CLIAMS:1. A method for screening anti-sebum actives comprising the steps of:
a) Seeding 3T3 L1 cells in 96 or more-welled micro titer plate;
b) Differentiating the cells and incubating them with said actives
c) Staining the cells with Nile red stain
d) Quantifying the fluorescence intensity by micro plate reader.

2. The method as claimed in claim 1, wherein said incubation is in DMEM high glucose media with 10% FBS and 100mM sodium pyruvate.
3. The method as claimed in claim 2, wherein after 48 hours of said incubation, differentiating media is added.
4. The method as claimed in claim 2, wherein said differentiating media is a high glucose DMEM with 10% FBS, 100mm sodium pyruvate, 0.115 µg/ml3-isobutyl-1-methylxanthine, 1 µM Dexamethasone.
5. The method as claimed in claim 1, whereinthe cells are further incubated for 72 hours.
6. The method as claimed in claim 5, wherein a trigger media for oil production is added after said 72 hours
7. The method as claimed in claim 6, wherein said trigger media is DMEM high glucose media with 10% FBS and insulin.
,TagSPECI:Field of invention
The present invention relates to a method for screening anti-sebum actives.
Background and prior art of the invention
Sebaceous glands made up of sebocytes produce sebum, which is skin oil and secreted to the skin surface. Excessive secretion of sebum leads to an undesirable skin condition referred to as “oily skin”. Oily skin is associated with a shiny, undesirable appearance and a disagreeable tactile sensation and affects various age groups. It has been also associated with the skin condition acne. Therefore, cosmetic products and methods that provide sebum control are highly desirable.
Screening methods of actives of such cosmetic products controlling sebum secretion are also known in the art. Such methods include both in vivo and in vitro methods. In vivo screening methods require human volunteers, who are provided with anti-sebum actives in a delivery vehicle. The delivery vehicle without the active serves as a placebo. It comprises measuring of sebum on skin face prior to initiation of study (also referred to as baseline). Volunteers are asked to apply the placebo on half of the face and the active with delivery base on the other half of the face. Presence of topical sebum is measured at fixed intervals. In vivo studies are conducted over a minimum period of 4 weeks. Besides the obvious disadvantage of requiring human volunteers, the duration of such studies also makes them less desirable. On the other hand in vitro studies are conducted either using primary sebocyte cell line or mouse pre-adipocyte cell line.
The drawbacks of the former include shorter life span, need of volunteers and fresh supply of stock culture, while that of the latter is inaccurate results due to false positives.
US006004751A discloses screening anti-sebum active by using primary rat sebocyte cells. It involves determining activators and inhibitors of sebum formation but fails to provide high throughput. Further, the rat primary cell lines have short life span, which makes them undesirable.
Further US2005/0053631A1, US6599936B1 and EP1765272B1 disclose in vitro assays for sebum using primary human sebocytes. As stated these cultures are prolonged in time, taking more than ten days and can evaluate only seventeen actives at a time, excluding controls. Moreover, they use radioactive labelling for quantification studies, thus requiring special safety measures.
Further sebocytes are difficult to culture in vitro for prolonged periods of time as there are no permanent sebocyte cell lines available commercially. In turn necessitating isolation of fresh sebocyte cells from human volunteers after a certain period of time. Thus such methods are time consuming and have low throughput.
In other prior art, mouse 3T3 L1 cells are differentiated to adipocytes and are used to screen anti-sebum actives as it is reported in literature that adipocytes bear resemblance to sebocytes in terms of common bio-synthetic pathways for oil production as reported in Smith and Thiboutot, 2007. However this method poses a problem with respect to quantifying oil production as different cells in the plate start differentiating at different points in time and burst open, releasing oil into the media. Such inconsistency of the existing methods for screening anti-sebum actives by way of standard deviation has been represented in Figure 1.
Therefore, there remains a need for a method of screening anti-sebum actives in vitro in a high-throughput, quantifiable manner and which bears good correlation with in vivo data.
Accordingly, the present invention provides a novel and improved in vitro method for screening anti-sebum actives.
Objects of the Invention
It is an object of the present invention to overcome the disadvantages of prior art methods.
It is another object of the present invention to provide a novel and improved in vitro method for screening anti-sebum actives.
It is another object of the present invention to provide a method which is high throughput and minimizes the chances of false positives.
It is yet another object of the present invention to provide a method wherein as many as thirty actives can be assayed at once.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method for screening anti-sebum active comprising the steps of:
a) Seeding cells in 96 or more-welled micro titer plate;
b) Differentiating the cells and incubating them with actives.
c) Staining the cells with Nile red stain
d) Quantifying the fluorescence intensity by micro plate reader.

BRIEF DESCRIPTIONS OF ACCOMPANYING DRAWINGS
Figure 1: High standard deviation observed in in vitro assay for actives screening
Figure 2: Decrease of sebum production in presence of niacinamide and UNIREDUCE
Figure 3: Decrease in sebum by niacinamidein vivomethod analysis.
Figure 4: Significant decrease of sebum in vivo with niacinamide as active
DETAILED DESCRIPTION OF THE INVENTION
Various in vitro and in vivo assays are known in the art for screening anti-sebum actives however such methods do not have high throughput and provides inconsistent results. It was surprisingly found by the present inventors that a method comprising the steps of:
a) Seeding 3T3 L1 cells in 96 or more-welled micro titer plate;
b) Differentiating the cells and incubating them with actives
c) Staining the cells with Nile red stain
d) Quantifying the fluorescence intensity by micro plate reader
has better accuracy and provides high throughput results.
It was found by that the addition of insulin in the differential media as reported in prior art led to unsynchronized differentiation of cells and hence oil production. In prior art insulin was added to both media, namely, the differentiation media and the trigger media. The present inventors have surprisingly found that by adding insulin to the trigger media only provides consistent tests results.
It was observed that because of this critical feature of the present invention whereinsulin growth factor is added at a predetermined time point all cells attained the differentiated state at around the same time.Therefore, the cells could be harvested for quantification at the time of maximum oil production for a sensitive and robust fluorescent signal.
The present invention can be used to screen various types of active ingredients for their anti-sebum activity. The invention is validated using appropriate positive and negative controls and also using in vivo model systems.
In a particular embodiment the method for screening anti-sebum activity of niacinamide was carried out in accordance with the present invention, comprising the steps of
1. Seeding 3T3 L1 cells on 96 well plates in DMEM high glucose media (Glucose 4500 mg/L) with 10% FBS and 100mM sodium pyruvate; cells are allowed to attach and become confluent.
2. Post 48 hours of incubation, differentiating media is added. The differentiating media consists of high glucose DMEM with 10% FBS, 100mm sodium pyruvate, 3-isobutyl-1-methylxanthine (IBMX) (0.115 µg/ml), Dexamethasone (1 µM).
3. The cells are further incubated for 72 hours and allowed to grow.
4. The trigger media for oil production is added, which is DMEM high glucose media (4500 mg/L) with 10% FBS, insulin (1µg/ml)
5. The cells start producing oil after 48 hours of incubation
6. Cells are fixed and stained with Nile red
7. The fluorescence signal is quantified from the bottom using micro plate reader to determine oil produced in adipocytes (Excitation 485 nm and emission 535 nm).

The invention is now illustrated by way of non-limiting examples
Examples:
Assay Results from in vitro analysis
1. 3T3 L1 cells were seeded in 96 well plates in DMEM high glucose media (Glucose 4500 mg/L) with 10% FBS and 100mM sodium pyruvate; cells were allowed to attach and become confluent.
2. Post 48 hours of incubation, differentiating media was added. The differentiating media consisted of high glucose DMEM with 10% FBS, 100mm sodium pyruvate, 3-isobutyl-1-methylxanthine (IBMX) (0.115 µg/ml), Dexamethasone (1 µM).
3. The cells were further incubated for 72 hours and allowed to grow.
4. The trigger media for oil production was added, which is DMEM high glucose media (4500 mg/L) with 10% FBS, insulin (1µg/ml)
5. The cells started producing oil after 48 hours of incubation
6. Cells are fixed and stained with Nile red
7. The fluorescence signal was quantified from the bottom using micro plate reader to determine oil produced in adipocytes (Excitation485 nm and emission 535 nm).

p values for niacinamide and UNIREDUCE show significant decrease in oil production (p< .05) when compared with control
Unireduce
4.99E-05
Niacinamide
9.16E-06

In Vivo formulation
In vivo formulation of the niacinamide gel is as follows
PHASE NAME OF RAW-MATERIAL % ADDN 200g 1000g
A Water 82.650 165.300 826.5
Niacinamide 4.0 8.0
Disodium EDTA 0.050 0.100 0.5
CarbopolUltrez 10 0.400 0.800 4.00
Pemulen TR2 0.100 0.200 1.00
B Glycerine 2.000 4.000 20.0
Propylene Glycol 3.000 6.000 30.0
Sorbitol 70% 1.000 2.000 10.0
D-Panthenol 0.100 0.200 1.00
Methyl Paraben 0.200 0.400 2.00
Water (FOR RINSING) 2.000 4.000 20.0
C Water 2.000 4.000 20.0
Triethanolamine (99%) 1.200 2.400 12.0
Water (FOR RINSING) 1.000 2.000 10.0
Total 100.000 200.000 1000.00

The gel was tested on human volunteers. Volunteers were asked to apply the gel twice a day 4 weeks on forehead and measurement of sebum production was done once a week using a Sebumeter.
Assay Results from In vivo method analysis
50% of the panelist showed over 40% decrease of oil production as illustrated in Figure 3.
In vivo sebum efficacy data
Parameter Sample Type Mean Diff CI_L CI_U t df p.value
Sebum NIAGEL two.sided 21.417 6.199 36.634 2.911 23 0.008

UNIREDUCE, an oil soluble bioactive complex to reduce sebum production in oily skinshowed sebum reduction by the in vitro method has prior art mentioning the ability of the product to decrease sebum in vivo thus correlating with the in vitro data. In vitro data for UNIREDUCE has a p-value of less than 0.01.

Therefore, bothUNIREDUCE and niacinamide showed reduction of sebum in vitro and in vivo. Further the p-values for both actives in vitro and in vivo are below 0.05 indicating that theyprovide significant reduction of sebum synthesis in vivo and in vitro.
It further indicates that the in vitro tests of the present process can be extrapolated to in vivomethods, thereforerendering it a reliable and beneficialin vitro screening technique for anti-sebum actives.