SYSTEMIC ABSORPTION OF TOPICALLY APPLIED SALICYLIC ACID

Salicylic acid was applied topically to the skin of rabbits and humans in order to study the pharmacokinetics of percutaneous absorption, and the effects of formulation and application variables on the rate and extent of absorption. Drug samples incorporated into hydrophilic ointment (USP} and hydrophilic ointment containing 10% urea, were applied to the shaved abdominal areas of female New Zealand rabbits for either four or eight hours. Drug samples used for the human studies were taken up in either hydrophilic ointment or in a polyethylene glycol/water solution. Blood samples were drawn from the rabbits over a twenty-four hour period and from humans over an eight-hour period. Absorption of salicylic acid was determined by fluorometric analysis. Pharmacokinetic analysis of (blood) data resulted from two treatments: the Wagner-Nelson methods; and a computerized, non-linear, least squares regression program NONLIN. The systemic absorption of salicylic acid from hydrophilic ointment was substantial in rabbits; however, incorporation of urea, 10% (w/w) effected no significant change. into the test ointment The effect of ointment contact time on serum levels showed that the absorption process was essentially complete within six hours. The elimination curve for salicylic acid applied to animals fed ii. during the test period exhibited an interesting and very substantial secondary peak not seen in fasted animals. The second peak may be due to biliary recycling. The systemic absorption of salicylic acid from polyethylene glycol/water solution applied to human skin was negligible, suggesting the formation of a salicylate-glycol complex. Plasma concentrations resulting from the application of salicylic acid in hydrophylic ointment to the intact skin of humans were less than 1 mg. However absorption from the sam~ system was increased considerably by disrupting the stratum corneum at the site of ointment application.

The epidermis is connectad to the dermis by a series of cohelike ridges or papillae. The dermis consists primarily of a network of densely packed, non-cellular, collagen and elastin fibers. It functions as a supportive system for both blood and lymph vessels as well as for muscle and nerve fibers.
In addition, the dermis contains eccrine, sweat, and sebaceous glands and numerous hair follicles. It varies in thickness from three to five mm., has a pH of about 7.1 to 7.3 and contains 603 to 70% water.
Beneath the dermis lies the subcutaneous fat layer which may be of varied thickness, functioning as the insulation and shock absorber for the internal organs of the body.
Several excellent reviews of the complete anatomy and physiology of the skin have been published (6)(7)(8).

B. Barrier Function of the St~tum Corneum
The epidermis, more specifically the stratum £.Q~ggy~L is generally recognized as the major barrier to the systemic absorption of drugs applied topically. Disruption of the integrity of the st~tu~ £Or.lliU!fil has been shown to increase the diffusion of phenol and of strontium chloride (9,10).
Disease states such as exfoliative dermatitis and psoriasis increase the _diffusion rates of sodium, zinc chloride, ammoniated mercury, and testosterone (11). The low permeability of the stratum corneum compared to the viable layers of the epidermis (10-9 to 10-13 cm2 sec-1 as compared to 10-6 cm2 sec-1) (12), characterize this layer as the principle barrier to diffusion. Through sequential stripping of the stratum £Q.£.!! §ill!! with adhesive tape, Blank (13) showed that its barrier properties remained intact ( until the lowest layers were removed. Scheuplein (14) and Idson (15)    Barr (31) and Idson (32) have published excellent reviews of percutaneous absorption which refer to much of the earlier vork in which salicylic acid was included.
Notable among these early works are those of Kimura (33) and Nogami (34) who were among the first to consider the effect of the vehicle on the rate and extent of absorption of salicylic acid.
Much of the work dealing specifically with salicylic acid or salicylates has been published within the past twenty years and can be conveniently divided into in vif£Q and in vivo studies.   The effect of hydration on the absorption of salicylic acid has been studied by Wurster   Higuchi ( 28) has stated that the driving force for percutaneous diffusion is the thermodynamic activity or activity coefficient. He has also shown that for weakly acidic drugs, such as salicylic acid, activity is inversely proportional to 10~". Arita et al. (49) and Marcus g1 al.   hours.
The dressing was left intact for a period of ten The procedure was repeated daily for five days.  (43)).
The template was centered on the adhesive surface of a strip of adhesive tape; the assembly was carefully inverted and applied to the previously shaved area of the animal. The rabbit was then placed in a restraining box for the duration of the experiment. Water was provided ad lib.i food was given or withheld depending on the test conditions for the particular study. Blood samples were drawn at specified intervals for twenty four hours following initial application of· the test ointment. All blood samples were allowed to clot, centrifuged at approximately 2000 RPM for ten minutes, and then at least one ml. of serum was drawn off and stored under refrigeration until analysis.
Since the proper determination of the pharmacokinetics of a drug usually involves the collection of blood samples

Spectrophotometric
The secondary assay for the determination of salicylic acid in blood relies on the purple color given by a salicylate-ferric ion complex. The procedure used is that developed by Trinder (58) , as modified by Biber and Rhodes In this procedure, mercuric chloride and hydrochloric acid are used to precipitate plasma proteins, ferric nitrate to generate the salicylic acid-ferric ion complex and the purple color on which the colorimetric analysis is based.

IV. RESULTS AND DISCUSSION
A. Animal Pharmacokinetics

Elimination Rate Constants
Elimination rate constants for salicylic acid were

Half Life
Half lives for each treatment were calculated from the elimination rate constants determined by plotting the l og salicylic acid concentration versus time and obtaining the slope of the elimination phase portion of the curve. Since salicylic acid has been shown (23) to follo w first ord er kinetics at low doses, half-life was calculated according to equation (6).
Equation (6) Calculated halr-lives are presented in Table 2. No statistical differences were shown between a n y treatments. Where; At Hr-1, and 0.306 Hr-1, respectively. Absorption rate constants for each treatment are shown in Table 3 and a dAt/V versus time plot is given in Figure 2.   data.
An example fit is shown in Figure 4. The absorption rate constants obtained by the NONLIN fits appear in Table   3.  However, it seems unlikely that the addition of ten percent urea would show any clinically significant increase in the absorption of topically applied salicylic acid.

Lag Time
The lag time may be defined as the time required after application for the first order absorption process to to begin. Extrapolation of the line generated from the log percent remaining versus time curve to the y axis will yield the estimated · lag time for each treatment. calculated lag times are shown in Table 4.
Lag times ·calculated from animals treated for eig ht hours with SA ointment and those treated with SAU ointment were significantly different at the 0.01 level. Similarly, significant differences were shown to exist for animals treated with SA ointment for eight hours compared with those treated with the same ointmen t for a four hour period.  Although no significant difference in areas under the curve could be demonstrated in animals treated with SAU ointment compared with those treated with salicylic acid alone, it is not possible to conclude that there is no effect of ointment on AUC. Because blood level data were obtained from only two animals treated with urea, the test statistic must necessarily be very large in order to show significance. Further work using more animals would be necessary to reach any conclusion regarding the effect of urea on the absorption of salicylic acid measured as the area under the blood level time curve.

Feeding Studies
In contrast to previous studies (43,44) in which animals were fasted throughout an experiment (i.e. for the   Values of ke and ka used for this prediction are those obtained from the normal volunteer treated for three hours.
The dose used was six times that used in the volunteer, corresponding to the six fold increase in the area of ointment application. The dosing interval, T, was every six hours. Figure 7 shows the predicted plasma salicylic acid versus time profile when the dosing regimen described above was carried out for forty eight hours. Steady state salicylic acid levels were attained after five doses and a concentration of approximately 60 mg% was predicted. This level is well above the lower level of toxicity, 20 tO 30mg.
While the values of ka and ke used for this model were those obtained from only one experiment, it is probable that these values approximate those which would be seen in psoriatic patients, therefore, the predicted plasma concentrations appear reasonable.
Although kinetic parameters have not been characterized in psoriatic patients, it is apparent from this work that patients treated chronically with salicylic acid containing ointments face the potential hazard of salicylate toxicity. (1) ™iis IRB has determined that the subjects in this activity are at risk. The risks are: local skin irratation, Tinnitus and the possibility of in-. fectio~ due to blood puncture • . (2) This IRB has determined to its satisfaction that the following saEeuards against the specified risks are adequate: All subjects will be carefully screened before testing for broken skin or history of blood c ::.-,;ula tion disorders, ulcers, or aspirin sensitivity. The tests wi:L be conducted in the University (Potter) Infirmary under the direc tion of an M.D., blood samples will be taken by a licensed medical laboratory technologist, utilizing the hest antiseptic techniques. (3) This !RB has determined t o its satisfaction that the risks to the subjects are so outweighed ~y the sum of the benefits to the subject and the importance of the ~~o wle<l ge to be gained as to warrant a decision to allow the subject to accept the risks. This determination is based upon the following benefits or reasons: The risks involved are small since toxic hazard is both unlikely and of minor nature. This IRB has determined to its satisfaction that legally effective informed consent is to obtained and properly documented in accordance with the requirements specified · in the regulation (45 CFR 46.3 (c) and 46.10). The informational statement to be given or read to each prospective subject before his participation in the activity is attached. (5) This I RB agrees to arrange for the continuing exchange of infornation 73 and advice between itself and the activity director on any matters affecting the rights and welfare of human subjects who participate in the activity. The specific instructions, advice, counsel and the conditions imposed by the !RB for the conduct of the activity are: .. -.:.
. . , ( ( The next scheduled meeting of the !RB for review of this activity is December 1976. The IRB may be called into an interim review session by the Chairman at the request of any member, an institutional official or the project director to consider any matter concerned with the rights and welfare of any subject~ (6) This IRB has determined to its satisfaction that this institution will have available the professional attention and facilit i es that may be needed for subjects who may suffer physical, psychological or other injury as a result of participation in the activity.  To examine and characterize the pharmacokinetics of salicylic acid absorption through the skin, in particular to define the rate and extent of drug absorption. An attempt to characterize interpatient variability will also be undertaken.
yreliminary Screening of Subjects All those being considered for involvement in this study will be healthy volunteers between the ages of twenty and fifty, with no broken skin or history of blood coagulation disorders, ulcers or aspirin sensi-75 tivity. In addition, any other medical abnormality which in the opinion of the medical consultant or investigators is likely to complicate the study will result in exclusion from the study. All subjects will be carefully interviewed and pertinent medical information gathered. All participants will be provided the opportunity of reading and havinz carefully explained the Informed Consent Form. In addition, participants will be provided the opportunity for further questioning of the investigators. Any subjects may withdraw from the study at any time during the investigation. Participants will be required to abstain from aspirin containinp, products for one week prior to the test period and all drugs and alcohol for 43 hours prior to the test period.
A one week "wash-out" period will be allowed between tests.
The tests will be conducted in the University (Potter) Infirmary ( under the direction of an M.D., blood samples will be taken by a licensed medical laboratory technologist.
Salicylic acid will be supplied in the form of a buffered aqueous solution in which the subject will soak his arm for a period of two hours. Blood samples, to be drawn by a licensed medical laboratory technologist, will be taken inunediately preceeding immersion in the test solution and at the following times from the time of immersion, 0.5 hours, 1.0, 1.5, 2.0, 4.0, and 3.0 hours. The test will require a lOml sample to be taken at each interval, for a total of 70mls of blood from each volunteer for each test. Participants will also be asked to provide a urine sample twenty-four hours following application of the test solution.
Minimum toxic levels of salicylic acid have been reported at be- (

Professor and Chairman of Pharmacy
Salicylic acid is a drug used in the treatment of several types of skin disorders. It is normally supplied to the patient in the form of an ointment or cream which is applied topically (i.e., directly to the affected areas of the skin.) The objective of this research project is to study the absorption characteristics of salicylic acid when applied to normal volunteers in the form of an aqueous (water) solution.
Should you volunteer to participate in this project you will be required to abstain from all drugs and alcohol for a period of 48 hours prior to each test, as well as abstaining from all aspirin containing compounds for one week prior to the beginning of the test period and for the duration of the study, approximately ten weeks. The test solution will be prepared by the investigators and will contain 3% salicylic acid in water. You will be asked to place your arm in a bath containing the test solution for a period of two hours. Blood samples will be taken during the course of topical application and at four and eight hours after the start of the test. You will be asked to supply urine samples for a period of 2'• hours following each treatment.
The possible effect of the salicylic acid test solution application is local skin irritation at the site of application of the test solution. Tinnitus (i.e., a ringing sensation in the ears) is the first sign of toxicity which rarely occurs .below salicylate levels of 20mg%. This study is designed not to exceed levels of 10mg%.
Should you volunteer for this project you will be free to withdraw from participation at any time during the investigation. You will not be i de ntified in any publication resulting from this work.

·r,
having carefully read and understood the above and having been given the opportunity of asking questions regarding the above project now give my informed consent to participation in the Study of the Percutaneous Absorption of Salicylic Acid, University of Rhode Island.

Date
Signed Witness