Hair foliculo malus estudio

Proposal n°: GT061212C Study n°: GT061212

EFFECT OF THE PRODUCT “EXTRACT 800B323.I” ON THE LIFESPAN OF HUMAN HAIR FOLLICLES

FINAL REPORT OF THE STUDY GT061212

Study promoter: Dr. Daniel Schmid MIBELLE AG BIOCHEMISTRY Bolimattstrasse 1 CH-5033 BUCHS Switzerland

Phone: +41 62 836 11 11 Fax: + 41 62 836 13 13 E-mail: [email protected]

Date: April 23rd, 2007

Study n° GT061212 Effect of the product “EXTRACT 800B323.I” MIBELLE AG. on the lifespan of human hair follicles BIOalternatives 1/15

The investigators and the author of this report hereby certify the validity of the data presented and attest their full agreement with the conclusions presented at the end of the report. Certified by: Name: Franck JUCHAUX Position: Study Director Date: April 23rd, 2007 Signature


1 - INTRODUCTION 3

2 - MATERIALS AND METHODS 3

2.1. BIOLOGICAL MODEL 3 2.2. TEST COMPOUND 3 2.3. PRELIMINARY CYTOTOXICITY ASSAYS 4 2.3.1. CELLS AND CULTURE CONDITIONS 4 2.3.2. CYTOTOXICITY ASSAY 4 2.4. HAIR FOLLICLES GROWTH 4 2.5. DATA MANAGEMENT 4

3 - RESULTS AND CONCLUSION 5

3.1. PRELIMINARY CYTOTOXICITY 5 3.2. EFFECTS ON HUMAN FOLLICLE GROWTH AND MORPHOLOGY 5

4 - TABLES 6

5 - FIGURES 13


1 - INTRODUCTION MIBELLE AG BIOCHEMISTRY has the product “Extract 800B323.I” which could have an effect on the lifespan of human hair follicles.

BIOalternatives carried out an experimental assay in order to study hair survival effects using human hair follicles obtained by microdissection from skin human fragments (face lifting). The viability, the length and the morphology of the hair root was analysed at various times. This model consisted in a whole isolated organ and represented a “natural” coculture model of normal cutaneous differentiated cells (keratinocytes, but also fibroblasts, melanocytes and the bulge region). Obviously, this model did not include blood vessels and this limited the detection of products active on the scalp via blood circulation. This model directly derives from the widely validated standard conditions described by Philpott. It is based on the immediate micro-dissection of human scalp samples, which makes it an experimentally heavy procedure, dependant on individual biological samples. It is necessary to point out that an optimal research protocol on hair requires intra-experiment analysis of sufficient amount of hair follicles (12 growing hair follicles per condition) and experiment reproducibility on several donors.

2 - MATERIALS AND METHODS

2.1. Biological model Assays on hair bulbs The hair bulbs used in this study were extracted from the microdissection of a human skin fragment from a female patient (plastic surgery, donor n°E4449).

2.2. Test compound

Test compound Stock solution Dilution Final tested concentration

Extract 800B323.I Batch n°150107

Expiry date : 07/2007 (GT061212/1)

Liquid supplied by the study promoter and stored at 4°C.

in culture medium 2% and 0.2%


2.3. Preliminary cytotoxicity assays

2.3.1. Cells and culture conditions - Cells: Normal human epidermal keratinocytes (NHEK, K015) used at 4th passage. - Culture medium : Keratinocyte SFM (Invitrogen 17005034), supplemented with

EGF 0.25 ng/ml, pituitary extract 25 µg/ml (Invitrogen 3700015) Gentamycin 25 µg/ml (Sigma G1397)

- Culture: at 37°C and 5% CO2

2.3.2. Cytotoxicity assay - plates: 96-well plate - cells/well: 10000 NHEK - concentrations tested: first concentration at 10%, then dilution factor 5 (see table 1) - replicates: 6 - treatment time: 168h (72h+96h) - Evaluation parameter: MTT hydrolysis assay and morphological observations

2.4. Hair follicles growth The hair bulbs were cultivated in William culture medium containing or not (control) the test compounds. The hair follicles growth was followed during 18 days with length measurements and photographs at day 0, day 4, day 14, day 16 and day 18 for each of them. For each condition, an excess of hair bulbs was isolated to reach the target-value of 12 hair follicles in the end. At day 14, two plates were labelled with propidium iodide and photographs were taken at day 14, day 16, day 18 and day 20.

2.5. Data management Raw data were processed with the statistical analyzer PRISM software (Graph Pad Software). The inter-group comparisons were performed by variance analysis (ANOVA) with multiple comparison test of Dunnett.


3 - RESULTS AND CONCLUSION

3.1. Preliminary cytotoxicity Table 1 A dedicated preliminary cytotoxicity was performed on human keratinocytes by MTT hydrolysis assay for the compound in order to determine, in accordance with the study promoter, the concentrations to be assayed (see paragraph 2.2).

3.2. Effects on human follicle growth and morphology Table 2 and Figure 1 The growth of hair follicles was normal and in proportion to results usually obtained in the laboratory. The treatment with “Extract 800B323.I” at 2% clearly decreased the growth of the hair follicles. At the second concentration (0.2%), the product did not modify the growth. Tables 3 to 8 and Figure 2 On the 14th day of culture, 4 hair follicles were labelled with propidium iodide. The other hair follicles growth was followed until complete apoptosis (day 20). In these experimental conditions, the growth of the control hair follicles was highly slowed down between the 14th and 20th day. At day 20 of culture, most of the hair follicles was in apoptosis and highly incorporated propidium iodide. The treatment with “Extract 800B323.I” at 2% clearly induced a blockage of the growth. At day 14, the hair follicles highly incorporated propidium iodide. At the second concentration of 0.2%, the growth was slowed down compared to the control from day 14. At day 20, the apoptosis was comparable to the control. To conclude, in these experimental conditions the effects of the product “Extract 800B323.I” did not show any lifespan extension of hair follicles. On the contrary, the treatment slowed down the growth and could even block the growth at concentration 2%. The product could therefore be used as hair follicles ‘anti-growth’ such as for example in deodorant or aftershave.


4 - TABLES OD540Extract 800B323.I dilution factor 5% 0 0.000128 0.0006 0.0032 0.016 0.08 0.4 2 10 0

0.401 0.396 0.383 0.403 0.405 0.396 0.460 0.003 0.003 0.4500.419 0.434 0.419 0.413 0.398 0.408 0.446 0.003 0.004 0.4560.418 0.411 0.417 0.355 0.410 0.458 0.470 0.003 0.013 0.4590.409 0.432 0.403 0.435 0.436 0.401 0.459 0.004 0.004 0.4230.419 0.449 0.458 0.438 0.429 0.450 0.472 0.005 0.007 0.4990.438 0.502 0.466 0.454 0.435 0.456 0.450 0.006 0.009 0.467

average 0.438 0.437 0.424 0.416 0.419 0.428 0.460 0.004 0.007viability (%) 100 100 97 95 96 98 105 1 2Observations + + + + + + +/-, spi 0 0

Morphological observations: +, normal population +/ -, growth reduction -, toxicity0, cell mortalityspi, spikes

Table 1: Effect of the product “Extract 800B323.I” on keratinocytes monolayers viability

(contact 168h). MTT assay and morphological observations.


Lengthening Day 0 - Day 4Treatment Conc. image n° Length D0

(µm)Length D4

(µm)Lengthening

(µm) Average sd % Control p

a4 1703 2397 694a5 1736 2698 962b1 1526 2458 931b2 1600 2346 747c1 1894 3115 1221c2 1848 2922 1075c3 1394 2180 787c5 2069 3084 1015c6 2011 3401 1390d2 1757 2580 823d5 1492 2523 1032a3 1587 2918 1332d1 1754 3135 1382b4 1490 2409 919b5 1577 2292 715d6 1792 2898 1106a2 1802 2174 372a3 1500 1779 280a6 1700 2142 442b2 1657 2058 401b3 1665 2051 386b4 1698 1964 266b6 1546 1600 54c3 1536 2007 471c5 1508 1849 340c6 1567 1944 378d1 1628 2082 454c1 1688 2063 375d4 1449 1915 466d5 1757 2099 342d3 1735 2093 358d2 1435 1588 153a3 1334 2353 1018a4 1814 3047 1232a6 1734 2714 981b1 1327 2213 885b5 1653 2724 1071b6 1757 2909 1153c1 1411 2541 1130c2 1467 2466 998c4 1641 2746 1104c5 1660 2607 946c6 2030 3187 1157a1 1310 2789 1479a2 1537 2513 976a5 1661 2754 1093d2 1881 3300 1419d6 1437 2645 1208

162 111 p>0.05

100 -

800 B 323 - 1

2% 346 113 34 p<0.01

0.2% 1116

Control - 1008 231

Table 2: effects of “Extract 800B323.I” on the hair follicles growth after 4 days (D4) of contact. Sd, standard deviation.



(µm)Length D7

(µm)Lengthening



21 p<0.01

0.2% 1559 200 99 p>0.05

800 B 323 - 1

2% 336 105

Control - 1579 348 100 -




(µm)Length D14

(µm)Lengthening



456 60 p<0.01

100 -

800 B 323 - 1

2% 348 121 13 p<0.01

0.2% 1585

Control - 2650 1275




(µm)Length D16

(µm)Lengthening


a4 1703 2938 1235a5 1736 3913 2177b1 1526 3866 2340b2 1600 3246 1646c1 1894 5172 3278c2 1848 4182 2334c3 1394 3423 2029c5 2069 4246 2177c6 2011 6319 4308d2 1757 4464 2707d5 1492 3796 2305d6 1792 5237 3445a2 1802 2191 389a3 1500 1788 289a6 1700 2178 478b2 1657 2071 415b3 1665 2038 373b4 1698 1995 296b6 1546 1615 69c3 1536 2004 467c5 1508 1841 333c6 1567 1879 312d1 1628 2097 469d2 1435 1597 162a3 1334 3212 1878a4 1814 3350 1535a6 1734 3037 1304b1 1327 3050 1722b5 1653 3627 1974b6 1757 3830 2073c1 1411 2856 1445c2 1467 2968 1501c4 1641 3017 1376c5 1660 2874 1214c6 2030 3616 1587d6 1437 4341 2903

14 p<0.01

0.2% 1709 461 68 p<0.01

800 B 323-1

2% 338 125

Control - 2498 835 100 -




(µm)Length D18

(µm)Lengthening


a4 1703 2755 1052a5 1736 3778 2041b1 1526 3206 1679b2 1600 3197 1597c1 1894 5033 3139c2 1848 4100 2252c3 1394 3364 1970c5 2069 4121 2052c6 2011 6456 4446d2 1757 4375 2618d5 1492 3638 2146d6 1792 5096 3305a2 1802 2018 216a3 1500 1707 207a6 1700 2005 305b2 1657 2098 441b3 1665 2077 411b4 1698 1775 76b6 1546 1460 -86c3 1536 1953 417c5 1508 1818 310c6 1567 1938 371d1 1628 1904 276d2 1435 1589 154a3 1334 2962 1627a4 1814 3266 1451a6 1734 2911 1177b1 1327 3036 1708b5 1653 3685 2032b6 1757 3718 1961c1 1411 2778 1366c2 1467 2931 1464c4 1641 2994 1353c5 1660 2904 1244c6 2030 3447 1417d6 1437 4233 2796

451 69 p<0.01

100 -

800 B 323-1

2% 258 156 11 p<0.01

0.2% 1633

Control - 2358 908




(µm)Length D20

(µm)Lengthening


a4 1703 2397 694a5 1736 2698 962b1 1526 2458 931b2 1600 2346 747c1 1894 3115 1221c2 1848 2922 1075c3 1394 2180 787c5 2069 3084 1015c6 2011 3401 1390d2 1757 2580 823d5 1492 2523 1032d6 1792 2898 1106a2 1802 2174 372a3 1500 1779 280a6 1700 2142 442b2 1657 2058 401b3 1665 2051 386b4 1698 1964 266b6 1546 1600 54c3 1536 2007 471c5 1508 1849 340c6 1567 1944 378d1 1628 2082 454d2 1435 1588 153a3 1334 2353 1018a4 1814 3047 1232a6 1734 2714 981b1 1327 2213 885b5 1653 2724 1071b6 1757 2909 1153c1 1411 2541 1130c2 1467 2466 998c4 1641 2746 1104c5 1660 2607 946c6 2030 3187 1157d6 1437 2645 1208

34 p<0.01

0.2% 1074 108 109 p>0.05

800 B 323-1

2% 333 126

Control - 982 204 100 -



5 - FIGURES

Figure 1: effects of the compound “Extract 800B323.I” at 2% and 0.2% on hair follicles growth at D0, D4 and D7 (Lens x4)

Control Extract 800B323.I

2% (P11) Extract 800B323.I

0.2% (P12)

D0

a3 a3 a3

D4

a3 a3 a3

D7

a3 a3 a3

D0: the compounds were put in contact with the hair follicles D4: after 4 days of contact D7: after 7 days of contact


Figure 2: effects of the compound “Extract 800B323.I” at 2% and 0.2% on hair follicles

growth at D14, D16, D18 and D20 (Lens x4).

Control Extract 800B323.I 2% (P11)

Extract 800B323.I 0.2% (P12)

a3a a3 a3 D14

a3a c1 a1

D16

c5a c5 c5

D18

c5 c5 c5


Figure 2 (continu): effects of the compound “Extract 800B323.I” at 2% and 0.2% on hair

follicles growth at D14, D16, D18 and D20 (Lens x4).

c5 c5 c5 D20

c5 c5 c5

D0: the compounds were put in contact with the hair follicles. D14: after 14 days of contact + propidium iodide labelling D16 after 16 days of contact D18 after 18 days of contact D20 after 20 days of contact + propidium iodide labelling

Hair foliculo malus estudio

Health & Medicine

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