with the booster session, but not with the regular session. In addition, a very slight rise in temperature, to about 37.1 or 37.2 for a few hours, was noted. Dizziness for a few minutes was also observed in a few patients, and the adjustable patient chair was reclined and the procedure was continued uneventfully. No diarrhea was observed, but softer stool was reported.

Protocol after the Sessions

[0139] Patients are asked to drink 2 liters of water per day throughout the day.

[0140] Patients were asked to walk or exercise for 25 minutes/day for the week after each session. This is intended to flush out dissolved fat byproducts.

[0141] Avoid fatty diet, wine, alcohol, for the first 48 hrs after the injections, to allow the blood to carry more of the fat byproducts to be excreted.

A Second Mode of Application—Creme Preparations

[0142] The present preparations may be provided in the form of a crème containing 1 ml of PF at a concentration of 1 mmol mixed with 1 ml of azone which is then massaged into the target area. Azone has been extensively studied using a range of permeants. Azone has been shown to enhance permeability through the skin by disrupting the organized lipid structure in the intercellular region of the stratum corneum. This process leads to increased lipid fluidity and enhanced drug diffusion. This is the reason PF was mixed with azone. Furthermore, a low current is then passed through the tissue area, which then drives the PF through the skin into the adipose tissues. PF may in this way come in contact with fat cells. The clinical results are show below and the details of each patient is documented.

TABLE-US-00007 TABLE 6 AMOUNT OF NUMBER OF REDUCTION PATIENT AGE SEX TIMES IN CM #1 37 M 6 3.5 cm above and below the umbilicus #2 40 F 16 9 cm above the umbilicus. 9 cm from midsection. 8 cm from waist #3 19 F 6 2 cm from each side of the upper thigh #4 32 F 7 8 cm from the right buttock & 9 cm from the left buttock #5 25 F 8 9 & 10 cm from each leg below the knees #6 44 F 12 7 cm above umbilicus and 10 cm from midsection #7 19 F 9 6 cm from left thigh and 8 cm from right thigh #8 24 F 30 1.5 size in bra size #9 44 F 20 6 cm above umbilicus and 9 cm below umbilicus #10 44 F 20 5 cm from the waist and 5 cm from midsection #11 38 F 16 10 cm above the umbilicus, 9.5 cm below the umbilicus and 5 cm from mid- section (love- handles) #12 30 F 8 10 cm from love handles and 6 cm below the umbilicus

Alternative Injection Protocol

[0143] The present example illustrates an alternative series of treatments that will comprise a treatment session for targeted fat reduction using the injectable preparation and/or composition of PF. In this example, a series of 20 small (0.1 to 0.2 ml) injections comprises a session of treatment. On average, a noticeable difference can be seen in a patient, as evidenced by a reduction in size, within 4 to 5 sessions. The measurable fat loss was 3 cm of fat loss in 5 sessions.

[0144] In eight patients treated with PF as described here, the levels of 3 indices such as serum cholesterol, LDL cholesterol, serum triglycerides in the blood were examined before and after treatment. All of these cholesterol parameters were reduced in these eight patient.

[0145] In a prior study with Mesotherapy, these cholesterol levels in blood were not reduced (Hexsel, Serra et al 2003).

[0146] For serum cholesterol, the average level for these cases was higher than the high limit of a normal range, and the average fell into normal range after treatment, which showed a statistically significant change. (p<0.01). For LDL cholesterol, serum triglycerides, although the change of pre-treatment and post-treatment did not show a statistically significant change, the extent of decrease was clinically meaningful in the management of the patient.

Example 9

Delivery Devices

[0147] The present example described particular devices and apparatus that may be used in the delivery and application of the various PF topical preparations and injectable preparations of the present invention.

[0148] Sauna belt: Uzap tummy, butt, thighs (osim)

[0149] Input 100-240 V about 56 Hz

[0150] 1.5 A 200VA

[0151] Output+24V−2.5 A

[0152] It has a remote with the following details:

[0153] Power consumption 60W

[0154] Operating voltage 24V d.c. 2.5 A

[0155] Alpha wave healthtronic muscle stimulator and exercise (the device that transmits electrical current to the pads and is used after/before application of the PF containing crème)

[0156] Model BM-303

[0157] Power supply DC6V (batteries UM-1×4)

[0158] Watts 0.6

[0159] Injection Device: Syringe Barrel with 90° angle needle.

[0160] Specific steps that were used in preparing the injectable preparations: 0.5 mg of PF was dissolved in 5 ml of injectable water rendering a concentration of 0.1 mg/ml. This was then mixed with 5 ml of Phosphatidyl Choline in a 20 ml syringe. The large needle was then replaced with a smaller gauge insulin needle and the 10 ml was injected into 20 different sites of a given area. 0.5 ml of the solution was injected into the stomach with about 1 cm separating each injection site.

[0161] Concentration of PF included into the topical preparation: 0.2 mg to 0.3 mg of PF were dissolved in 1 ml of “Azone liquid”. This mixture was then massaged into the target area. Azone has been extensively studied using a range of permeates. The chemical formula for azone is C.sub.18H.sub.35NO. It is colorless, slightly yellowish, or transparent, and is oil based. Heavy metal content in this compound is less than 0.001%. Azone for the present study was purchased from Nan Jing Long Tan chemical company, China. Azone has been shown to enhance permeability through the skin by disrupting the organized lipid structure in the intercellular region of the stratum corneum. This process leads to increased lipid fluidity and enhanced drug diffusivity. This is the reason PF was chosen to be mixed with azone. A low current of 2.5 Amp is then passed through pads placed on the skin of the desired treatment area, which then drives the PF of the crème/lotion through the skin into the adipose tissues. In this manner, PF is allowed to come in contact with the fat cells.

Example 10

Nanosphere Formulations

[0162] Many different preparations of nanospheres may be used in the practice of the present preparations and compositions. The most effective formulations will then be identified.

[0163] Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) for the topical application to the skin are made of lipids such as glycerol behenate (Compritol 888 ATO), glycerol palmitostearate (Percirol ATO 5), or the wax, acetyl palmitate. For NLC, at room temperature liquid lipids such as medium chain triglycerides (Miglyol 812) are added. Alternatively, oleic acid belonging to the frequently used penetration enhancers in semisolid vehicles applied to the skin may enhance drug uptake further. Mean particle size ranges from 50 to 1000 nm. Nanodipsersions contain 5 to 40% lipid. The higher concentrated preparations are of semisolid appearance and are cosmetically acceptable as they are. Depending on the mode and concentration of the lipid, 0.5 to 5% surfactant have to be added for physical stabilization of the particles. For dermal use, the preparations may also include, by way of example, the following: Poloxamer 188; Polysorbate 80; Lecithin; Tyloxapol; TegoCare 450 (polyglycerol methylglucose distearate); Miranol Ulta C32 (sodium cocoamphoacetater); or saccharose fatty acid ester.

[0164] To facilitate dermal application, fluid dispersions which are obtained when the lipid content is low (<10%) can be incorporated into a crème or gel base which does not include dissolution or aggregation of SLN particles. Photon correlation spectroscopy and differential scanning calorimetry results have not changed over a storage period of 6 months.

Example 11

Fat Reduction Kits

[0165] Component Pieces that would be a part of a kit according to the present example include: [0166] 1. 1 ml ampule of PF and Azone at a concentration of 0.2 mg/ml [0167] 2. device like the Alpha Wave healthtronic muscle stimulator & exercise. This a small device used by physiotherapist to stimulate muscles after a sports injury by passing a low density current that causes muscle contraction. Due to the low voltage current of about 2.5 Amp, a current will be transmitted that will drive the PF through the skin and into the adipose tissue after the azone disrupted the organized lipid layer.
It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.

BIBLIOGRAPHY

[0168] The following references are specifically incorporated herein by reference. [0169] Asaadi, M., A. P. Salas, et al. (2004). Mesoplasty: a new approach to non-surgical liposculpture. American Society of Plastic Surgery, Philadelphia, Pa., Oct. 9 to 13, 2004. [0170] Ablon G., Preliminary experience with mesotherapy utilizing phosphatidylcholine. American Society for Dermatologic Surgery-American College of Mohsmicrographic surgery and cutaneous oncology combined annual meeting, 2005. [0171] Rittes, P. G. (2001). Dermatologic Surgery 27(4): 391-392 [0172] Hexsel, D., M. Serra, et al. (2003). J Drugs Dermatol 2(5): 511-8. [0173] Guedes Rittes, P. (2003). Aesthetic Plastic Surgery 27(4): 315-318. [0174] Rotunda, A. M. (2004). Journal of the American Academy of Dermatology 50(3S): 160-160. [0175] Rotunda, A. M., H. Suzuki, et al. (2004). Dermatologic Surgery 30(7): 1001-1008. [0176] Moy L S. Phosphatidylcholine injections. A study measuring decreased subcutaneous fat thickness. American Society for Dermatologic Surgery and the American Society of Mohsmocrographic surgery and cutaneous oncology combined annual meeting, San Diego, Calif., Sep. 30 to Oct. 3, 2004. [0177] Rullan P, Hexsel D. Phosphatidylcholine injections for lipolysis of neck and jowls: 50 case presentation. American Society for Dermatologic Surgery-American College of Mohsmicrographic surgery and cutaneous oncology combined annual meeting, Oct. 27 to 31, 2005. [0178] Duncan, D. I. and F. Hasengschwandtner (2005). Aesthetic Surgery Journal 25(5): 530-543. [0179] EP1748780/W02005112942 [0180] EP1021191/W09917712 [0181] Duncan, et al (2005) Aesthetic Surgery, 25(5):530-543. [0182] Hexsel, et al (2005) Otolaryngologic Clinics of North America, 38(5):119-29. [0183] Jones, et al (1999) International Journal of Pharmaceutics 177(2): 137-159. [0184] Le Maire, et al (2000) BBA-Biomembrances 1508 (1-2): 86-111. [0185] Navder (1997) Life Sciences 61(19): 1907-1914. [0186] Rittes (2001) Dermatologic Surgery 27(4): 391-392. [0187] Rose, et al (2005) Journal of Cosmetic and Laser Therapy, 7(1):17-19. [0188] Salti, et al (2007) Dermatologic Surgery, 34(1):60-66. [0189] Vedamurthy (2007) Indian J Dermatol Venereol Leprol 73(1): 60-2. [0190] Jenning, et al (2000) International Journal of Pharmaceutics 199(2): 167-177. [0191] Lombardi, et al (2005) Journal of Controlled Release 110(1): 151-163. [0192] Schafer-Korting, et al (2007) Advanced Drug Delivery Reviews 59(6): 427-443. [0193] Wissing, et al (2001) Pharmazie 56(10): 783-6.