Effects of oral cyclosporine and cremophor-EL on microvascular dynamics and the barrier function of skin in the nude rat.

Studies were conducted to asses the impact of the oral administration of cyclosporine and cremophor-EL on parameters important to the per-cutaneous penetration of compounds through nude rat skin. The two parameters chosen were dermofluorometric response to injected fluorescein (reflecting vascularity and perfusion) and trans-epidermal water loss (reflecting barrier function). Differences were seen in the elimination phase of the dermofluormeter curves at 7 and 14 days. Some of these changes persisted in animals treated with cremophor-EL for 21 days but were not evident in the cyclosporine treated group. After cessation of treatment with other compound differences were again noted at 7 and 14 days. The change seen were thought to reflect vasodilatory responses consequent to cremophor-EL stimulated release of modulators superimposed on the nephrotoxic effects of cyclosporine. Trans-epidermal water loss consistently lowers from the flanks than from abdomen of animals regardless of treatment regimen with the exception of animals given cyclosporine for 7 days. At that time there was no significant difference between the flank and abdominal readings.

EFFECTS OF ORAL CYCLOSPORINE AND CREMOPHOR-EL ON MICROVASCULAR DYNAMICS AND THE BARRIER FUNCTION OF SKIN IN THE NUDE RAT by Warren William Jederberg A thesis submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Master of Science in Pharmacology Department of Pharmacology and Toxicology The University of Utah August 1987 Copyright C Warren W. Jederberg 1987 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a thesis submitted by Warren William Jederberg This thesis has been read by each member of the following supervisory committee and by mc~jorit)' vote has been found to be satisfactory. I I I ~ynn K. Persh~~ THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING AI)PIlOVAL To the Graduate Council of the University of Utah: I have read the thesis of Warren Wi lliam J ederberg in its final form and have found that (1) its format, citations, and biblio­graphic style are consistent and acceptable; (2) its illustrative materi­als including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the Supervisory Committee and is ready for submission to the Graduate School. DaLe Approved for the Major Department ~bb Chair/Dean Approved for the Graduate Council B. G. Dick, Jr. Dean of The Graduate School ABSTRACT Studies were conducted to assess the impact of the oral administration of cyclosporine and cremophor-EL on parameters important to the percutaneous penetration of compounds through nude rat skin. The two parameters chosen were dermofluorometric response to injected fluorescein (reflecting vascularity and perfusion) and transepidermal water loss (reflecting barrier function). Differences were seen in the elimination phase of the dermofluorometer curves at 7 and 14 days. Some of these changes persisted in animals treated with cremophor-EL for 21 days but were not evident in the cyclosporine treated group. After cessation of treatment with either compound differences were again noted at 7 and 14 days. The changes seen were thought to reflect vasodilatory responses consequent to cremophor-EL stimulated release of modulators superimposed on the nephrotoxic effects of cyclosporine. Transepidermal water loss was consistently lower from the flanks than from abdomen of animals regardless of treatment regimen with the exception of animals given cyclosporine for 7 days. At that time there was no significant difference between the flank and abdominal readings. TABLE OF CONTENTS ABSTRACT ... LIST OF TABLES LIST OF FIGURES. ACKNOWLEDGEMENTS INTRODUCTION . . . METHODS AND MATERIALS. Animals . . . . . . Anesthesia. . . . . . . . . . Instruments . . . Dermofluorometer . Evaporimeter . . Thermometer. . . . Data Analysis . . . . . Dermofluorometry . Transepidermal Water Loss. Reagents .. RESULTS ......... . Dermofluorometer Studies. Transepidermal Water Loss . . DISCUSSION . APPENDICES . A. B. SUMMARY DERMOFLUOROMETER DATA FOR NAIVE ANIMALS . . . . . . . . SUMMARY DERMOFLUOROMETER DATA FOR ANIMALS ON ORAL CYCLOSPORINE ... iv vii viii ix 1 3 3 3 4 4 4 5 5 5 6 7 8 8 22 32 39 40 43 C. SUMMARY DERMOFLUOROMETER DATA FOR ANIMALS ON ORAL CREMOPHOR-EL .......... 53 D. SUMMARY DERMOFLUOROMETER DATA FOR ANIMALS OFF ORAL CYCLOSPORINE . . . . . . . . . 62 E. SUMMARY DERMOFLUOROMETER DATA FOR ANIMALS OFF ORAL CREMOPHOR-EL . . 69 F. TRANSEPIDERMAL WATER LOSS DATA. . REFERENCES . . . . . . . . . . . . . . . vi . 76 90 LIST OF TABLES Table 1. Dermofluorometry of Naive Nude Rats . . . . . . . . 2. Dermofluorometry of Nude Rats On Oral Cyclosporine and Cremophor-EL for 3 Days . · · 3. Dermofluorometry of Nude Rats On Oral Cyclosporine and Cremophor-EL for 7 Days . · · 4. Dermofluorometry of Nude Rats On Oral Cyclosporine and Cremophor-EL for 14 Days. · · 5. Dermofluorometry of Nude Rats On Oral Cyclosporine and Cremophor-EL for 21 Days. · 6. Dermofluorometry of Nude Rats Off Oral Cyclosporine and Cremophor-EL for 7 Days 7. Dermofluorometry of Nude Rats Off Oral Cyclosporine and Cremophor-EL for 14 Days. · · · · · · · · · · · · · · · · · · 8. Overall Evaluation of Dermofluorometer Profile for Rats On Oral Cyclosporine and Cremophor-EL . . . . 11 · · · . · · 15 · · . · · 16 · · · 17 · · · · 19 · 20 · · · · · 21 . 33 LIST OF FIGURES Figure 1. 2. 3. Dermofluorometry Of Abdominal Sites On Three Naive Nude Rats. Dermofluorometry Of Abdominal And Flank Sites On Three Naive Nude Rats. Dermofluorometer Calibration Curve For Fluorescein . . . . . . . . . . 4. Tissue Elimination Half-Life Values for Nude Rats On Oral Cyclosporine ,and Cremphor-EL . . . . . . . . . . 5. 6. Transepidermal Water Loss Measurements On Flanks And Abdomens On Rats On Oral Cyclosporine . . . . . . . . . . . Transepidermal Water Loss Measurements On Flanks And Abdomens On Rats Off Oral Cyclosporine . . . . . . . . . . . 10 12 23 25 28 30 ACKNOWLEDGEMENTS The author wishes to express his gratitude to the faculty and staff of the Department of Pharmacology and Toxicology, University of Utah for their fine instruction and aid. Special thanks to Gerald G. Krueger, James W. Gibb, and Lynn K. Pershing for serving as the graduate committee. These studies were greatly facilitated by assistance of Rebecca L. Conklin, and Lyssa D. the Division of Dermatology. My family is greatly appreciated for the expert Lambert of their kind patience and loving support during my graduate studies and during this project. Acknowledgement is given to the U.S. Army and the Department of Defense for funding my academic training at the University of Utah. INTRODUCTION Numerous studies have been conducted to measure the percutaneous chemicals in penetration humans and of in beneficial or harmless animals (1,2,3,4,5). Some models have also been developed to measure the percutaneous penetration of toxic compounds (6,7). Only limited data exist regarding the capacity of these models to predict skin penetration" in man. Previous studies have shown that the skin of densely haired animals (mice, rats, rabbits, guinea pigs) tended to be highly permeable, while the permeability properties of the skin of the pig, monkey, and dog were more comparable to that of man (4,6,7,8). Recently Wojciechowski, Pershing, and Huether et al. detailed the development of the nude rat skin sandwich flap model for studying the percutaneous absorption of various compounds (9). Briefly, the animals were prepared in three stages; the graft is placed under the skin (dermis up), after two weeks both the graft and the overlaying skin are freed except for the caudal area, later the flap vasculature is microsurgically isolated and the flap translocated to the animals back. Cyclosporine A is used throughout the stages when xenografts are made to reduce graft failure. Studies using this model have shown that A enhances the percutaneous absorption of have suggested that cyclosporine may alter function of the skin (10). 2 cyclosporine compounds and the barrier Because cyclosporine is always administered in cremophor in the skin-flap model, he studies described in this manuscript were undertaken to differentiate the effects of cyclosporine and vehicle (cremophor-EL) on two parameters that influence percutaneous absorption. Dermofluorometry has been used since the 19405 to assess viability and perfusion of skin flaps (11,12,13). Dermofluorometry was used to evaluate the perfusion dynamics of skin in animals on cyclosporine with cremophor-EL (CS) and cremophor-EL (CL) alone. The dynamics in dermofluorometry profiles of animals on both substances were compared with those from animals which were naive with regard to either. In parallel to some of the dermofluorometry studies, the barrier function of the skin was assessed by measurements of transepidermal water 1055 (TEWL). It is thought that TEWL relates to the extent of saturation of the granular layer and the stratum corneum and represents an assessment of the overall barrier function of the skin (14,15,16,17). METHODS AND MATERIALS Animals Outbred congenitally athymic (nude) rats were used because of their depressed immune system and their partial­to- complete hairlessness. Initial breeding pairs were obtained from the animal production facility of the National Cancer Institute (Frederick, Maryland). The local colony was expanded by mating male rats homozygous for nude with female rats heterozygous for nude. Typically, experimental rats weighed 200-300 g at the initiation of experiments. Most of the animals on cyclosporine had flaps which were unusable for other purposes. Anesthesia Animals were anesthetized with Ketamine BeL (KetalarR , Parke-Davis, Morris Plains, NJ 07950, 100 mg/ml) administered intraperitoneally at a dose of 0.01 mg/10 g animal weight. During the experiment further doses (0.05 ml) were administered as necessary to maintain adequate anesthesia. 4 Instruments Dermofluorometer Surface fluorescence was measured using the Fluoroscan Surface Fluorometer (Santa Barbara Technology, Santa Barbara, CAl. Incandescent light is passed through a blue filter (470 nm) and conducted via fiberoptics (wavelength 450-500 nm) to the surface. The fluorescein molecules present emit light in the yellow-green spectrum. This fluorescence is carried along the same fiberoptic pathway and passed through a narrow bandpass yellow-green filter (510-600 nm). The light is then detected by a photomultiplier tube and amplified (11). Evaporimeter The instrument used to measure transepidermal water loss was Model EP 1C (8ervomed, P.O. Box 129, 8-51101 KINNA, Stockholm, Sweden). The instrument had been calibrated as recommended by the manufacturer within 90 days of use in the studies described. Transepidermal water loss is measured by two detectors at a set distance from each other within the detector head. Integrated circuitry calculates the water loss (g/m2/hr), the partial pressure of water vapor, and the relative humidity. 5 Thermometer Core body temperature readings were made using a YSI­Tele- thermometer (Model 44TD, Yellow Springs Instrument Co., Inc., Yellow Springs, DH) attached to a rectal probe. Data Analysis Because some sample sizes and variances were unequal, data sets were compared using the Student's t-test (18) for independent means or Welch's v test (19,20). Slopes and their standard errors for regions of the response curve were calculated as suggested by Colton (18) from log-linear regression of the dermofluorometer time curves. The elimination time (tl/2) was calculated according to Silverman (21) as tl/2=ln 2/(2.303 Kel). In any sample set (i.e., on cyclosporine 3 days, nongrafted abdomen) potential outliers were identified by inspection. Such values were tested against all observations for that sample set using the Z-test (20). Values which resulted in a significant Z score (> 1.9651 or < -1.9651, representing a 95% confidence interval) were deleted from the set if review of the notes evidenced an error in procedure. Values from experiments where an error had been made (i.e., injection leaked) were not used in summary data nor in statistical analysis. Dermofluorometry To measure the appearance and disappearance of 6 dye, the left femoral vein was exposed and fluorescein injected (8 mg/kg). Readings from the skin at various anatomical sites were taken before injection and at various time intervals in areas that were circumscribed with a felt marker. At each time point, core temperature was recorded and the dermofluorometer reading on its calibration surface made. Body temperature was kept within 10 C of 370 C. When the null readings of the instrument were greater than 5 milliwatts away from 500, adjustment was made and the new setting recorded. Dermofluorometer values were converted to Dermofluorometer Index (D. I.) values by dividing the reading (milliwatts) by the control reading (zero time). Values for each time point for each animal in each status category (i.e., minus cremophor-El for 7 Days, Flank) were averaged. The means, standard deviations, standard errors, number of animals, and number of observations were tabulated and used for analysis. Transepidermal Water Loss Readings (water loss, water vapor pressure, and relative humidity) were made and recorded before and after dermofluorometry and at other appropriate times as indicated by the experimental protocol. Readings were not recorded for water loss until such readings had stabilized (usually about 30 seconds). Evaporimeter readings were recorded and means, 7 standard deviations, standard errors, number of animals, and number of observations recorded. Reagents Cyclosporine A (SandimmuneTH I.V., 250 mg/5 ml, Lot No. 10047, Sandoz LTD, Basle, Switzerland) was administered by mixing 0.22 ml with 150 ml of sterile drinking water. Water was checked on alternate days to assure adequate availability and drinking (average consumption was 11 mg/kg daily). Cremophor-EL (Lot No. 4852774, Sandoz LTD, Basle, Switzerland) was administered in the same manner as the cyclosporine. Fluorescein (FluoresciteR , 10%, Alcon Laboratories, Inc., Fort Worth, TX) was diluted with sterile saline and administered via intraperitoneal injection at a dose of 8 mg/kg body weight. RESULTS Dermofluorometer Studies As a control for all dermofluorometry assays, the procedure was performed on three animals that had never been exposed to either cyclosporine in cremophor-EL or cremophor-EL alone. The results of readings taken at nongrafted abdominal sites on three animals are given in Figure 1 and Table 1. Each point represents the· mean of three readings for each animal at each time point. The curve is very similar to that reported by Graham (11) for non-nude animals of comparable size. The figure illustrates the variability and general response pattern seen with this technique. Figure 2 illustrates the results of averaged readings taken on the flank of the same three naive animals compared with the averaged readings for the abdominal sites. Figure 2 has been divided into three regions (A, B, and C). Region A represents the appearance of fluorescein in the tissue and is mathematically characterized by a positive slope, Region B represents saturation of the tissue compartment and is characterized by a plateau in the curve, Region C represents elimination of the dye from the tissue and is characterized by a negative slope (Kel). 9 Figure 1. Dermofluorometry Of Abdominal Sites On Three Naive Nude Rats. The mean D.l. for each of three animals is represented by (.) for NW1 J ( • ) for NW2 J and (. ) for NW3. The mean of all three is represented ( ). 10 • ~ ,....., .c- Q) ~~ Q) So... g -l..L Ie'+- 0 C 0 ........ () ~.~ C E 0 So... • ~'+- (f) Q) ........ :::J C • ~~ • (J) E • l- • • !!? •• xapUJ Jal9WOJonlJOWJao Time: o 1 3 5 7 10 12 14 16 20 25 30 45 60 75 90 105 120 Slope Up: Slope Down: tl/2 Plateau Length: d Table 1. Dermofluorometry of Naive Nude Rats Abdomen Mean D. I.& 1.00 16.95(1.27)c 21.00(1.30) 23.37(1.64) 24.85(1.83) 24.40(2.98) 23.87(2.74) 25.43(4.47) 23.87(3.41) 24.03(2.55) 24.85(4.33) 25.49(4.73) 22.87(3.85) 17.54(2.04) 14.18(2.16) 11.63(3.31) 9.80(3.37) 8.22(3.94) 0.027(0.005) -0.006(0.001) 50.16 min. 19.33(1.16) Nb 3 3 3 3 3 3 3 3 3, 3 3 3 3 3 3 3 3 3 Flank Mean D. I. N 1.00 3 16.85(5.64) 3 19.22(5.30) 3 22.01(7.11) 3 24.84(11.55) 3 19.78(5.85) 3 20.23(6.03) 3 20 . 82 ( 6 . 09 ) 3 22.84(8.48) 3 21.62(7.53) 3 19.81(5.97) 3 20.18(4.89) 3 18.86(2.80) 3 16.43(4.33) 3 14.26(3.61) 3 12.46(5.06) 3 8.92(3.04) 3 6.55(2.51) 3 0.028(0.000) -0.006(0.001) 50.16 min. 33.67(9.29)* aD.I. = Dermofluorometer Index (Reading at given timejReading at zero time). bNumber of animals represented by mean. cMean(standard deviation). dMean length in minutes (standard deviation). *p<0.025 compared with abdominal site. 11 12 Figure 2. Dermofluorometry Of Abdominal And Flank Sites On Three Naive Nude Rats. Abdominal sites (. .), and flank sites (8--------.-). Regions for analysis marked A (appearance of fluorescein in tissue), B (plateau or stabilization of fluorescence level), and C (elimination of fluorescein from site), 3O ,A 20 x Q) "'C ~ 10 L 8 Q) ....f.-J ~ 6 e 5 § - 4 t.e- 0 E 3 L Q) (:) 2 8 c \ ,a.. ~ ',.--~-------- " " 11...., " ' ...... " ........ 10~~~----~----~~----~----~----~--~~--~ o 15 30 45 60 75 90 105 120 Time (Minutes from Injection of Fluorescein) ~ w 14 None of the values from flanks at individual time points were significantly different than those of the abdominal sites (Table 1). Since no differences were evident between flank and abdominal sites, only data from abdominal sites is presented. Table 2 presents the results of dermofluorometer readings taken after animals had been taking oral cyclosporine and cremophor-EL for 3 days. Only the elimination phase of the curve for animals on cyclosporine showed significant differences. There were no significant differences between CL and CS or between CL and naive; this may result from the small sample size for CL (N=2). The characteristics of the dermofluorometry curve for animals on both cyclosporine and cremophor-EL for 7 days showed significant differences from naive as well as between CS and CL (Table 3). In animals receiving CS for 7 days there were significant decreases in amount of fluorescein seen in regions A and B of the DF response curve, while response seen in CL treated group from naive and CS treated animals predominantly elimination phase (decreases in region C). differed in the Data presented in Table 4 demonstrate that these differences persisted but were reduced by 14 days of treatment with either CS or CL. Further, there were differences in the plateau phase seen in both the CS and CL treated animals. Table 2. Dermofluorometry of Nude Rats on Oral Cyclosporine and Cremophor-EL for 3 Days. Naive Cyclosporine Cremophor-EL Time: Mean D. I.a Nb Mean D.I. N Mean D.I. 0 1.00 3 1.00 6 1.00 1 16.95(1.27)c 3 16.33(5.30) 5 23.43(11.41) 3 21.00(1.30) 3 23.34(9.67) 6 28 .. 16(7.69) 5 23.37(1.64) 3 25.24(11.52) 6 33.10(11.46) 7 24.85(1.83) 3 25.98(11.48) 6 31.02(10 .. 44) 10 24.40(2.98) 3 26.42(11.58) 6 33.75(12.37) 12 23.87(2.74) 3 25.96(12.21) 6 34.97(11.92) 14 25.43(4.47) 3 26.12(10.42) 6 33.95(9.69) 16 23.87(3.41) 3 27.27(12.00) 6 34.05(8.42) 20 24.03(2.55) 3 24.96(13.43) 6 33.07(7.82) 25 24.85(4.33) 3 22.37(12.47) 6 33.39(10.20) 30 25.49(4.73) 3 22.24(10.63) 6 30.13(8.16) 45 22.87(3.85) 3 16.59(7.92)* 6 25.89(6.52) 60 17.54(2.04) 3 12.80(6.43)* 6 19.61(6".91) 75 14.18(2.16) 3 9.61(5.79)* 5 12.78(4.83) 90 11.63(3.31) 3 7.04(3.64)* 6 9.01(3.10) 105 9.80(3.37) 3 5.35(3.22)* 6 8.00{2.69) 120 8.22{3.94) 3 4.00(2.62)* 6 4.85(1.20) Slope Up: 0.027(0.005) 0.020(0.058) 0.016(0.006) Slope Down: -0.006(0.001) -0.008{0.000) -0.009(0.001) tl/2 50.16 min. 37.62 min. 33.44 min. Plateau Length: d 19.33(1.16) 12.67(4.97)* 15.00(7.07) aD.I. = Dermofluorometer Index (Reading at given time/Reading at zero time). bNumber of animals represented by mean. cMean(standard deviation). dMean length in minutes (standard deviation). * p<0.05 compared with value of naive animals. + p<0.05 compared with animals on cyclosporine. 15 N 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Time: o 1 3 5 7 10 12 14 16 20 25 30 45 60 75 90 105 120 Slope Up: Slope Down: tl/2 16 Table 3. Dermofluorometry of Nude Rats on Oral Cyclosporine and Cremophor-EL for 7 Days. Naive Mean D. I.8 1.00 16.95(1.27)c 21.00(1.30) 23.37(1.64) 24.85(1.83) 24.40(2.98) 23.87{2.74) 25.43{4.47) 23.87(3.41) 24.03(2.55) 24.85(4.33) 25.49{4.73) 22.87(3.85) 17.54(2.04) 14.18{2.16) 11.63{3.31) 9.80(3.37) 8.22(3.94) 0.027(0.005) -0.006(0.001) 50.16 min. Cyclosporine Cremophor-EL Nb Mean D.I. N Mean D.I. N 3 1.00 3 11.64(2.36)* 3 14.91{3.04)* 3 15.88(2.42)* 3 17.06(2.78)* 3 17.72(2.73)* 3 17.74(3.35)* 3 18.22(3.17)* 3 17.60(5.19)* 3 19.93(3.41)* 3 18.96(3.23)* 3 16.36{4.03)* 3 15.55(5.79)* 3 13.35(4.12)* 3 12.14(5.69) 3 11.02(6.03) 3 9.40(6.06) 3 8.80{6.91) 0.019(0.005) -0.003(0.000) 100.33 min. 5 1.00 2 5 20.86(4.60) 2 5 21.91(1.85)+ 2 5 22.51(1.82)+ 2 5 23.44(2.35)+ 2 5 22.43(2.65) 2 5 22.51(0.41) 2 5 21.68(1.59) 2 4 19.42(1.38) 2 5 19.38(1.01) 2 5 17.14(1.92)* 2 5 15.04(0.37)* 2 5 7.69(0.13)*+ 2 5 5.68(0.17)*+ 2 5 3.46(0.93)*+ 2 5 2.60(0.57)*+ 2 5 2.07(0.53)*+ 2 5 1.77(0.80)*+ 2 0.010(0.003) -0.011(0.001) 27.36 min. Plateau Length: d 19.33(1.16) 19.60(9.40) 10.00(0.00)*+ aD.I. = Dermofluorometer Index (Reading at given time/Reading at zero time). bNumber of animals represented by mean. CMean(standard deviation). dMean length in minutes (standard deviation). * p<0.05 compared with value of naive animals. + p<0.05 compared with animals on cyclosporine. 17 Table 4. Dermofluorometry of Nude Rats on Oral Cyclosporine and Cremophor-EL for 14 Days. Naive Cyclosporine Cremophor-EL Time: Mean D. I.a Nb Mean D.I. N Mean D.I. N 0 1.00 3 1.00 5 1.00 4 1 16.95(1.27)c 3 9.91(4.68)* 5 21.36(7.61)*+ 4 3 21.00(1.30) 3 11.04(2.94)* 5 26.27(11.51)*+4 5 23.37(1.64) 3 14.33(5.49)* 5 27.85(13.02)+ 4 7 24.85(1.83) 3 14.94(7.19)* 5 31.83(15.79)*+4 10 24.40(2.98) 3 14.62(4.54)* 5 30.01(15.86)*+4 12 23.87(2.74) 3 15.16(7.71)* 5 30.30(15.10)*+4 14 25.43(4.47) 3 11.50(2.68)* 4 29.68(14.12)+ 4 16 23.87(3.41) 3 14.43(7.59)* 5 31.66(16.63)*+4 20 24.03(2.55) 3 15.32(9.64)* 5 26.59(15.47)+ 4 25 24.85(4.33) 3 15.06(5.53)* 5 24.19(13.57)+ 4 30 25.49(4.73) 3 13.45(6.38)* 5 22.83(15.40)+ 4 45 22.87(3.85) 3 12.23(4.65)* 5 16.00(12.75)* 4 60 17.54(2.04) 3 12.38(6.12)* 5 10.38(8'.33)* 4 75 14.18(2.16) 3 11.55(6.79) 5 7.45(7.01)*+ 4 90 11.63(3.31) 3 10.19(6.08) 5 7.12(4.61)* 2 105 9.80(3.37) 3 8.87(7.41) 5 4.02(3.70)*+ 4 120 8.22(3.94) 3 7.86(5.61) 5 3.47(3.19)*+ 4 Slope Up: 0.027(0.005) 0.032(0.006) 0.012(0.004) Slope Down: -0.006(0.001) -0.003(0.000) -0.009(0.000) tl/2 50.16 min. 100.33 min. 33.44 min. Plateau Length: d 19.33(1.16) 58.60(32.90)* 11.25(6.19)*+ aD.I. = Dermofluorometer Index (Reading at given time/Reading at zero time). bNumber of animals represented by mean. cMean(standard deviation). dMean length in minutes (standard deviation). * p<0.05 compared with value of naive animals. + p<0.05 compared with animals on cyclosporine. 18 After 21 days of treatment, the differences between CS treated and naive animals began to disappear (Table 5). Yet, the decreases between naive animals and those treated with CL persisted. Also, some of the decrease between CS treated and CL treated animals continue in regions A and B. To evaluate the duration of the altered DF profile induced by CS or CL treatment, the dermofluorometry response was evaluated following cessation of treatment with either agent. In Table 6 the results of studies performed on animals 7 days following removal from oral treatment with either compound are presented. No differences were noted between the CS treated group and the naive groups in regions A, B, or C. Significant differences between the CL treated group and naive groups were still present (Table 6). Significant differences also persisted in the individual time points seen in regions A and C of the CL curve. The slopes in these regions are not statistically different but are parallel. Removal of animals from CS for 14 days, resulted in no significance differences in the DF profile compared with naive animals. However, the animals treated with CL still demonstrate the same differences in the region B and region C of the response curve (Table 7). The above responses in abdominal site skin were also observed in grafted abdominal sites and flanks (Appendix A, B, C, and D). Table 5. Dermofluorometry of Nude Rats on Oral Cyclosporine and Cremophor-EL for 21 Days. Naive Cyclosporine Cremophor-EL Time: Mean D. I.a Nb Mean D.I. N Mean D.I. 0 1.00 3 1.00 8 1.00 1 16.95(1.27)c 3 13.96(5.46)* 8 9.97(6.65)*+ 3 21.00(1.30) 3 18.26(6.26)* 8 13.07(6.30)* 5 23.37(1.64) 3 21.17(7.55) 8 13.34(4.67)*+ 7 24.85(1.83) 3 21.49(6.58)* 8 16.01(5.59)*+ 10 24.40(2.98) 3 22.19(7.39) 8 17.99(8.65)* 12 23.87(2.74) 3 23.32(8.17) 7 16.57(5.45)*+ 14 25.43(4.47) 3 24.13(7.90) 8 16.20(6.68)*+ 16 23.87(3.41) 3 23.24(6.17) 8 17.36(9.72)*+ 20 24.03(2.55) 3 23.07(8.21) 8 17.72(6.73)*+ 25 24.85(4.33) 3 20.99(7.63) 8 17.30(8.37)* 30 25.49(4.73) 3 20.20(8.01)* 8 16.23(6.82)* 45 22.87(3.85) 3 15.65(7.97)* 8 12.69(2.91)* 60 17.54(2.04) 3 12.00(5.99)* 7 9.61(2'.98)* 75 14.18(2.16) 3 10.13(5.37) 7 7.39(2.10)* 90 11.63(3.31) 3 7.64(4.90)* 8 5.78(1.81)* 105 9.80(3.37) 3 6.02(3.32) 8 3.71(2.16)* 120 8.22(3.94) 3 5.18(3.07) 6 2.78(1.12)* Slope Up: 0.027(0.005) 0.017(0.005) 0.027(0.004) Slope Down: -0.006(0.001) -0.007(0.000) -0.009(0.000) tl/2 50.16 min. 42.00 min. 33.44 min. Plateau Length: d 19.33(1.16) 22.71(15.37) 24.67(5.69) aD.I. = Dermofluorometer Index (Reading at given time/Reading at zero time). bNumber of animals represented by mean. cMean(standard deviation). dMean length in minutes (standard deviation). * p<0.05 compared with value of naive animals. + p<0.05 compared with animals on cyclosporine. 19 N 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Time: o 1 3 5 7 10 12 14 16 20 25 30 45 60 75 90 105 120 Slope Up: Slope Down: tl/2 20 Table 6. Dermofluorometry of Nude Rats Off Oral Cyclosporine and Cremophor-EL for 7 Days. Naive Mean D. I.& 1.00 16.95(l.27)c 21.00(1.30) 23.37(1.64) 24.85(1.83) 24.40(2.98) 23.87(2.74) 25.43(4.47) 23.87(3.41) 24.03(2.55) 24.85(4.33) 25.49(4.73) 22.87(3.85) 17.54(2.04) 14.18(2~16) 11.63(3.31) 9.80(3.37) 8.22(3.94) 0.027(0.005) -0.006(0.001) 50.16 Min. Cyclosporine Cremophor-EL Nb Mean D.I. N Mean D.I. N 3 1.00 3 14.51(6.12) 3 18.90(8.45) 3 19.11(6.66) 3 20.50(5.67) 3 21.75(6.57) 3 22.60(5.88) 3 21.94(5.59) 3 21.60(6.51) 3 21.60(6.44) 3 20.87(6.54) 3 19.61(5.76) 3 16.27(4.35) 3 12.97(5.01) 3 10.52(5.43) 3 8.28(5.03) 3 6.75(4.43) 3 6.10(4.54) 0.015(0.004) -0.006(0.000) 50.16 min. 8 1.00 4 8 19.25(6.48)+ 4 8 22.08(4.79)+ 4 8 25.31(5.06)+ 4 8 24.37(3.84)+ 4 8 27.02(8.93)+ 4 7 24.00(4.63) 4 8 25.78(9.16) 4 8 26.48(5.70) 4 8 24.91(5.35) 4 8 24.03(3.89) 4 8 22.70(3.02)+ 4 8 15.52(5.39)* 4 8 11.21(4~17)* 4 8 7.77(2.94)* 4 8 4.28(1.75)*+ 4 8 3.33(1.23)*+ 4 8 2.72(1.23)*+ 4 0.015(0.004) -0.011(0.001) 27.36 min. Plateau Length:d 19.33(1.16) 22.88(10.01) 22.00(3.56) aD.I. = Dermofluorometer Index (Reading at given time/Reading at zero time). bNumber of animals represented by mean. cMean(standard deviation). dMean length in minutes (standard deviation). * p<O.05 compared with value of naive animals. + p<0.05 compared with animals on cyclosporine. Table 7. Dermofluorometry of Nude Rats Off Oral Cyclosporine and Cremophor-EL for 14 Days. Naive Cyclosporine Cremophor-EL Time: Mean D. I.a Nb Mean D.I. N Mean D.I. 0 1.00 3 1.00 5 1.00 1 16.95(1.27)c 3 16.01(3.93) 5 14.91(3.52) 3 21.00(1.30) 3 22.40(3.04) 5 22.48(0.88) 5 23.37(1.64) 3 24.58(7.16) 5 22.46(0.36) 7 24.85(1.83) 3 26.56(8.06) 5 20.99(2.71) 10 24.40(2.98) 3 23.18(10.37) 5 20.14(3.33) 12 23.87(2.74) 3 32.32(12.67)* 3 18.57(4.85)*+ 14 25.43(4.47) 3 29.18(8.06) 5 16.13(9.58)* 16 23.87(3.41) 3 30.35(9.37) 5 19.02(6.19)*+ 20 24.03(2.55) 3 28.21(9.14) 5 16.49(5.95)*+ 25 24.85(4.33) 3 24.61(10.43) 5 16.70(3.11)*+ 30 25.49(4.73) 3 23.43(10.47) 5 17.41(0.98)*+ 45 22.87(3.85) 3 18.59{10.34) 5 13.92(1.81)* 60 17.54(2.04) 3 13.48(8.54)* 5 10.23(0'.53)*+ 75 14.18(2.16) 3 12.31(7.19) 5 6.50(0.71)*+ 90 11.63(3.31) 3 9.61{6.56) 5 5.36(1.51)*+ 105 9.80(3.37) 3 7.59(7.53) 4 4.36(1.21)* 120 8.22(3.94) 3 5.44(4.59) 5 2.47(0.46)* Slope Up: 0.027{0.005) 0.035(0.010) 0.044(0.026) Slope Down: -0.006{0.001) -0.007(0.000) -0.009(0.001) tl/2 50.16 min. 43.00 min. 33.44 min. Plateau Length: d 19.33(1.16) 19.00(5.83) 21.00(5.66) aD.I. = Dermofluorometer Index (Reading at given time/Reading at zero time). bNumber of animals represented by mean. cMean{standard deviation). dMean length in minutes (standard deviation). * p<0.05 compared with value of naive animals. + p<0.05 compared with animals on cyclosporine. 21 N 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 22 To explore the possibility that the plateau (region B) observed in these curves was not due to self-quenching (3) a standard concentration response curve for the dermofluorometer was evaluated. The calibration setting used routinely (500 milliwatts) was originally chosen empirically. The linearity of the response curves was determined at 500 milliwatts and at 250 milliwatts to evaluate instrument drift during the assay. As seen from Figure 3 the dermofluorometric response to different concentrations of fluorescein was linear over the range of 10 to 400 ng/ml. The correlation coefficient at both calibration settings was greater than 0.99. Readings on distilled water were the same as seen on the skin of animals prior to injection of fluorescein. Figure 4 plots the elimination half-life values (tl/2) as calculated from the slopes of elimination. The tl/2 was shorter than naive for animals on CL at all time points. At 7 and 14 days of treatment with CS the tl/2S were approximately double that of naive (Figure 4, Tables 3 and 4) . Transepidermal Water Loss As a noninvasive measure of barrier function transepidermal water loss measurements were made. Figures 5 and 6 show the results of measurements of transepidermal 23 Figure 3. Dermofluorometer Calibration Curve For Fluorescein. Dermofluorometer readings in milliwatts vs concentration of fluorescein. Mean readings with fluorometer set at 500 milliwatts (tt). Regression of 500 milliwatt data ( ). Mean readings with fluorometer set at 250 milliwatts «». Regression of 250 milliwatt data (-----) . ~ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ , \ \ \ \ \ \ \ , \ \ • \ , \ , \ \ \ 0, \ \ \ \ \ , \ \ \ \ \ \ \ \ , \ \ '0 \ \ \ \ \ \ \ o \ \ , o • 0E, -.:::to) No.. a C 0-­NO) o (f) ~~ ~O ::J aLL N o 00 --~--~~--~----~--------~--~----~-----+o 8 ~ 8 ~ 8 ~ 8 g 8 ~ 8 ~ 8 ~ 0 ~ ~ ~ ~ ~ ~ ~ ~ ~ N N - - 5U!PP9~ J9l9WOJonlJoWJ90 llPM~ I I ~W 24 Figure 4. Tissue Elimination Half-Life Values Rats On Oral Cyclosporine and Cremophor-EL. naive animals ( ), for CS treated animals and CL treated animals (0------0). Values were from observed slopes by the following equation: 2/(2.303 Kel). Numbers of animals used calculation are beside each point. For Value 25 Nude for (.------.. ) , calculated tl/2 = In for each 26 U) N ~ • 14] ..- I , I I , I \ I \ I \ I \ ,I \ \ I \ I , I , rol ~ rp £"... I \ I \ I \, ,I \ , \, I -+-' I \, I C I \ I (]) \ I .,c,:,b" f'4m)/ N.. - -E+- ' t + rd ",,' I .,"" "",' II G) -",," I ,." I ~ ,," I ,.,,"" I I- .,'" I ,"" I ,.' I "",' I 4- If) ,,"'.'" Vi .. ~ 0 '" dJ ..- III \1 \ + (fJ II \\ >-. II \\ rd I \ I \ 0 I , I , Lf)1 a, '& £". ' ... ' ... I + " ........ ........ ........ II I .... , ,I .... , ....... , " , ........ ........ ,I .......... I ...... .... , l..D. N (Y) + 0 0 0 0 0 0 0 0 0 0 0 0 ..- 0 0" co ["..... '-.D U'") ~ (Y) N ..- ..- ..- (S8+nu ~ W) l/l+ 10 ~+6U8l 27 water loss for animals on and off oral cyclosporine for various time periods. The numbers beside the bars represent the number of animals used to calculate the mean value for the plotted data collected at both abdominal and flank skin sites. Though no statistically significant (p>0.05) differences were noted at the same skin site with CS treatment, there were consistent significant differences (mean difference = 3.02, standard deviation = 2.40) between abdominal and flank skin site (p<0.05) at all time points except at day 7 of CS treatment. As was evidenced from the data, TEWL measurements could differentiate between anatomic sites (abdomen vs flank) but did not indicate differences resulting from treatment with es. Therefore, evaluation of the sensitivity of the evaporimeter to alterations in barrier function was ascertained with serial tape strippings (3M brand cellophane tape; 3M, St Paul, MN) of the skin site. Fifteen to twenty tape strippings were necessary to see significant alteration in barrier function as detectable by water loss measurements (data not shown). 28 Figure 5. Transepidermal Water Loss Measurements On Flanks And Abdomens On Rats On Oral Cyclosporine. Numbers beside bars represent number of animals for which bar represents mean. Abdominal sites (open bars), and flank sites (hatched bars). Vertical lines represent standard errors of the means. * p<O.05, ** p<O.005 Abdomen vs Flank. 12 ~ 11 ~ ..c 10 . ~ NE 9 "0" )8 '-..../ -f--J 7 c Cl) 6 E ~ 5 :j ~ 4 ~ 3 ~ 2 ~1 29 6** 7* 5~'* O~~~~~~~~~~~ Naive 3 7 1Ll 21 Days On Oral Cyclosporine 30 Figure 6. Transepidermal Water Loss Measurements On Flanks And Abdomens On Rats Off Oral Cyclosporine. Numbers beside bars represent number of animals for which bar represents mean. Abdominal sites (open bars), and flank sites (hatched bars). Vertical lines represent standard errors of the means. * p<0.05, ** p<0.005 Abdomen vs Flank. 12 ~ 11 L.. ..c 10 N"'" E 9 "0-)8 '-"" -t-J 7 c Cl) 6 E ~ 5 ::l ~ 4 ~ 3 ~ 2 ~1 31 7* 6**' 8** O~~~~~~~~~~~ Naive -3 -7 -14 -21 Days Off Oral Cyclosporine DISCUSSION To facilitate discussion, Table 8 summarizes the statistical analysis of all the dermofluorometry data. In our studies, changes in hemodynamics of DF profile in animals on either CS in CL or CL alone were not evident by 3 days (Table 8). While other studies have reported elevated renin activity in animals receiving cyclosporine for three days (22, 23, 24, 25) the influence of vehicle was not assessed. Batlle et ale (26) reported no change in glomerular with CS. filtration rate in rats for three days treated In our studies both CS and CL treated animals demonstrated a shortened length of plateau and clearance of fluorescein dye was shortened (the CL change was not statistically significant Table 2). Since there is no difference between the CS and CL data, the agent responsible could not be delineated. Following seven days of treatment, distinct differences in the DF profile were observed between untreated animals and those receiving CS (regions A and B, Table 8). The overall DF profiles in CS and CL treated animals were decreased (Table 8). Such an alteration would be consistent with renal damage and decreased fluorescein clearance caused by partially compensated vaso- 33 Table 8. Overall Evaluation of Dermofluorometer Profile for Rats on Oral Cyclosporine and Cremophor-EL. Region A Days of Exposure +3 +7 +14 +21 -7 -14 CS v~ Nla1v~ Individual Points Oa 0 0 0 Slope Up 0 0 0 0 0 0 CL v~ Ha;1ve Individual Points 0 0 + 0 0 Slope Up 0 0 0 0 0 0 CL vs CS Individual Points 0 + + + 0 Slope Up 0 0 0 0 0 0 Region B Days of Exposure +3 +7 +14 +21 -7 -14 CS vs Naive Individual Points 0 0 0 0 Length a + a a 0 CL vs Naive Individual Points a a + 0 Length a 0 a a CL vs CS Individual Points a 0 + 0 Length a 0 0 0 0 Region C Days of Exposure +3 +7 +14 +21 -7 -14 CS vs Naiv~ Individual Points 0 a 0 a Slope Down a a a a 0 a CL vs Naive Individual Points 0 Slope Down 0 0 a 0 a 0 CL vs CS Individual Points 0 0 0 Slope Down 0 a a 0 a a bSign indicates direction (+ for higher, for lower) of statistically significant differences (p<O. 05) . 34 dilation from CL stimulated mediator release (24, 26, 27, 28, 29, 31, 32). While vasodilator responses to histamine have been noted in rat skin, there is also evidence that vascular response diminishes with chronic exposure (34,35), suggesting that other mechanisms may be involved in the sustained response seen in our studies. The changes in DF profile noted at seven days of CS treatment continue through fourteen days. Following twenty-one days of CS treatment the DF profiles return to those measured in naive animals. The data from CL treated animals, however, continued to differ significantly from untreated animals. Seven and fourteen days after cessation of drug or vehicle treatment the DF profile returns to that observed in naive rats with the exception of the changes seen in region C in CL treated animals. The most striking and consistent feature in the modified DF profiles presented was enhancement of elimination (region C) of fluorescein following treatment with CL. The prolonged duration of the alteration following cessation of treatment remains to be elucidated. It is unclear why this alteration in region C produced by CL was absent in the profiles of animals removed from CS, since CL is also present in the drug treated animals. If the enhanced elimination was the result of mediator release from the microvascular endothelium, one would have expected the response to diminish rapidly in the absence of 35 stimulant (28, 29). The striking changes in tissue elimination half-life values seen at 7 and 14 days (Figure 4) are consistent with the reduced kidney function resulting from the known nephrotoxicity of CS. We have no explanation for the return to normal of the tl/2 values with animals on CS for 21 days. The tl/2 values for animals on CL did not show such dramatic changes over time, though CL has also been demonstrated to cause alterations in kidney function (31). Overall the alterations seen could be explained by a combination of reduced glomerular filtration due to the nephrotoxicity (23, 24, 31, 36, 37, 38, 39) of CS which was compensated to various degrees by the direct or indirect release of modulators of vascular tone by CL. This "Ying­Yang" situation could have resulted in the nonsignificant data observed in CS treated animals and the significant response in CL treatments. As noted by Wassef, Cohen, and Langer (32) the oral administration of CS in rats results in highly variable systemic concentrations of the drug. This same investigative group found that the route of administration of CS that resulted in the least systemic variability of drug was via subcutaneous injection (even here the variability was 60%). The impact of such variability in the studies reported needs to be explored. Unfortunately, few studies have utilized vehicle (CL) to differentiate the CS effect. At least one study has concluded that CL containing solutions should avoided when exploring the influence of cyclosporine A 36 (31) be on kidney function owing to the renal vasoconstriction effects of CL alone. Our data support the contention that differentiation of the CL effects from the CS effects would be difficult but necessary to a complete understanding of the drug effects on vascular parameters. Other studies which examined the effect of CL on hemodynamics have demonstrated that the oxethylated components of the vehicle were responsible for the release of histamine and consequent changes in hemodynamics (31) in dogs and (34, 34) in rats. It has also been suggested that the antidiuretic effect of cremophor-EL in rats may be the result of laxative properties (35). A common observation in our studies was the development of diarrhea among cyclosporine treated animals, but not the among cremophor treated groups. To address problems seen in these studies it is suggested that similar studies to those reported in this manuscript be conducted in sex and weight matched animals and administering the drug or its vehicle only subcutaneously. An effort could be made to find another vehicle than CL. If such an alternate is not available, sequential studies are suggested. By starting the animals on CL alone and performing the assays, and then adding CS, 37 the impact of the two agents on perfusion dynamics might have a better chance of being delineated. The effects of histamine, leukotrienes and other modulators of vascular tone on DF profiles could provide insight into the differential effect we see between CS and CL. Since CS alters lipid and carbohydrate metabolism in the rat (25, 38), it was hypothesized that chronic administration of CS might result in sufficient variance in the lipid component of skin to result in changes in water loss. Recent studies (40, 41) have shown that diet can affect the lipid composition of the skin and, therewith, transepidermal water loss. The barrier function of the skin is a result of the lipid composition of the stratum corneum (14, 41, 42, 43, 44, 45, 46, 47, 48). While the transepidermal water loss data clearly demonstrate differences between anatomic sites regardless of treatment with CL or CS, no clear pattern of modification of barrier function was seen (Figures 4 and 5). This may have resulted from an insufficient dose of CS to cause significant changes and/or sufficient essential fatty acids in the rat chow mask changes. Samples of skin from animals that had been exposed to CS for various times have been submitted for fourier-transformed infrared analysis (46). Future experiments should continue this approach and be combined with studies to explore the impact of various dosing regimens. A more sensitive way to measure the changes may stripping) be to examine the change in damage necessary to see significant changes using various time on CL and CS. in 38 (tape­TEWL APPENDICES APPENDIX A SUMMARY DERMOFLUOROMETER DATA FOR NAIVE ANIMALS 41 Summary Dermofluorometer Data for Naive Animals (Abdominal Site) Timea Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 3 9 1 lS.95 1.27 0.73 3 9 3 21.00 1.30 0.75 3 9 5 23.37 1.S4 0.94 3 9 7 24.85 1.83 1.0S 3 9 10 24.40 2.98 1.72 3 9 12 23.87 2.74 1.58 3 9 14 25.43 4.47 2.58 3 9 16 23.87 3.41 1.97 3 9 20 24.03 2.55 1.47 3 9 25 24.85 4.33 2.50 3 9 30 25.49 4.73 2.73 3 9 45 22.87 3.85 2.22 3 9 60 17.54 2.04 1.18 3 9 75 14.18 2.16 1.25 3' 9 90 11.63 3.31 1.91 3 9 105 9.80 3.37 1.95 3 9 120 8.22 3.94 2.28 3 9 a Time in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. Summary Dermofluorometer Data for Naive Animals (Flank Site) 42 Timea Mean D.l. Std. Dev. b Std. Err.c Nd (Obs)e o 1.00 1 16.85 3 19.22 5 22.01 7 24.84 10 19.78 12 20.23 14 20.82 16 22.84 20 21.62 25 19.81 30 20.18 45 18.86 60 16.43 75 14.26 90 12.46 105 8.92 120 6.55 N/A 5.64 5.30 7.11 11.55 5.85 6.03 6.09 8.48 7.53 5.97 4.89 2.80 4.33 3.61 5.06 3.04 2.51 N/A 3.25 3.06 4.10 6.67 3.38 3.48 3.51 4.90 4.35 3.45 2.83 1.62 2.50 2.09 2.92 1.75 1.45 3 9 3 9 3 9 3 9 3 9 3 9 3 9 3 9 3 9 3 9 3 9 3 9 3 9 3 9 3' 9 3 9 3 9 3 9 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. APPENDIX B SUMMARY DERMOFLUOROMETER DATA FOR ANIMALS ON ORAL CYCLOSPORINE 44 Summary Dermofluorometer Data for Animals On Oral Cyclosporine 3 Days (Abdominal Site) Timea Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 6 24 1 16.33 5.30 2.37 5 21 3 23.34 9.67 3.95 6 24 5 25.24 11.52 4.70 6 24 7 25.98 11.48 4.69 6 24 10 26.42 11.58 4.73 6 24 12 25.96 12.21 4.98 6 24 14 26.12 10.42 4.25 6 24 16 27.27 12.00 4.90 6 24 20 24.96 13.43 5.48 6 24 25 22.37 12.47 5.09 6 24 30 22.24 10.63 4.34 6 24 45 16.59 7.92 3.23 6 24 60 12.80 6.43 2.63 6' 24 75 9.61 5.79 2.59 5 21 90 7.04 3.64 1.48 6 24 105 5.35 3.22 1.31 6 24 120 4.00 2.62 1.07 6 24 a Time in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 45 Summary Dermofluorometer Data for Animals On Oral Cyclosporine 7 Days (Abdominal Site) Times. Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 5 21 1 11.64 2.36 1.06 5 21 3 14.91 3.04 1.36 5 21 5 15.88 2.42 1.08 5 21 7 17.06 2.78 1.24 5 21 10 17.72 2.73 1.22 5 21 12 17.74 3.35 1.50 5 21 14 18.22 3.17 1.42 5 21 16 17.60 5.19 2.60 4 15 20 19.93 3.41 1.53 5 21 25 18.96 3.23 1.45 5 21 30 16.36 4.03 1.80 5 21 45 15.55 5.79 2.59 5 21 60 13.35 4.12 1.84 5· 21 75 12.14 5.69 2.54 5 21 90 11.02 6.03 2.70 5 21 105 9.40 6.06 2.71 5 21 120 8.80 6.91 3.09 5 21 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 46 Summary Dermofluorometer Data for Animals On Oral Cyclosporine 14 Days (Abdominal Site) Timea Mean D.I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 5 25 1 9.91 4.68 2.10 5 25 3 11.04 2.94 1.32 5 25 5 14.33 5.49 2.45 5 25 7 14.49 7.19 3.22 5 25 10 14.62 4.54 2.03 5 25 12 15.16 7.71 3.45 5 25 14 11.50 2.68 1.34 4 19 16 14.43 7.59 3.40 5 25 20 15.32 9.64 4.31 5 25 25 15.06 5.53 2.47 5 25 30 13.45 6.38 2.85 5 25 45 12.23 4.65 2.08 5 25 60 12.38 6.12 2.74 5" 25 75 11.55 6.79 3.04 5 25 90 10.19 6.08 2.72 5 25 105 8.87 7.41 3.31 5 25 120 7.86 5.61 2.51 5 25 aTime in minutes from I. V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 47 Summary Dermofluorometer Data for Animals On Oral Cyclosporine 21 Days (Abdominal Site) Timea Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 8 18 1 13.96 5.46 1.93 8 18 3 18.26 6.26 2.21 8 18 5 21.17 7.55 2.67 8 18 7 21.49 6.58 2.32 8 18 10 22.19 7.39 2.61 8 18 12 23.32 8.17 3.09 7 15 14 24.13 7.90 2.79 8 18 16 23.24 6.17 2.18 8 18 20 23.07 8.21 2.90 8 18 25 20.99 7.63 2.70 8 18 30 20.20 8.01 2.83 8 18 45 15.65 7.97 2.82 8 18 60 12.00 5.99 2.26 7' 15 75 10.13 5.37 2.03 7 15 90 7.64 4.90 1.73 8 18 105 6.02 3.32 1.17 8 18 120 5.18 3.07 1.25 6 12 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 48 Summary Dermofluorometer Data For Animals On Oral Cyclosporine 3 Days (Flank Site) Timea Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 2 6 1 13.86 0.35 0.25 2 6 3 14.60 5.24 3.71 2 6 5 19.35 2.17 1.53 2 6 7 22.34 1.64 1.16 2 6 10 23.52 2.70 1.91 2 6 12 22.97 3.74 2.65 2 6 14 22.18 2.68 1.90 2 6 16 22.88 0.49 0.35 2 6 20 21.93 3.53 2.50 2 6 25 26.34 6.09 4.31 2 6 30 22.65 2.94 2.08 2 6 45 19.22 9.86 6.97 2 6 60 17.40 8.90 6.29 2 6 75 15.03 6.86 4.85 2 6 90 13.11 6.42 4.54 2 6 105 9.98 6.23 4.41 2 6 120 9.97 7.49 5.30 2 6 a Time in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 49 Summary Dermofluorometer Data For Animals On Oral Cyclosporine 14 Days (Flank Site) Timea Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 4 12 1 8.93 3.75 1.88 4 12 3 10.21 5.65 2.82 4 12 5 9.82 4.84 2.42 4 12 7 10.80 4.10 2.05 4 12 10 13.49 6.19 3.09 4 12 12 12.79 6.36 3.18 4 12 14 9.69 2.85 1.65 3 9 16 12.15 5.58 2.79 4 12 20 11.71 6.03 3.02 4 12 25 12.25 6.18 3.09 4 12 30 11.72 6.42 3.21 4 12 45 11.25 5.96 2.98 4 12 60 10.88 6.71 3.35 4 12 75 10.55 7.19 3.59 4 12 90 9.56 6.66 3.33 4 12 105 8.67 6.74 3.37 4 12 120 7.54 6.55 3.27 4 12 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 50 Summary Dermofluorometer Data For Animals On Oral Cyclosporine 21 Days (Flank Site) Timea Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 4 12 1 7.87 0.81 0.40 4 12 3 11.72 3.70 1.85 4 12 5 12.01 3.03 1.51 4 12 7 11.55 2.27 1.13 4 12 10 11.82 2.53 1.26 4 12 12 13.57 4.12 2.06 4 12 14 13.47 5.03 2.51 4 12 16 14.67 6.73 3.36 4 12 20 13.96 6.74 3.37 4 12 25 15.23 6.93 3.46 4 12 30 13.57 4.83 2.42 4 12 45 14.06 6.00 3.00 4 12 60 14.79 8.92 4.46 4 12 75 13.78 11.67 5.83 4 12 90 10.98 9.60 4.80 4 12 105 9.75 8.6 4.30 4 12 120 10.38 11.71 5.85 4 12 a Time in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 51 Summary Dermofluorometer Data For Animals On Oral Cyclosporine 3 Days (Grafted Abdominal Site) Times. Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 4 12 1 19.23 6.53 3.26 4 12 3 23.17 7.52 3.76 4 12 5 28.44 11.46 5.73 4 12 7 28.09 12.11 6.05 4 12 10 29.30 12.84 6.42 4 12 12 27.01 11.08 5.54 4 12 14 28.90 12.66 6.33 4 12 16 27.28 12.07 6.03 4 12 20 27.66 13.01 6.51 4 12 25 23.96 9.46 4.73 4 12 30 22.18 9.86 4.93 4 12 45 17.15 7.28 3.64 4 12 60 13.97 7.37 3.69 4 12 75 9.59 3.83 1.91 4 12 90 6.86 3.34 1.67 4 12 105 5.67 2.76 1.38 4 12 120 4.26 1.77 0.88 4 12 aTime in minutes from I. V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 52 Summary Dermofluorometer Data For Animals On Oral Cyclosporine 21 Days (Grafted Abdominal Site) Timea Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 5 15 1 21.09 2.21 0.99 5 15 3 23.83 6.22 2.78 5 15 5 27.30 4.91 2.20 5 15 7 28.14 6.02 2.69 5 15 10 28.88 6.11 2.73 5 15 12 27.77 7.89 3.95 5 15 14 26.76 7.34 3.28 5 15 16 26.14 8.57 3.83 5 15 20 22.86 9.10 4.07 5 15 25 22.18 7.78 3.48 5 15 30 19.32 6.15 2.75 5 15 45 14.27 4.59 2.05 5 15 60 9.25 3.40 1.52 5 15 75 6.56 3.96 2.29 3 9 90 4.43 1.88 0.84 5 15 105 3.42 1.52 0.68 5 15 120 2.23 0.71 0.36 4 12 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. APPENDIX C SUMMARY DERMOFLUOROMETER DATA FOR ANIMALS ON ORAL CREMOPHOR-EL 54 Summary Dermofluorometer Data For Animals On Oral Cremophor-EL 3 Days (Abdominal Site) Timea Mean D.l. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 2 6 1 23.43 11.41 8.07 2 6 3 28.16 7.69 5.44 2 6 5 33.10 11.46 8.10 2 6 7 31.02 10.44 7.32 2 6 10 33.75 12.37 8.75 2 6 12 34.97 11.92 8.43 2 6 14 33.95 9.69 6.85 2 6 16 34.05 8.42 5.95 2 6 20 33.07 7.82 5.53 2 6 25 33.39 10.20 7.21 2 6 30 30.13 8.16 5.77 2 6 45 25.89 6.52 4.61 2 6 60 19.61 6.91 4.89 2 6 75 12.78 4.83 3.42 2 6 90 9.01 3.10 2.19 2 6 105 8.00 2.69 1.90 2 6 120 4.85 1.20 0.85 2 6 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 55 Summary Dermofluorometer Data For Animals On Oral Cremophor-EL 7 Days (Abdominal Site) Timea Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 2 6 1 20.86 4.60 3.26 2 6 3 21.91 1.85 1.31 2 6 5 22.51 1.82 1.29 2 6 7 23.44 2.35 1.66 2 6 10 22.43 2.65 1.87 2 6 12 22.51 0.41 0.29 2 6 14 21.68 1.59 1.12 2 6 16 19.42 1.38 0.98 2 6 20 19.38 1.01 0.72 2 6 25 17.14 1.92 1.36 2 6 30 15.04 0.37 0.26 2 6 45 7.69 0.13 0.09 2 6 60 5.68 0.17 0.12 2 6 75 3.46 0.93 0.66 2 6 90 2.60 0.57 0.40 2 6 105 2.07 0.53 0.37 2 6 120 1.77 0.80 0.57 2 6 a Time in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 56 Summary Dermofluorometer Data For Animals On Oral Cremophor-EL 14 Days (Abdominal Site) Times. Mean D.l. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 4 12 1 21.36 7.61 3.80 4 12 3 26.27 11.51 5.76 4 12 5 27.85 13.02 6.51 4 12 7 31.83 15.79 7.90 4 12 10 30.01 15.86 7.93 4 12 12 30.30 15.10 7.55 4 12 14 29.68 14.12 7.06 4 12 16 31.66 16.63 8.31 4 12 20 26.59 15.47 7.74 4 12 25 24.19 13.57 6.79 4 12 30 22.83 15.40 7.70 4 12 45 16.00 12.75 6.38 4 12 60 10.38 8.33 4.17 4 12 75 7.45 7.01 3.50 4 12 90 7.12 4.61 3.26 2 6 105 4.02 3.70 1.85 4 12 120 3.47 3.19 1.60 4 12 a Time in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 57 Summary Dermofluorometer Data For Animals On Oral Cremophor-EL 21 Days (Abdominal Site) Timea Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e a 1.00 N/A N/A 3 9 1 9.97 6.65 3.84 3 9 3 13.07 6.30 3.64 3 9 5 13.34 4.67 2.70 3 9 7 16.01 5.59 3.23 3 9 10 17.99 8.65 4.99 3 9 12 16.57 5.45 3.14 3 9 14 16.20 6.68 3.86 3 9 16 17.36 9.72 5.61 3 9 20 17.72 6.73 3.89 3 9 25 17.30 8.37 4.83 3 9 30 16.23 6.82 3.94 3 9 45 12.69 2.91 1.68 3 9 60 9.61 2.98 1.72 3 9 75 7.39 2.10 1.21 3 9 90 5.78 1.81 1.04 3 9 105 3.71 2.16 1.25 3 9 120 2.78 1.12 0.64 3 9 a Time in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 58 Summary Dermofluorometer Data For Animals On Oral Cremophor-EL 3 Days (Grafted Abdominal Site) Timea Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 2 6 1 20.64 5.22 3.69 2 6 3 24.69 6.94 4.91 2 6 5 23.69 16.94 11.98 2 6 7 28.64 11.35 8.02 2 6 10 29.77 9.64 6.82 2 6 12 29.70 9.14 6.46 2 6 14 29.04 9.96 7.04 2 6 16 29.18 7.87 5.57 2 6 20 29.70 11.15 7.89 2 6 25 28.15 11.46 8.10 2 6 30 22.44 5.27 3.73 2 6 45 20.15 8.50 6.01 2 6 60 14.51 5.64 3.99 2 6 75 10.46 3.01 2.13 2 6 90 8.43 3.51 2.48 2 6 105 6.10 3.51 2.48 2 6 120 3.86 1.62 1.14 2 6 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 59 Summary Dermofluorometer Data For Animals On Oral Cremophor-EL 7 Days (Grafted Abdominal Site) Time& Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 2 6 1 14.30 1.70 1.20 2 6 3 18.83 5.42 3.83 2 6 5 19.58 6.73 4.76 2 6 7 20.43 7.29 5.16 2 6 10 20.78 4.32 3.05 2 6 12 18.10 5.16 3.65 2 6 14 16.80 5.12 3.62 2 6 16 16.05 6.76 4.78 2 6 20 15.41 3.54 2.50 2 6 25 14.05 4.06 2.87 2 6 30 11.95 1.72 1.22 2 6 45 7.49 1.08 0.76 2 6 60 4.50 0.71 0.50 2 6 75 3.37 0.76 0.54 2 6 90 2.68 0.84 0.59 2 6 105 2.06 0.56 0.39 2 6 120 1.63 0.53 0.37 2 6 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 80 Summary Dermofluorometer Data For Animals On Oral Cremophor-EL 14 Days (Grafted Abdominal Site) Times. Mean D.I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 3 9 1 18.07 3.94 2.28 3 9 3 19.17 3.50 2.02 3 9 5 18.98 1.12 0.65 3 9 7 19.35 0.69 0.40 3 9 10 18.28 2.41 1.39 3 9 12 17.73 1.85 1.07 3 9 14 18.55 2.91 1.68 3 9 18 18.78 2.65 1.53 3 9 20 16.00 2.78 1.60 3 9 25 15.43 2.35 1.35 3 9 30 13.59 3.57 2.06 3 9 45 9.96 3.64 2.10 3 9 60 6.54 2.65 1.53 3 9 75 4.75 2.08 1.20 3 9 90 4.84 1 3 105 2.78 0.82 0.47 3 9 120 2.08 0.46 0.27 3 9 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 61 Summary Dermofluorometer Data For Animals On Oral Cremophor-EL 21 Days (Grafted Abdominal Site) Timea Mean D.l. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 3 9 1 11.69 1.16 0.67 3 9 3 21.59 11.15 6.44 3 9 5 16.41 1.94 1.12 3 9 7 17.22 2.68 1.55 3 9 10 19.51 2.06 1.19 3 9 12 18.95 3.10 1.79 3 9 14 17.78 4.60 2.66 3 9 16 18.06 4.35 2.51 3 9 20 19.33 1.83 1.06 3 9 25 17.19 4.45 2.57 3 9 30 15.90 5.59 3.23 3 9 45 11.17 4.48 2.59 3 9 60 8.48 3.85 2.22 3 9 75 6.11 2.94 1.70 3 9 90 2.69 1.91 1.10 3 9 105 3.52 1.46 0.85 3 9 120 2.74 1.01 0.58 3 9 a Time in minutes from I. V. injection of 8 mg/kg Fluorescein. bStandard Deviation. C Standard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. APPENDIX D SUMMARY DERMOFLUOROMETER DATA FOR ANIMALS OFF ORAL CYCLOSPORINE 63 Summary Dermofluorometer Data For Animals Off Oral Cyclosporine 7 Days (Abdominal Site) Timea Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 8 36 1 14.51 6.12 2.16 8 36 3 18.90 8.45 2.99 8 36 5 19.11 6.66 2.36 8 36 7 20.50 5.67 2.00 8 36 10 21.75 6.57 2.32 8 36 12 22.60 5.88 2.22 7 30 14 21.94 5.59 1.98 8 36 16 21.60 6.51 2.30 8 36 20 21.60 6.44 2.28 8 36 25 20.87 6.54 2.31 8 36 30 19.61 5.76 2.04 8 36 45 16.27 4.35 1.54 8 36 60 12.97 5.01 1.77 8 36 75 10.52 5.43 1.92 8 36 90 8.28 5.03 1.78 8 36 105 6.75 4.43 1.57 8 36 120 6.10 4.54 1.60 8 36 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 64 Summary Dermofluorometer Data For Animals Off Oral Cyclosporine 14 Days (Abdominal Site) Times. Mean D.I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 5 15 1 16.01 3.93 1.76 5 15 3 22.40 3.04 1.36 5 15 5 24.58 7.16 3.20 5 15 7 26.56 8.06 3.61 5 15 10 23.18 10.37 4.64 5 15 12 30.32 12.67 7.32 3 9 14 29.18 8.06 3.61 5 15 16 30.35 9.37 4.19 5 15 20 28.21 9.14 4.09 5 15 25 24.61 10.43 4.66 5 15 30 23.43 10.47 4.68 5 15 45 18.59 10.34 4.62 5 15 60 13.48 8.54 3.82 5 15 75 12.31 7.19 3.21 5 15 90 9.61 6.56 2.93 5 15 105 7.59 7.53 3.76 4 12 120 5.44 4.59 2.05 5 15 aTime in minutes from I. V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 65 Summary Dermofluorometer Data For Animals Off Oral Cyclosporine 21 Days (Abdominal Site) Times. Mean D. I. Std. Dev. b Std. Err. c Nd (Obs)e 0 1.00 N/A N/A 3 9 1 21.29 3.03 2.14 2 6 3 23.61 5.95 3.44 3 9 5 25.08 6.35 3.67 3 9 7 23.93 3.58 2.07 3 9 10 24.16 3.40 1.97 3 9 12 24.31 2.85 1.65 3 9 14 25.75 3.74 2.16 3 9 16 24.34 2.30 1.33 3 9 20 24.37 3.26 1.88 3 9 25 23.75 2.50 1.45 3 9 30 20.74 4.98 2.87 3 9 45 17.22 6.13 3.54 3 9 60 13.88 5.71 3.30 3 9 75 11.47 7.02 4.05 3 9 90 10.50 8.42 4.86 3 9 105 7.09 5.72 3.30 3 9 120 5.78 5.00 2.88 3 9 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 66 Summary Dermofluorometer Data For Animals Off Oral Cyclosporine 7 Days (Flank Site) Time&- Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 5 15 1 10.78 5.79 2.59 5 15 3 12.72 5.08 2.27 5 15 5 12.86 6.21 2.78 5 15 7 14.39 7.19 3.22 5 15 10 15.08 6.59 2.95 5 15 12 15.60 5.09 2.55 4 12 14 14.21 5.80 2.59 5 15 16 13.95 5.09 2.28 5 15 20 14.78 5.51 2.46 5 15 25 14.69 4.51 2.02 5 15 30 13.76 4.13 1.85 5 15 45 12.43 3.64 1.63 5 15 60 10.67 2.28 1.02 5 15 75 9.06 2.69 1.20 5 15 90 7.78 3.04 1.36 5 15 105 6.43 2.58 1.16 5 15 120 5.27 2.39 1.07 5 15 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 67 Summary Dermofluorometer Data For Animals Off Oral Cyclosporine 7 Days (Grafted Abdominal Site) Times. Mean D.l. Std. Dev. b Std. Err .. c Nd (Obs)e 0 1.00 N/A N/A 2 6 1 21.85 4.19 2.96 2 6 3 27.01 4.10 2.90 2 6 5 24.93 1.00 0.71 2 6 7 26.56 1.26 0.89 2 6 10 25.22 3.29 2.33 2 6 12 24.26 3.36 2.37 2 6 14 21.44 1.79 0.56 2 6 16 22.12 3.94 2.79 2 6 20 20.42 4.68 3.31 2 6 25 17.45 3.47 2.45 2 6 30 15.30 2.15 1.52 2 6 45 9.87 0.34 0.24 2 6 60 6.51 0.85 0.60 2 6 75 4.67 0.94 0.67 2 6 90 2.98 0.49 0.35 2 6 105 2.42 0.34 0.24 2 6 120 1.89 0.62 0.44 2 6 a Time in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 88 Summary Dermofluorometer Data For Animals Off Oral Cyclosporine 21 Days (Grafted Abdominal Site) Timea Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 2 8 1 15.82 10.78 7.81 2 8 3 18.88 14.42 10.19 2 8 5 21.42 14.25 10.08 2 8 7 17.83 9.84 8.98 2 8 10 18.74 9.78 8.90 2 8 12 17.37 8.57 8.08 2 8 14 19.22 10.70 7.58 2 8 18 18.17 9.55 8.75 2 6 20 17.88 8.44 5.97 2 8 25 17.31 8.55 6.05 2 8 30 13.45 3.91 2.77 2 6 45 10.54 0.35 0.25 2 8 60 8.43 0.40 0.28 2 8 75 8.20 1.89 1.20 2 8 90 5.03 1.86 1.31 2 8 105 3.68 1.67 1.18 2 8 120 3.09 1.44 1.02 2 8 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. APPENDIX E SUMMARY DERMOFLUOROMETER DATA FOR ANIMALS OFF ORAL CREMOPHOR-EL 70 Summary Dermofluorometer Data For Animals Off Oral Cremophor 3 Days (Abdominal Site) Timea Mean D. I. Std. Dev. b Std. Err.e Nd (Obs)e 0 1.00 N/A N/A 2 6 1 18.15 15.34 10.85 2 6 3 25.63 20.55 14.53 2 6 5 31.08 22.75 16.08 2 6 7 33.20 23.05 16.30 2 6 10 35.93 20.84 14.73 2 6 12 38.47 21.02 14.87 2 6 14 40.98 20.06 14.18 2 6 16 41.43 13.06 9.23 2 6 20 40.12 5.26 3.72 2 6 25 43.38 3.23 2.28 2 6 30 39.77 1.32 0.93 2 6 45 34.85 1.91 1.35 2 6 60 22.42 1.53 1.08 2 6 75 15.38 5.26 3.72 2 6 90 10.55 4.31 3.05 2 6 105 7.50 2.12 1.50 2 6 120 5.07 1.74 1.23 2 6 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. eStandard Error of the Mean. d Number of Animals. eNumber of Observations in Calculations. 71 Summary Dermofluorometer Data For Animals Off Oral Cremophor-EL 7 Days (Abdominal Site) Timea Mean D. I. Std. Dev. b Std. Err.c Nd (Obs )e 0 1.00 N/A N/A 4 12 1 19.25 6.48 3.24 4 12 3 22.08 4.79 2.39 4 12 5 25.31 5.06 2.53 4 12 7 24.37 3.84 1.92 4 12 10 27.02 8.93 4.46 4 12 12 24.00 4.63 2.32 4 12 14 25.78 9.16 4.58 4 12 16 26.48 5.70 2.85 4 12 20 24.91 5.35 2.68 4 12 25 24.03 3.89 1.94 4 12 30 22.70 3.02 1.51 4 12 45 15.52 5.39 2.70 4 12 60 11.21 4.17 2.09 4 12 75 7.77 2.94 1.47 4 12 90 4.28 1.75 0.88 4 12 105 3.33 1.23 0.62 4 12 120 2.72 1.23 0.61 4 12 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 72 Summary Dermofluorometer Data For Animals Off Oral Cremophor-EL 14 Days (Abdominal Site) Times. Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 2 6 1 14.91 3.52 2.49 2 6 3 22.48 0.88 0.62 2 6 5 22.46 0.36 0.26 2 6 7 20.99 2.71 1.91 2 6 10 20.14 3.33 2.36 2 6 12 18.57 4.85 3.43 2 6 14 16.13 9.58 6.77 2 6 16 19.02 6.19 4.38 2 6 20 16.49 5.95 4.21 2 6 25 16.70 3.11 2.20 2 6 30 17.41 0.98 0.69 2 6 45 13.92 1.81 1.28 2 6 60 10.23 0.53 0.37 2 6 75 6.50 0.71 0.50 2 6 90 5.36 1.51 1.06 2 6 105 4.36 1.21 0.86 2 6 120 2.47 0.46 0.33 2 6 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 73 Summary Dermofluorometer Data For Animals Off Oral Cremophor-EL 3 Days (Grafted Abdominal Site) Timea Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 2 6 1 18.42 14.43 10.21 2 6 3 18.88 10.24 7.24 2 6 5 21.75 8.84 6.25 2 6 7 21.35 7.11 5.03 2 6 10 24.10 7.46 5.28 2 6 12 20.43 2.22 1.57 2 6 14 24.48 8.51 6.02 2 6 16 23.38 5.13 3.63 2 6 20 21.42 1.35 0.96 2 6 25 19.71 1.46 1.04 2 6 30 17.25 0.35 0.25 2 6 45 12.54 1.30 0.92 2 6 60 8.86 1.21 0.86 2 6 75 5.96 1.17 0.83 2 6 90 4.85 0.67 0.47 2 6 105 3.38 0.01 0.01 2 6 120 2.35 0.04 0.03 2 6 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 74 Summary Dermofluorometer Data For Animals Off Oral Cremophor-EL 7 Days (Grafted Abdominal Site) Timea Mean D.I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 3 9 1 15.67 2.36 1.36 3 9 3 18.04 2.69 1.55 3 9 5 16.74 2.69 1.55 3 9 7 17.98 2.89 1.67 3 9 10 20.13 0.63 0.37 3 9 12 20.39 0.59 0.34 3 9 14 18.59 2.57 1.48 3 9 16 18.22 3.33 1.92 3 9 20 15.84 1.40 0.81 3 9 25 13.76 2.76 1.59 3 9 30 12.57 3.46 2.00 3 9 45 8.34 2.32 1.34 3 9 60 5.78 2.49 1.44 3· 9 75 3.63 1.24 0.72 3 9 90 2.96 1.29 0.75 3 9 105 2.02 0.77 0.45 3 9 120 1.54 0.44 0.26 3 9 aTime in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. 75 Summary Dermofluorometer Data For Animals Off Oral Cremophor-EL 14 Days (Grafted Abdominal Site) Times. Mean D. I. Std. Dev. b Std. Err.c Nd (Obs)e 0 1.00 N/A N/A 2 6 1 19.93 0.10 0.07 2 6 3 22.94 3.95 2.79 2 6 5 24.21 2.02 1.43 2 6 7 23.36 1.42 1.00 2 6 10 24.73 0.39 0.27 2 6 12 21.73 2.05 1.45 2 6 14 20.15 2.52 1.78 2 6 16 19.10 2.08 1.47 2 6 20 20.97 3.17 2.24 2 6 25 16.67 0.17 0.12 2 6 30 15.36 4.24 3.00 2 6 45 12.79 4.33 3.06 2 6 60 8.72 2.82 1.99 2 6 75 6.01 2.71 1.92 2 6 90 5.13 3.65 2.58 2 6 105 3.71 2.03 1.44 2 6 120 3.48 1.84 1.30 2 6 a Time in minutes from I.V. injection of 8 mg/kg Fluorescein. bStandard Deviation. cStandard Error of the Mean. dNumber of Animals. eNumber of Observations in Calculations. APPENDIX F TRANSEPIDERMAL WATER LOSS DATA 77 Transepidermal Water Loss Data For Naive Nude Rats Date Animal Status Site WE P ReI. Hum. 04/03/87 3BR NAIVE ABDl NG 14.0 05.3 17.0 04/15/87 3BR NAIVE ABDl NG 15.3 07.8 23.0 05/05/87 Nl NAIVE ABDl NG 05.S 11.2 3S.0 05/05/87 N2 NAIVE ABDl NG 04.7 11.3 37.0 05/05/87 N3 NAIVE ABDl NG 05.9 10.2 35.0 05/05/87 N4 NAIVE ABDl NG OS.3 11.0 39.0 05/05/87 N5 NAIVE ABDl NG OS.3 11.4 34.0 05/13/87 NWl NAIVE ABDl NG OS.9 12.1 42.0 05/13/87 NWl NAIVE ABDl NG 02.S 10.8 37.0 05/14/87 NW2 NAIVE ABDl NG OS.l 08.3 28.0 05/14/87 NW2 NAIVE ABDl NG 05.8 08.8 28.0 04/17/87 SDR NAIVE ABD2 NG 20.0 OS.7 20.0 05/05/87 Nl NAIVE ABD2 NG 10.9 11.1 35.0 05/05/87 N2 NAIVE ABD2 NG 05.9 10.1 34.0 05/05/87 N3 NAIVE ABD2 NG 05.5 09.8 32.0 05/05/87 N4 NAIVE ABD2 NG 10.S 11.3 34.0 05/05/87 N5 NAIVE ABD2 NG 08.5 10.7 32.0 05/13/87 NWl NAIVE ABD2 NG 07.0 12.8 43.0 05/13/87 NWl NAIVE ABD2 NG 04.1 11.0 37.0 05/14/87 NW2 NAIVE ABD2 NG 12.5 09.7 29.0 04/02/87 3BR NAIVE Flank L OS.3 05.9 19.0 04/03/87 3BR NAIVE Flank L 08.1 04.9 19.0 04/14/87 3BR NAIVE Flank L 02.7 08.2 22.0 04/15/87 3BR NAIVE Flank L 07.0 08.3 23.0 04/17/87 SDR NAIVE Flank L 03.S 05.1 16.0 04/02/87 WJl NAIVE Flank L 05.S 05.8 21.0 04/02/87 WJ2 NAIVE Flank L 02.3 05.S 19.0 04/02/87 WJ3 NAIVE Flank L 05.3 05.7 21.0 04/06/87 WJ3 NAIVE Flank L 11.5 08.S 31.0 04/0S/87 WJ4 NAIVE Flank L 02.9 08.S 28.0 04/02/87 3BR NAIVE Flank R 13.0 07.3 21.0 04/03/87 3BR NAIVE Flank R OS.9 05.5 21.0 04/15/87 3BR NAIVE Flank R 14.S 10.2 30.0 04/17/87 SDR NAIVE Flank R 04.8 OS.2 21.0 05/13/87 NWl NAIVE Flank R 05.0 10.1 39.0 05/13/87 NW1 NAIVE Flank R 02.4 10.7 39.0 05/14/87 NW2 NAIVE Flank R 01.8 OS.9 25.0 05/14/87 NW2 NAIVE Flank R 04.7 08.0 27.0 04/02/87 WJl NAIVE Flank R 04.3 05.7 20.0 04/02/87 WJ2 NAIVE Flank R 03.9 05.9 20.0 04/02/87 WJ3 NAIVE Flank R 03.8 05.8 19.0 04/0S/87 WJ3 NAIVE Flank R OS.O 09.2 39.0 04/0S/87 WJ4 NAIVE Flank R 08.9 09.0 30.0 78 Transepidermal Water Loss Data for Nude Rats Given Oral Cyclosporine for 3 Days Date Animal Status Site WE P ReI. Hum. 03/26/87 1AR +CS 3D ABD1 NG 07.1 05.2 21.0 06/08/87 . D1 +CS 3D ABD1 NG 16.5 12.0 54.0 06/08/87 D2 +CS 3D ABD1 NG 07.1 10.6 38.0 06/08/87 OH1 +CS 3D ABD1 NG 12.4 10.2 34.0 06/08/87 OH1 +CS 3D ABD1 NG 05.6 10.6 35.0 06/08/87 OH2 +CS 3D ABD1 NG 09.2 10.2 38.0 06/08/87 OH2 +CS 3D ABD1 NG 08.5 11.4 38.0 03/26/87 Z8 +CS 3D ABD1 NG 16.4 06.4 23.0 03/26/87 Z8 +CS 3D ABD1 NG 10.5 07.6 25.0 06/08/87 OH1 +CS 3D ABD2 NG 15.1 11.1 34.0 06/08/87 OH1 +CS 3D ABD2 NG 05.2 10.6 36.0 06/08/87 OH2 +CS 3D ABD2 NG 07.7 10.3 35.0 06/08/87 OH2 +CS 3D ABD2 NG 09.1 12.4 38.0 06/08/87 D1 +CS 3D Flank 03.6 10.2 33.0 06/08/87 D2 +CS 3D Flank 03.2 10.0 36.0 06/08/87 OH1 +CS 3D Flank 03.9 09.1 32.0 06/08/87 OH1 +CS 3D Flank 04.1 10.1 32.0 06/08/87 OH2 +CS 3D Flank 06.8 10.2 40.0 06/08/87 OH2 +CS 3D Flank 08.9 12.5 40.0 04/05/87 WJ1 +CS 3D Flank L 03.9 07.6 30.0 04/05/87 WJ2 +CS 3D Flank L 04.7 07.8 28.0 04/09/87 WJ3 +CS 3D Flank L 02.0 05.5 20.0 04/09/87 WJ4 +CS 3D Flank L 02.8 05.2 19.0 04/05/87 WJ1 +CS 3D Flank R 05.7 08.4 32.0 04/05/87 WJ2 +CS 3D Flank R 04.1 08.2 32.0 04/09/87 WJ3 +CS 3D Flank R 02.6 05.3 18.0 04/09/87 WJ4 +CS 3D Flank R 03.0 05.3 17.0 79 Transepidermal Water Loss Data for Nude Rats Given Oral Cyclosporine for 7 Days Date Animal Status Site WE P ReI. Hum. 06/12/87 Dl +CS 7D ABDl NG 06.3 11.3 34.0 06/12/87 Dl +CS 7D ABDl NG 11.7 12.9 35.0 06/12/87 OHl +CS 7D ABDl NG 10.4 10.7 38.0 04/10/87 WJl +CS 7D ABDl NG 09.5 05.1 21.0 04/10/87 WJl +CS 7D ABDl NG 07.8 07.5 29.0 04/10/87 WJ2 +CS 7D ABDl NG 09.2 06.2 24.0 04/13/87 WJ3 +CS 7D ABDl NG 07.2 06.5 02.2 04/13/87 WJ3 +CS 7D ABDl NG 08.3 05.4 18.0 06/12/87 Dl +CS 7D ABD2 NG 04.9 13.4 35.0 06/12/87 Dl +CS 7D ABD2 NG 11.4 13.1 35.0 06/12/87 OHl +CS 7D ABD2 NG 08.2 12.1 41.0 04/10/87 WJl +CS 7D ABD2 NG 05.4 05.5 23.0 04/10/87 WJl +CS 7D ABD2 NG 05.7 07.0 25.0 04/10/87 WJ2 +CS 7D ABD2 NG 05.7 05.8 23.0 04/10/87 WJ2 +CS 7D ABD2 NG 03.9 05.0 20.0 04/13/87 WJ3 +CS 7D ABD2 NG 07.4 05.4 17.0 04/13/87 WJ3 +CS 7D ABD2 NG 07.0 07.3 24.0 06/12/87 Dl +CS 7D Flank 04.4 10.5 39.0 06/12/87 Dl +CS 7D Flank 07.7 10.5 33.0 06/12/87 OHl +CS 7D Flank 02.6 10.1 38.0 04/10/87 WJl +CS 7D Flank L 09.0 07.2 27.0 04/10/87 WJ2 +CS 7D Flank L 05.1 05.7 21.0 04/10/87 WJ2 +CS 7D Flank L 10.5 07.8 26.0 04/13/87 WJ3 +CS 7D Flank L 04.2 04.9 19.0 04/13/87 WJ3 +CS 7D Flank L 06.1 08.1 22.0 04/14/87 WJ4 +CS 7D Flank L 11.8 05.9 20.0 04/10/87 WJ2 +CS 7D Flank R 03.7 05.4 21.0 04/13/87 WJ3 +CS 7D Flank R 04.9 05.5 19.0 04/14/87 WJ4 +CS 7D Flank R 08.0 06.2 20.0 80 Transepidermal Water Loss Data for Nude Rats Given Oral Cyclosporine for 14 Days Date Animal Status Site WE P ReI. Hum. 04/17/87 WJ1 +CS 14D ABD1 NG 04.6 04.6 16.0 04/17/87 WJ1 +CS 14D ABD1 NG 06.1 04.9 17.0 04/17/87 WJ2 +CS 14D ABD1 NG 07.3 05.2 17.0 04/17/87 WJ2 +CS 14D ABD1 NG 06.1 04.5 15.0 04/20/87 WJ3 +CS 14D ABD1 NG 14.4 05.7 22.0 04/20/87 WJ3 +CS 14D ABD1 NG 14.5 05.7 23.0 04/20/87 WJ4 +CS 14D ABD1 NG 11.4 06.3 25.0 04/20/87 WJ4 +CS 14D ABD1 NG 06.7 05.8 23.0 04/17/87 WJ1 +CS 14D ABD2 NG 04.7 05.0 17.0 04/17/87 WJ1 +CS 14D ABD2 NG 05.9 05.1 17.0 04/17/87 WJ2 +CS 14D ABD2 NG 05.1 04.9 16.0 04/17/87 WJ2 +CS 14D ABD2 NG 05.3 04.5 14.0 04/20/87 WJ3 +CS 14D ABD2 NG 16.0 08.4 28.0 04/20/87 WJ3 +CS 14D ABD2 NG 14.0 06.7 . 22.0 04/20/87 WJ4 +CS 14D ABD2 NG 10.4 06.7 22.0 04/20/87 WJ4 +CS 14D ABD2 NG 08.2 06.6 24.0 04/17/87 WJ1 +CS 14D Flank L 03.8 04.6 16.0 04/17/87 WJ1 +CS 14D Flank L 03.4 04.9 19.0 04/17/87 WJ2 +CS 14D Flank L 06.9 05.1 20.0 04/17/87 WJ2 +CS 14D Flank L 04.6 05.5 17.0 04/17/87 WJ1 +CS 14D Flank R 01.7 04.0 14.0 04/17/87 WJ1 +CS 14D Flank R 02.3 05.6 20.0 04/17/87 WJ2 +CS 14D Flank R 04.6 04.4 17.0 04/17/87 WJ2 +CS 14D Flank R 05.4 05.2 16.0 04/20/87 WJ3 +CS 14D Flank R 06.0 06.0 21.0 04/20/87 WJ4 +CS 14D Flank R 08.2 06.0 21.0 04/20/87 WJ4 +CS 14D Flank R 04.7 05.7 22.0 81 Transepidermal Water Loss Data for Nude Rats Given Oral Cyclosporine for 21 Days Date Animal Status Site WE P Rei. Hum. 04/14/87 1MB +CS 21D ABDl NG 14.9 07.2 22.0 04/24/87 WJl +CS 21D ABDl NG 06.0 05.9 21.0 04/24/87 WJl +CS 21D ABDl NG 12.7 07.6 25.0 04/24/87 WJ2 +CS 21D ABDl NG 03.4 06.2 22.0 04/24/87 WJ2 +CS 21D ABDl NG 03.8 06.3 22.0 04/27/87 WJ3 +CS 21D ABDl NG 06.3 08.4 27.0 04/27/87 WJ3 +CS 21D ABDl NG 06.9 11.0 40.0 04/27/87 WJ4 +CS 21D ABDl NG 07.3 09.4 31.0 04/27/87 WJ4 +CS 21D ABDl NG 07.7 08.2 29.0 04/24/87 WJl +CS 21D ABD2 NG 11.3 07.1 24.0 04/24/87 WJ2 +CS 21D ABD2 NG 02.7 05.9 20.0 04/24/87 WJ2 +CS 21D ABD2 NG 03.3 06.6 21.0 04/27/87 WJ3 +CS 21D ABD2 NG 06.1 08.5 27.0 04/27/87 WJ3 +CS 21D ABD2 NG 06.9 08.5 . 30.0 04/27/87 WJ4 +CS 21D ABD2 NG 06.1 09.1 28.0 04/27/87 WJ4 +CS 21D ABD2 NG 03.4 09.0 29.0 04/14/87 lMH +CS 21D Flank L 05.0 07.2 20.0 04/24/87 WJl +CS 21D Flank L 02.6 07.1 26.0 04/24/87 WJl +CS 21D Flank L 07.8 06.4 20.0 04/24/87 WJ2 +CS 21D Flank L 02.6 06.1 19.0 04/24/87 WJ2 +CS 21D Flank L 02.5 05.2 21.0 04/27/87 WJ3 +CS 21D Flank L 03.7 07.4 29.0 04/27/87 WJ3 +CS 21D Flank L 02.7 07.6 28.0 04/27/87 WJ4 +CS 21D Flank L 02.7 07.8 30.0 04/27/87 WJ4 +CS 21D Flank L 07.5 09.3 38.0 04/24/87 WJl +CS 21D Flank R 06.5 07.3 23.0 04/24/87 WJl +CS 21D Flank R 07.1 06.8 21.0 04/24/87 WJ2 +CS 21D Flank R 01.3 05.4 21.0 04/24/87 WJ2 +CS 21D Flank R 01.7 06.1 19.0 04/27/87 WJ3 +CS 21D Flank R 05.0 08.2 30.0 04/27/87 WJ3 +CS 21D Flank R 02.6 08.1 29.0 04/27/87 WJ4 +CS 21D Flank R 03.2 08.3 30.0 04/27/87 WJ4 +CS 21D Flank R 03.1 07.6 28.0 82 Transepidermal Water Loss Data for Nude Rats Given Oral Cyclosporine for 28 Days Date Animal Status Site WE P ReI. Hum. 05/01/87 WJI +CS 28D ABD1 NG OS.5 09.3 30.0 05/01/87 WJ2 +CS 28D ABD1 NG 10.1 08.9 26.0 05/01/87 WJ1 +CS 28D ABD2 NG 07.9 07.8 25.0 05/01/87 WJ2 +CS 28D ABD2 NG 05.4 08.1 24.0 04/16/87 31XH +CS 28D Flank L 08.9 05.8 19.0 05/01/87 WJl +CS 28D Flank L 03.4 08.9 27.0 05/01/87 WJ2 +CS 28D Flank L 04.5 09.3 26.0 04/16/87 31XH +CS 28D Flank R 08.4 05.2 16.0 05/01/87 WJ1 +CS 28D Flank R 04.7 07.8 37.0 05/01/87 WJ2 +CS 28D Flank R 05.7 07.7 26.0 83 Transepidermal Water Loss Data for Nude Rats Off Of Oral Cyclosporine for 1 Day Date Animal Status Site WE P ReI. Hum. 04/30/87 OH1 -CS 1D ABD1 NG 11.3 10.4 35.0 04/30/87 OH2 -CS lD ABD1 NG 13.S 12.8 33.0 04/15/87 OH3 -CS 1D ABD1 NG 09.0 07.3 23.0 05/02/87 WJl -CS lD ABDl NG 08.S 07.2 39.0 05/02/87 WJ2 -CS 1D ABD1 NG 10.0 07.2 27.0 04/28/87 WJ3 -CS lD ABDl NG 08.3 09.9 30.0 04/28/87 WJ4 -CS 1D ABD1 NG OS.l 08.4 26.0 04/15/87 Z8 -CS lD ABD1 NG OS.4 07.9 29.0 04/30/87 OB1 -CS lD ABD2 NG 08.6 09.5 32.0 05/02/87 WJl -CS lD ABD2 NG 06.1 07.9 31.0 05/02/87 WJ2 -CS lD ABD2 NG 05.4 06.9 24.0 04/28/87 WJ3 -CS lD ABD2 NG 04.9 09.2 29.0 04/28/87 WJ4 -CS lD ABD2 NG 05.4 08.3 23.0 04/30/87 OBl -CS 1D Flank L 05.7 09.0 30.0 04/30/87 OB2 -CS lD Flank L 09.2 10.6 31.0 04/30/87 OH2 -CS 1D Flank L 07.4 09.4 30.0 04/15/87 OH3 -CS 1D Flank L 03.6 06.2 19.0 04/28/87 WJ3 -CS lD Flank L 01.3 08.4 34.0 04/28/87 WJ4 -CS 1D Flank L 02.6 07.8 24.0 04/15/87 Z8 -CS 1D Flank L 06.6 06.2 22.0 04/30/87 OB1 -CS 1D Flank R 05.0 09.1 30.0 04/15/87 OH3 -CS lD Flank R 03.4 06.5 21.0 05/02/87 WJ1 -CS 1D Flank R 04.8 06.9 33.0 05/02/87 WJ2 -CS 1D Flank R 02.9 07.1 27.0 04/28/87 WJ3 -CS 1D Flank R 04.2 07.8 28.0 04/28/87 WJ4 -CS 1D Flank R 03.6 07.5 24.0 04/15/87 Z8 -CS 1D Flank R 06.1 06.2 24.0 • 84 Transepidermal Water Loss Data for Nude Rats Off Of Oral Cyclosporine for 2 Days Date Animal Status Site WE P ReI. Hum. 05/01/87 OH1 -CS 2D ABD1 NG 09.5 10.0 36.0 05/01/87 OH2 -CS 2D ABD1 NG 11.1 10.7 34.0 04/16/87 OH3 -CS 2D ABD1 NG 10.0 09.2 24.0 05/03/87 WJ1 -CS 2D ABD1 NG 08.0 06.4 27.0 05/03/87 WJ2 -CS 2D ABD1 NG 08.1 06.0 23.0 04/29/87 WJ3 -CS 2D ABDl NG 10.7 11.6 38.0 04/29/87 WJ4 -CS 2D ABD1 NG 13.5 16.0 44.0 04/16/87 Z8 -CS 2D ABD1 NG 06.9 05.7 21.0 05/01/87 OH1 -CS 2D ABD2 NG 09.2 09.5 34.0 05/01/87 OH2 -CS 2D ABD2 NG 10.7 10.4 31.0 05/03/87 WJ1 -CS 2D ABD2 NG 06.8 07.6 30.0 05/03/87 WJ2 -CS 2D ABD2 NG 04.7 06.0 21.0 04/29/87 WJ3 -CS 2D ABD2 NG 07.2 09.8 30.0 04/29/87 WJ4 -CS 2D ABD2 NG 10.7 11.5 . 34.0 05/01/87 OH1 -CS 2D Flank L 05.1 08.9 33.0 05/01/87 OH2 -CS 2D Flank L 06.6 09.3 31.0 04/16/87 OH3 -CS 2D Flank L 04.4 06.9 22.0 04/29/87 WJ3 -CS 2D Flank L 03.7 08.2 37.0 04/29/87 WJ4 -CS 2D Flank L 04.0 09.2 27.0 04/16/87 Z8 -CS 2D Flank L 04.0 05.0 19.0 05/01/87 OH1 -CS 2D Flank R 05.0 07.5 31.0 05/01/87 OH2 -CS 2D Flank R 07.4 07.7 26.0 04/16/87 OH3 -CS 2D Flank R 06.1 05.2 16.0 05/03/87 WJ1 -CS 2D Flank R 07.3 05.6 27.0 05/03/87 WJ2 -CS 2D Flank R 03.8 05.9 23.0 04/29/87 WJ3 -CS 2D Flank R 04.3 08.5 33.0 04/29/87 WJ4 -CS 2D Flank R 03.5 08.5 26.0 04/16/87 Z8 -CS 2D Flank R 07.3 05.0 17.0 85 Transepidermal Water Loss Data for Nude Rats Off Of Oral Cyclosporine for 3 Days Date Animal Status Site WE P ReI. Hum. 05/02/87 OH1 -CS 3D ABD1 NG 11.4 OS.l 32.0 05/02/87 OH2 -CS 3D ABD1 NG 09.1 08.3 30.0 04/17/87 OH3 -CS 3D ABD1 NG 04.2 05.5 15.0 04/30/87 WJ3 -CS 3D ABD1 NG OB.7 10.8 3S.0 04/30/87 WJ4 -CS 3D ABD1 NG 09.0 10.5 30.0 04/17/87 Z8 -CS 3D ABDl NG OB.4 07.B 21.0 05/02/87 OHl -CS 3D ABD2 NG 10.8 09.B 32.0 04/17/87 OH3 -CS 3D ABD2 NG 03.1 05.5 15.0 04/30/87 WJ3 -CS 3D ABD2 NG OS.8 11.4 42.0 04/30/87 WJ4 -CS 3D ABD2 NG 10.2 09.B 30.0 04/17/87 Z8 -CS 3D ABD2 NG 13.1 OB.l 20.0 04/17/87 OH3 -CS 3D Flank L 03.5 04.B 14.0 04/30/87 WJ3 -CS 3D Flank L 04.9 08.4 ·33.0 04/30/87 WJ4 -CS 3D Flank L 03.7 08.B 29.0 04/17/87 Z8 -CS 3D Flank L OB.1 04.7 17.0 05/02/87 OHl -CS 3D Flank R 05.8 08.4 28.0 05/02/87 OH2 -CS 3D Flank R 05.4 07.0 2B.0 04/17/87 OH3 -CS 3D Flank R 02.5 05.4 15.0 04/30/87 WJ3 -CS 3D Flank R OB.2 08.5 39.0 04/30/87 WJ4 -CS 3D Flank R 05.1 08.B 30.0 04/17/87 Z8 -CS 3D Flank R 03.3 04.8 18.0 86 Transepidermal Water Loss Data for Nude Rats Off Of Oral Cyclosporine for 4 Days Date Animal Status Site WE P ReI. Hum. 05/03/87 OHl -CS 4D ABDl NG 08.1 07.8 28.0 05/03/87 OH2 -CS 4D ABDl NG 09.5 08.1 28.0 05/05/87 WJl -CS 4D ABDl NG 05.7 10.6 37.0 05/05/87 WJ2 -CS 4D ABDl NG 04.2 09.4 31.0 05/03/87 OHl -CS 4D ABD2 NG 12.7 08.0 23.0 05/03/87 OH2 -CS 4D ABD2 NG 09.3 08.1 25.0 05/05/87 WJl -CS 4D ABD2 NG 09.8 10.2 29.0 05/05/87 WJ2 -CS 4D ABD2 NG 03.3 09.1 30.0 05/03/87 OHl -CS 4D Flank R 06.0 05.9 21.0 05/03/87 OH2 -CS 4D Flank R 05.6 08.5 21.0 05/05/87 WJl -CS 4D Flank R 06.4 09.0 34.0 05/05/87 WJ2 -CS 4D Flank R 02.0 09.0 34.0 87 Transepidermal Water Loss Data for Nude Rats Off Of Oral Cyclosporine for 7 Days Date Animal Status Site WE P ReI. Hum. 05/06/87 OH2 -CS 7D ABDl NG 08.4 07.4 23.0 04/21/87 OH3 -CS 7D ABDl NG 04.3 07.2 23.0 05/08/87 WJ2 -CS 7D ABDl NG 06.7 07.8 28.0 05/08/87 WJ2 -CS 7D ABDl NG 07.1 09.2 33.0 05/04/87 WJ3 -CS 7D ABDl NG 06.1 05.9 26.0 05/04/87 WJ3 -CS 7D ABDl NG 08.5 07.3 28.0 05/04/87 WJ4 -CS 7D ABDl NG 03.1 07.1 28.0 05/04/87 WJ4 -CS 7D ABDl NG 03.3 08.1 33.0 04/21/87 Z8 -CS 7D ABDl NG 06.1 08.3 26.0 04/17/87 lMH -CS 7D ABD2 NG 09.7 05.7 19.0 05/06/87 OH2 -CS 7D ABD2 NG 07.4 09.1 29.0 04/21/87 OH3 -CS 7D ABD2 NG 04.5 08.7 24.0 05/08/87 WJ2 -CS 7D ABD2 NG 09.2 10.0 31.0 05/08/87 WJ2 -CS 7D ABD2 NG 05.3 07.6 . 26.0 05/04/87 WJ3 -CS 7D ABD2 NG 07.2 07.7 30.0 05/04/87 WJ3 -CS 7D ABD2 NG 06.9 08.6 27.0 05/04/87 WJ4 -CS 7D ABD2 NG 05.5 08.3 31.0 05/04/87 WJ4 -CS 7D ABD2 NG 02.7 07.4 28.0 04/17/87 lMH -CS 7D Flank L 02.9 06.4 18.0 04/21/87 OH3 -CS 7D Flank L 03.1 06.7 24.0 04/21/87 Z8 -CS 7D Flank L 03.7 06.7 26.0 04/17/87 lMH -CS 7D Flank R 07.0 05.8 18.0 05/06/87 OHl -CS 7D Flank R 06.1 06.1 24.0 05/06/87 OH2 -CS 7D Flank R 06.6 06.0 19.0 04/21/87 OH3 -CS 7D Flank R 02.9 06.7 23.0 05/08/87 WJ2 -CS 7D Flank R 03.2 07.1 27.0 05/04/87 WJ3 -CS 7D Flank R 03.1 06.7 31.0 05/04/87 WJ3 -CS 7D Flank R 03.9 07.0 30.0 05/04/87 WJ4 -CS 7D Flank R 02.5 06.1 27.0 05/04/87 WJ4 -CS 7D Flank R 01.7 06.7 32.0 04/21/87 Z8 -CS 7D Flank R 03.7 07.6 27.0 88 Transepidermal Water Loss Data for Nude Rats Off Of Oral Cyclosporine for 14 Days Date Animal Status Site WE P ReI. Hum. 05/13/87 OHl -CS 14D ABDl NG 08.7 12.1 37.0 05/13/87 OB2 -CS 14D ABDl NG 04.7 13.0 38.0 04/28/87 OB3 -CS 14D ABDl NG 04.8 09.3 27.0 05/11/87 WJ3 -CS 14D ABDl NG 07.5 11.7 40.0 05/11/87 WJ4 -CS 14D ABDl NG 07.0 12.3 37.0 05/13/87 OBl -CS 14D ABD2 NG 09.9 12.3 37.0 05/13/87 OH2 -CS 14D ABD2 NG 06.5 12.9 37.0 04/28/87 OB3 -CS 14D ABD2 NG 06.4 08.7 26.0 05/11/87 WJ3 -CS 14D ABD2 NG 11.3 12.1 40.0 05/11/87 WJ4 -CS 14D ABD2 NG 07.1 11.1 35.0 04/28/87 OB3 -CS 14D Flank L 04.2 08.3 26.0 04/28/87 Z8 -CS 14D Flank L 06.0 08.5 27.0 05/13/87 OHl -CS 14D Flank R 06.4 11.2 35.0 05/13/87 OH2 -CS 14D Flank R 05.3 12.1 34.0 04/28/87 OH3 -CS 14D Flank R 03.4 08.6 26.0 05/11/87 WJ3 -CS 14D Flank R 03.1 10.3 39.0 05/11/87 WJ4 -CS 14D Flank R 03.5 10.3 37.0 89 Transepidermal Water Loss Data for Nude Rats Off Of Oral Cyclosporine for 21 Days Date Animal Status Site WE P ReI. Hum. 04/09/81 5BP -CS 21D ABDl NG 08.6 05.1 19.0 OS/20/87 OHl -CS 21D ABD1 NG 10.9 13.0 53.0 OS/20/87 OH2 -cs 21D ABDl NG 08.7 14.2 45.0 05/18/87 WJ3 -CS 21D ABDl NG 07.7 11.0 40.0 05/18/87 WJ4 -CS 21D ABDl NG 09.1 12.7 41.0 OS/20/87 OH2 -CS 21D ABD2 NG 09.5 13.7 47.0 05/18/87 WJ3 -CS 21D ABD2 NG 08.2 11.4 37.0 05/18/87 WJ4 -CS 21D ABD2 NG 09.3 12.3 39.0 OS/20/87 OHl -cs 21D Flank 06.7 10.1 43.0 OS/20/87 OH2 -CS 21D Flank 05.3 10.4 38.0 05/18/87 WJ3 -CS 21D Flank 03.9 10.4 41.0 05/18/87 WJ4 -CS 21D Flank 03.1 10.7 37.0 REFERENCES 1. Bartek MJ, Labudde JA, Maibach HI: Skin permeability in vivo: comparison in rat, rabbit, pig and man. J Invest Dermatol 58:114-123, 1972. 2. Reifenrath WG, Hill JA, Robinson PB, Hcvey DL, Akers WA, Anjo DM, Maibach HI: Percutaneous absorption of carbon 14 labeled insect repellents in the hairless dog. J Environ Pathol Toxicol 4:249-256, 1980. 3. Stoughton RB: Animal models for in vitro percutaneous absorption. 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