Final Report

KGL #5669

TABLE OF CONTENTS
I. OBJECTIVE.........................................................................................................2 II. EXPERIMENTAL DESIGN.................................................................................2 A. General Considerations................................................................................2 B. Panelist Selection...........................................................................................3 Light Source (Solar Simulator)............................................................................3 D. MED Determination ......................................................................................4 E. Test Products and Treatment Procedures ...................................................4 F. Expert Graders Evaluations..........................................................................5 G. Minolta Chromameter L* a* b* ......................................................................6 H. Cortex Technology DermaLab Water Loss Meter.........................................7 I. Data Analysis...................................................................................................8 III. RESULTS..........................................................................................................8 A. Panelist Accountability...................................................................................8 B. Expert Grader Scores....................................................................................9 C. Minolta Chromameter ............................................................................10 D. DermaLab Water Loss Probe..................................................................12 IV. CONCLUSIONS..............................................................................................13 V. RECORD RETENTION....................................................................................13 Appendix A: Appendix B: Appendix C: Appendix D: Appendix E: Appendix F: Calendar of Events Weather Information Demographic Data Expert Grader Data Chromameter Data Water Loss Data

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I.

OBJECTIVE

To determine if the use of a topically applied test formulation can diminish the sunburn response which is induced when the skin is photo-insulted with 2 MED's of UV-B. II. EXPERIMENTAL DESIGN A. General Considerations This study was conducted under the supervision of Kays Kaidbey, M.D. and Gary Grove, Ph.D., at the Skin Study Center in Broomall, Pennsylvania. Copies of Dr. Kaidbey and Dr. Groves curriculum vitae are on file with the Sponsor. In conducting this study, we followed the general guidelines recommended in Good Clinical Practices (GCP) and Good Laboratory Practices (GLP) as well as the COLIPA Efficacy Testing Guidelines. This study was conducted from August 19, 2004 to August 27, 2004. A calendar of events outlining the schedule of treatments and evaluative procedures that were followed is attached as Appendix A. The daily weather records covering this time as extracted from newspaper reports were recorded and are attached as Appendix B. A more detailed account issued by the US Weather Bureau can be provided upon the Sponsors request. Briefly, this study was a single-blind, controlled, randomized study which determined the effects of a test material on skin photo-insulted with 2 MED's of UV-B. . The panelists reported first for MED determination and then returned to the laboratory the following week for Baseline assessments and to receive a 2 MED dose on 4 of the 6 sites. They returned the following day for additional evaluations and to commence twice daily product treatments. The panelists then returned each day for the remainder of the week for additional assessments and treatments (no treatment on Friday).

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B. Panelist Selection The volunteers for this study were selected from a pool of healthy women who were between 18-55 years of age and must have been willing to comply with the requirements of this experimental design. Each candidate was interviewed to make certain that they had no medical problems and were not using concomitant medications that might interfere with the study results. Women who were either pregnant or breast-feeding were also excluded from participating in this study. Each volunteer signed a consent form after being informed as to her obligations and risks that might be encountered as a participant in this study. The selected panelists were advised of the general nature of this study and were instructed not to "tamper" with the sites in any way. Prior to testing, all candidates were assessed by Ms. Angelit Barnes, for suitability to be included on the panel. Any individuals with cuts, scratches or any clinical signs of erythema on the lower back were excluded at that time. Qualified panelists were assigned a panelist number in the order of their admittance to the study panel. Light Source (Solar Simulator) This is a 150-watt compact xenon arc source equipped with a UVreflecting dichroic mirror and a 1mm thick Schott WG-320 filter to produce simulation of the solar spectrum. A 1mm thick UG5 filter is added to remove reflected heat and remaining visible radiation (Berger, D.S.: Specification and design of solar ultraviolet simulators. J. Invest. Dermatol. 53:192-199, 1969). Warm up time of the lamp before use is 20-25 minutes. Total irradiance at skin level is measured with a calibrated Eppley Thermopile and the UVB component is monitored with a Robertson-Berger sunburn meter (R-B meter). The size of the irradiated field is approximately a 1 cm diameter circle.

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D. MED Determination One test area serves for determining each panelists Minimal Erythema Dose (MED). The MED is determined by exposing several unprotected skin sites (1cm in diameter) over the mid back to a series of exposures from the solar simulator that were done in 25% increments. The MED is defined as the time of exposure required to produce minimally perceptible erythema 22 + 4 hours after exposure. The MED of the panelists unprotected skin was determined the week prior to the actual testing. Visual grading of the MED is done under standardized lighting conditions when the panelists returned to the testing facility approximately 24 hours after irradiation. E. Test Products and Treatment Procedures The test material utilized in this study which was a biocatalyst that had been filtered through 5 micron and 1 micron filters, then irradiated with UV and filtered through activated charcoal was supplied by the Sponsor in a bottle and labeled as follows: Sample A Three sites (approximately 1 x 1) were marked on both the left and right lower back (6 sites total) by Ms. Barnes. After completing the visual grades and instrument measurements on Day 1, two times each individuals MED dose was administered to 4 of the 6 test sites. On Day 2, panelists reported to the Skin Study Center for additional visual assessment by the Expert Grader and for instrumental measurements using the DermaLab Water Loss Probe and Minolta Chromameter. After these assessment/measurements were completed, twice daily treatments commenced. Treatment was done in a randomized fashion for the following: 1 site on each side of the back: 2 MED + Sample A 1 site on each side of the back: 2 MED + no treatment 1 site on each side of the back: Control (no 2 MED + no treatment)

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The test product was applied to the designated sites at the lab in the morning and in the afternoon by Mrs. Marie Windle. Approximately 0.1cc of the test material was applied to the assigned sites and rubbed into the skin using a cotted finger. Additional product (approximately 0.1cc) was also applied to a Band-Aid Brand bandage which was placed over the site. During the afternoon visit, the morning bandages were removed and product was again applied directly to the skin and covered with additional product on a fresh bandage. Care was taken to prevent cross-contamination of the treatment sites during application and throughout the treatment/measurement phase of the study.

F. Expert Graders Evaluations Ms. Barnes served as the Expert Grader for this study. Assessments were done on Day 1 (prior to 2 MED) and again on Days 2, 3, 4 prior to treatment and on Day 5. Erythema 0 = 1 = 2 = 3 = 4 = No erythema Minimally visible erythema Deeper erythema with clear, distinct margins Intense erythema and edema Vesicular or blistering reaction

To maintain the Expert Grader's blindness to products/scores, the visual assessments were conducted in an area that was physically separated from the treatment room. The data was manually recorded by the Expert Grader on a worksheet.

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G. Minolta Chromameter L* a* b* Skin surface color was measured instrumentally using reflectance techniques based on the standardized tristimulus system recommended by CIE. The specific model employed for such measurements was the Minolta CR-200 Chromameter that has an 8mm measuring area using the illuminant conditions of D65 which most closely approximates normal daylight conditions. This is a hand held device that is gently placed against the surface to be color characterized. When triggered, a pulsed xenon light source flashes and this light is reflected off the surface and measured back into the device. Within the device, there are 6 silicon photocells that are filtered to detect primary stimulus values for red, green and blue wavelengths of light. For color readings, the values are translated into the L*a*b* coordinates whose spacing correlates closely with color changes perceived by the human eye. This is an internationally recognized convention for numerically expressing color differences established by the C.I.E. (Commission International de LEclairage). The L* value represents the density value from black to white. The a* and b* values represent the color axes ranging from green to red and from blue to yellow, respectively. Higher a* values along the red-green axis are an indication that a site is more irritated. [Babulak, S.W., Rhein, L.D., Scala, D.D., Simion, A.F. and Grove, G.L., Quantitation of Erythema in a Soap Chamber Test Using the Minolta Chroma (Reflectance) Meter: Comparison of Instrumental Results with Visual Assessments, J. Soc. Cosmet. Chem. 37:475-479, 1986.] On Day 1 prior to 2 MED exposure, Days 2, 3 and 4 prior to treatment and again on Day 5, three sets of L*, a* and b* readings from each of the test sites were taken by Mrs. Trish Alfano with the assistance of Mrs. Nancy Bates and the average value was computed for each site.

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H. Cortex Technology DermaLab Water Loss Meter All water loss measurements were taken following a 15-30 minute acclimation period in a controlled environment with the relative humidity maintained at less than 50% and temperature maintained at 70 + 2oF. This instrument is based on the vapor pressure gradient estimation method as designed by Nilsson and initially utilized by the Servo Med Evaporimeter. There are slight dimensional differences and the sensor technology is greatly improved in the DermaLab TEWL probe but the underlying principles of the measurement remain the same. Both probes contain two sensors which measure the temperature and relative humidity at two fixed points along the axis normal to the skin surface. This arrangement is such that the device can electronically derive a value that corresponds to evaporative water loss expressed in gm/m2hr. The DermaLab Modular System with TEWL Probe is more fully described in: Grove, G.L., M.J. Grove, C. Zerweck and E. Pierce: Comparative metrology of the evaporimeter and the DermaLab TEWL probe. Skin Res. & Tech. 5:18, 1999. Grove, G.L., M.J. Grove, C. Zerweck and E. Pierce: Computerized evaporimetry using the DermaLab TEWL probe. Skin Res. & Tech. 5:9-13, 1999. The guidelines established for using the Servo Med Evaporimeter as described by Pinnagoda [Pinnagoda, J., R.A. Tupker, T. Anger and J. Serup. Guidelines for transepidermal water loss (TEWL) measurement. In: Contact Dermatitis 1990: 22:164-178] are quite appropriate for the DermaLab TEWL Probe as well. The data from the DermaLab Modular System is completely computerized and continuously communicates with its PC through a serial port using an RS-232C cable and associated cyberDERM, inc. software for the Evaporimeters. We use the application program entitled C_BASIX_ which captures the water loss data from the attached evaporimeter at a sampling rate of 5 inputs/second. These inputs are graphed as a real time display on the computer monitor. The extracted value refers to the average evaporative water loss rate collected over a twenty second interval once steady state conditions have been achieved. These are directly transferred to an Excel spread sheet file using a DDE link.

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At each session, duplicate water loss readings were taken from each test site and electronically recorded using a spreadsheet format based on Excel 7.0 software which computed the average value for each test site. These values are also manually recorded on a worksheet that serves as a back-up in case there are problems with the computerized records. On Day 1, prior to 2 MED exposure, Days 2, 3 and 4 prior to treatment and again on Day 5, evaporative water loss measurements were obtained from each of the test sites by Ms. Barnes and electronically recorded using a spreadsheet format based on Excel software that computes the average value for each test site. These values were also manually recorded on a worksheet that serves as a back-up in case there were problems with the computerized records. Such measures provide a noninvasive method for determining the barrier function of the stratum corneum. Damage leads to a disruption of the barrier that is accompanied by elevated water loss rates. I. Data Analysis Dr. Grove was responsible for devising a sorting template based on Excel 2000 spreadsheet software and implemented on the IBM clone desktop computer. The sorted data was tabulated and arranged in order of panelist number for each point of evaluation. In creating these tables, column averages were computed, but only to give a preliminary look at the findings. Due to the small sample size in this study, a full statistical analysis of the findings cannot be performed. The means were calculated to ascertain any trends in the data which would suggest that further study is warranted.

III. RESULTS A. Panelist Accountability Six panelists were recruited for this study, all of whom were accepted onto the study panel. Appendix C contains a listing of the selected panelists along with their age and sex. This table also provides a listing of the actual UVB doses (2 MEDs) used to challenge the skin. All of the panelists were able to successfully complete the study. There were no missed visits and we have no reason to believe that the panelists were not fully compliant with the requirements of the study.

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Final Report B. Expert Grader Scores

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Appendix D contains the tabulated and sorted erythema scores from Baseline and Days 2, 3, 4 and 5. These results are graphically summarized in the figure shown below:

As expected the mean erythema scores increased due to the skin becoming redder which is the classic response of the skin to a dose of UV known to induce a sunburn. Over time, the degree of erythema did decrease and there are trends in the data that strongly suggest that this is happening to a greater extent with Sample A than the control.

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C. Minolta Chromameter The tabulated and sorted data for the L*, a* and b* measurements obtained with the Minolta Chromameter on Days 1, 2, 3, 4 and 5 are enclosed as Appendix E. These results are graphically summarized in the figures shown below:

For the a* readings which are a measure of redness, we see patterns that are consistent with the Expert Graders visual scores. First, there is an dramatic increase in redness at all of the skin sites exposed to 2 MEDs of UV followed by a gradual decline. Although the differences are modest, this does seem to be happening at a greater rate in those irradiated sites that are being treated with Sample A.

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A similar pattern is seen in the L* readings which is most likely a reflection that the sites are a darker due to increased redness and perhaps some immediate tanning. Again over time, the values are returning to a more normal range with the rate seeming to be somewhat quicker in those sites treated with Sample A as shown below:

There are no readily apparent changes in the b* readings which are measuring the blue to yellow color components of the skin as shown below:

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D. DermaLab Water Loss Probe Appendix F contains the tabulated and sorted TEWL rates from Baseline and Days 2, 3, 4 and 5. These results are graphically summarized in the figure shown below

We found no appreciable changes in the TEWL during this time period. Although it appears that the TEWL rates are slightly elevated in both treated sites, we feel that this is more likely due to the fact that these sites were being partially occluded by the dressings used to cover the treated sites. We also know from other studies using this experimental design that dramatically elevated water loss rates as a result of barrier disruption do not occur until the skin begins to peel which is usually Day 8 or later.

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IV. CONCLUSIONS On the basis of the information collected during the course of this study, we feel that it is reasonable to conclude that Sample A can measurably reduce the appearance of redness of sunburned skin. That this occurred was suggested by both the Expert Graders ratings of visual erythema and the instrumental measures provided by the a* readings of the Minolta Chromameter. Given the small panel size, it is impossible to evaluate the significance of these findings but these data do suggest that treating with Sample A can be clinically effective. V. RECORD RETENTION Please be advised that the records for this study will remain on file at KGL, Inc. (or a remote storage site) for a period of 1 year from the issue date of the final report and then destroyed unless we are notified otherwise by the Sponsor using the form accompanying this report.

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