Cyclic AMP / Lipase / Caffeine:

A short review of the biochemical mechanisms of lipolysis shows the essential steps of this phenomenon, where it is possible to intervene in order to stimulate the lipid catabolism.
These reserve lipids are stored as Triglycerides (fatty acid esters of glycerol).
Primary lipolysis consists of the splitting of the Triglycerides into glycerol and free fatty acids.
The diagram, on page 3, shows the various steps that lead to lipolysis.
The triglyceride lipase, key enzyme of lipolysis, must be activated. Thus an inactive pro-enzyme turns into active piase through the protein kinase mediated phosphate group transfer.
The protein kinase itself, however, needs an activator which is cyclic adenosine mono phosphate (cAMP). The quantity of cycilic AMP, a general purpose cellular messenger, is maintained at a constant level, through precise regulation mechanisms.

Cyclic AMP depends on two enzymes

Adenylcyclase which manufactures cyclic AMP
Phosphodiesterase, which destroys it.
One knows that certain substances, such as caffeine or pheophylline inhibit the latter enzyme which leads to an increase in intercellular concentration of cyclic AMP and thus, through the described cascade, to stimulate lipolysis.

The free fatty acids thus generated are then transported to mitochondria (respiratory organelles) where they enter the oxidation cycle that transforms them into carbon dioxide gas and water for total elimination.

Description of cyclolipase
In order to stimulate lipolysis we have chosen to favor an approach of multiple attack against subcutaneous fat. Therefore we call upon the key molecules of this process:

• 0.025% Cyclic AMP
• 0.1% Lipase
• 0.5% Caffeine

The chosen recipient formula allows us to stabilize cyclic AMP and lipases over the time, maintaining the enzymes at a high level of activity.

Cyclic AMP, of biotechnological origin, is irreplaceable as a substrate for protein kinase which activates directly the triglyceride lipase.

For the first time it is possible to incorporate this molecule into a slimming product. The lipases are lipolytic enzymes, also obtained by fermentative processes on an industrial scale. The lipases reinforce the lipolytic potency of adipocyte lipases. To improve the synergy with the principal components of cyclic AMP/lipase we have included caffeine which slows down the cellular inactivation of cyclic AMP (see diagram on page 3).

Efficacy Tests

Lipolytic activity on adipocytes : Hydrolysis of Triglycerides
In the aftermath of triglyceride lipase action it is possible to assay the resulting metabolites, such as glycerol.
The stimulating effect of the tested product is obtained by comparing the basic level of glycerol production with the quantity released in presence of increasing concentrations of the active ingredient.

Principle of the Method
The adipose tissue is incubated with buffer containing either the cyclicAMP/lipase/caffeine at various concentrations, or ISOPROTERENOL as positive control, or buffer alone.
After 90 minutes the media are harvested and the released glycerol is determined with the aid of a Böhringer Mannheim quantification kit (experimental details in the appendix).Results
The following figure shows the quantity of glycerol released into the medium by the andipocytes. One notices a remarkable stimulation effect on lipolysis by increasing concentrations of cyclinAMP/lipase/caffeine. This effect compares favorably with the positive control of isoproterenol, a powerful adrenergic molecule.

Visualization of the disappearances of Triglycerides in human adipocytes after treatment with cyclinAMP/lipase/caffeine

Aim of the study
To demonstrate visually the disappearance of Triglycerides in andipocytes

Principle
Human andipocytes are harvested on biopsies of plastic surgery. Adipose tissue is dissociated by collagenese in order to obtain isolated adipocytes in suspension in a medium of survival and coloration. Specific colorants allow us to identify the Triglycerides contained in the cells, other colorants label the cell nucleus (DNA) which makes it possible to distinguish the cells from fat globules. Photographic pictures of the the various preparations (control, placebo, cyclinAMP/lipase/caffeine at various concentrations) are obtained under the microscope with fluorescence accessory.

In vivo demonstration of the slimming effect
SUMMARY : study carried out by spincontrol – the complete document can be provided upon request.
The slimming effect was evaluated by echography over a four week period.Eleven gemale subjects, selected on the basis of strict criteria of inclusion and exclusion, participated in this test.

The gel containing 4% of cyclinAMP/lipase/caffeine was applied twice a day, in the morning and evening, until complete penetration of the product. For each subject, the thickness of the adipose infiltration, was evaluated and compared to an untreated site, on the external side of each thigh.

The total duration of the experiment was four weeks from the first application of the gel.
The echographic measurements were carried out:

• On day zero : measurement of the three parameters before application. - thickness of the adipose tissue - centimetric measurement of each thigh - weight of each volunteer.
• On day fourteen: Measurement of the thickness, centimetric perimeter and weight after 14 days application
• On day twenty eight: Same measurement after 28 days of treatment.

Results
1. Variation of adipose tissue thickness
The analysis of the echographic results, demonstrate that the adipose tissue thickness decreased significantly by 12.2% during the four weeks of treatment compared to D0 and the non treated site.(insert echographic measurement graph)

2. Centimetric measurements
compared to D0 the treated side perimeter decreased by 5mm after 14 days and 12mm after 28 days of application compared to the control site which decreased by 4mm after 14 days and 7mm after 28 days.(insert centimetric measurements chart)

Appendix A
Lipolytic activity on adipocytes

Assay of released glycerol
Adipose tissue is cut into small fragments of 120+20 mg. The pieces are maintained in a buffer all along the test; after rinsing, they are incubated in a Krebs Ringer bicarbonate buffer during 2 hours at 37°C, in 5% CO2 the contact product/cells occur during this incubation.

Various controls are carried out

• Negative controls : adipose tissue alone
• buffer alone
• product+buffer (without adipose tissue)
• Positive controls : Isoproterenol (catecholamine) at 31.5 10-6% (with adipose tissue)

After incubation, the adipose tissue fragments are removed carefully. The proteins of the buffer are precipitated with a solution of trichloroacetic acid, the concentration of glycerol is then determined with the help of a Böhringer Mannheim kit.

The concentrations of glycerol obtained are corrected for the initial weight of the adipose tissue present in each reaction tube.

Appendix B
Visualization of the lipolytic effect on human adipocytes by specific coloration

Aim of the study
To make visible the disappearance of Triglycerides in adipocytes

Principle
Human adipocytes are harvested from biopsies of plastic surgery. The adipose tissue is dissociated by collagenase in order to obtain isolated adipocytes in suspension in a medium of survival and coloration. Specific colorants label the cell nucleus (DNA) which makes it possible to distinguish the cells from fat globules. Photographic pictures of the various preparations (control, placebo, cyclicAMP/lipase/caffeine at various concentrations) are obtained under a microscope with fluorescence accessory.

Material and Methods

• Culture medium #1 (dissociation medium of the adipocytes)
• MEM Medium without phenol red, to which 50 IU/ml of penicillin and 50 ml streptomycin are added.
  Culture medium #2 (incubation medium)
• Medium # 1 with 0.5% (w/ v) of dilapidated BSA added.

Isolation of adipocytes

• The adipose tissue from human biopsies is dissociated with 0.1% (w/ v) collagenase in medium #1.
• The cell suspension of adipocytes is filtered, then rinsed in medium #2.

Incubation protocol

• CyclinAMP/lipase/caffeine and its exipient (placebo) are diluted directly in medium #2 at 2.5 and 5%.
• Theophylline is tested in parallel as a positive control at the concentration of 10-4 M.

The products are placed in contact with the appropriate number of cells for 2 hours at 37°C. In parallel, incubations of “controls without product” are also carried out. Each experimental condition is done in triplicate
.
Evaluation of lipolytic activity
At the end of the incubation period, the adipocytes are washed with medium #2 and incubated for one hour at room temperature in a solution of isopropanol/water (6/4, v/v) containing 30mg/ml of red oil and 10 ml of Hoechst 33258.

Red oil is a specific colorant for Triglycerides, that can be observed with or without fluorescence. Hoechst 33258 is a colorant of DNA that indicates the presence of cell nuclei.

The adipocytes are then rinsed with medium #2 and observed under optical microscope in while light or in epifluorescence (excitation at 360 nm and emission at 470 nm for the Hoechst colorant; excitation at 495 nm and emission at 525 nm for red oil).