Percutaneous Absorption of Nicotinic Acid and Derivatives

PERCUTANEOUS ABSORPTION OF NICOTINIC ACID AND DERIVATIVES RICHARD B. STOUGHTON, M.D.,* WILLIAM E. CLENDENNING, M.D.f AND DORIS KRTJSE, B.S4

We have utilized a biologic reaction (vaso- derive definite information regarding the route dilation) to measure penetration through the of penetration. The initial presence or absence epidermis of nicotinic acid and seven closely of a follicular or perifollicular reaction pattern may suggest a primarily transfollicular or transepidermal route but is rarely conclusive. Second,

related derivatives. We have also determined the partition coefficients and the absolute solubiities of these substances in water and ether. A correlation is shown between the ease of percutaneous absorption of a given compound and the absolute

the reactivity of an individual subject often varies somewhat from day to day and may depend

upon such variable factors as temperature,

and relative solubilities of these compounds in humidity, season, and the psychic state. ether and water. We feel, however, that useful information

Many technics have been devised for the can be derived from the employment of this detection and measurement of percutaneous method. The drawbacks would seem to be outabsorption (1, 2). One of those which can be weighed by the advantages of using an undisused in vivo on human skin is the biologic method. turbed in vivo system and the knowledge that This depends upon the ability of a topically the development of a reaction to the substance applied substance to reach a specific structure applied indicates with certainty that penetration (blood vessel, gland, smooth muscle, etc.) into the coriuni has taken place. Some agents are resulting in a reaction which can be measured or biologically active in concentrations as dilute visualized by surface observation. Vasodilation as 10 which gives a very sensitive index for and vasoconstriction are good examples of such quantitation. Furthermore, this method permits reactions and a variety of substances can induce investigation of regional differences in percu-

them. Thus, if a known compound such as taneous absorption and allows one to attempt nicotinic acid is applied to the surface of the correlation between these differences and the skin and we observe erythema restricted to the anatomical structure of the region under study. Walzer (3) utilized this method in showing site of application, we surmise that the compound has reached blood vessels in the corium. percutaneous absorption of antigens. First, a site In order to estimate how much of any com- on the skin was passively sensitized. After 24 to pound has penetrated it is necessary to know 48 hours the antigen was rubbed in at another how much of the substance is required to give site. The development of an urticarial reaction a minimal detectable reaction. This can be done in the sensitized area was considered evidence by intradermal injections of serial dilutions of that the antigen had entered the circulation. It the compound under study until the minimal is questionable however whether the "barrier concentration which will give a visible reaction area" was not damaged by the rather vigorous is determined.

method of inunction employed.

Disadvantages of this method are twofold. Shelley and Melton (4) inuncted acetylcholine First, as with other methods, it is difficult to and piocarpine nitrate. They then detected * Director of Dermatology, Western Reserve sweating by the starch paper-iodine method.

Atropine SO4 and Hyoscine hydrobromide were f Senior Resident in Dermatology, University effective topically in inhibiting sweating. A Hospitals, Cleveland 6, Ohio. Laboratory Assistant, Department of Derma- concentrated (18%) aqueous solution of hyoscine tology, Western Reserve University Cleveland 6, hydrobromide resulted in local anhidrosis which Ohio. persisted for weeks after the application (5). Supported by grants from The Army Chemical The biologic effects of the application of histaCenter, Maryland and Department of Health Education and Welfare, A 2196. mine were studied by these same workers (6). Presented at the Twenty-first Annual Meeting of The Society for Investigative Dermatology, Factors accelerating the penetration of histamine such as the vehicle, the concentration, and the Inc., Miami Beach, Florida, June 14, 1960. University, Cleveland 6, Ohio.

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Finally, a word should be said concerning It was found that the use of a cream as the the probable mechanism of action of the nicoform of the active substance were determined.

vehicle, increasing the concentration of the active agent, and using free histamine base as opposed to the salt, would all enhance absorption. They then went one step further and evaluated

tinates. Fulton, Farber, and Moreci (14) have applied concentrated solutions of esters of nicotithe acid to the everted hamster cheek pouch. No qualitative or quantitative differences were

the effect of 54 topical antipruritic preparations noted with respect to the vasomotor effect

on intradermally-induced histamine pruritus (dilation) among the esters studied (methyl, (7). None of the compounds tested had any ethyl, hexyl, tetrahydrofurfuryl). The characsignificant effect suggesting the relative imperme- teristics of this vasodilator response suggested both a direct action of the nicotinate on the ability of normal skin to these agents. Michelfelder and Peck (8) studied the absorp- vascular smooth muscle and mediation by a local tion of pyribenzamine through both intact and nerve conduction mechanism. It did not seem damaged skin by noting the effect of the inuncted likely that the responses initiated were dependent

antthistaminic on intradermal injections of upon tissue injury with resultant release of histamine.

Monash (9) used the topical application of local anesthetics (time required for anesthesia) and histamine (appearance of the triple response)

in his work on the location of the superficial "barrier area". The penetration of adrenaline through the skin has been studied by Nichols (10) using a method quite similar to the one we have employed. Adren-

aline on intradermal injection produced consistent blanching at concentrations of 10—6. Creams with adrenaline incorporated at concentrations of 1:5000 and 1:100 did not elicit visible reactions following inunction. It was concluded that if adrenaline from a 1 in 5000 or 1 in 100 cream passed into the skin, it did so in a concentration below 1 in 1,000,000. Recently there have been a number of reports on the response of normal and abnormal human subjects to the inunction of tetrahydrofurfuryl

histamine-like products. PROCEDURE AND RESULTS

1. Intradermal Injection of Nicotinic Acid and Derivatives

Procedure: Intradermal injections were done in 5 subjects. Serial dilutions (by a factor of 10 from 10-' to 10—10) in normal saline were employed. The

injections were made of the volar surface of the forearm and on the back. .05 ml. of each dilution was injected. Normal saline was used as the control. Detection of erythema greater than that produced by the saline injection was considered a positive reaction. Results: Nicotinie acid and the derivatives used

in this study were all approximately equal in their ability to produce erythema following intradermal injection. At the higher concentrations employed, erythema persisted for several hours following injection. Extremely dilute nicotinate (Trafuril, Ciba) (11, 12, 13). The solutions (10) of all the agents produced trannormal response is erythema and edema. Atopics sient but definite erythema. Dilutions of 10-10 are abnormal in their response (either no reaction elicited no visible reaction from any of the series or blanching) in 50 to 70% of those tested. There (See Table I).

is positive correlation between this abnormal reaction to tetrahydrofurfural nicotinate and the

acetyicholine blanch phenomenon in many

.

Topical

Application of Nicotinic Acid and Derivatives

Procedure: Serial dilutions in 95% alcohol of the compounds under study were applied to the back and volar surface of the forearm of 5 subjects. .01 ml. of each dilution was dropped on the skin from a pipette and lightly spread over a circountered in a recent series of patients with cular area approximately 2 cm. in diameter by 3 diseases unrelated to acute rheumatic fever or rotary movements of the tip of the pipette. Care the atopic diathesis (13). While the percentage was taken not to scratch or damage the superficial of abnormal responses in these conditions is epidermis. Minimal concentrations producing high it would hardly seem of the magnitude to visible erythema were considered the end-point. warrant much enthusiasm for the use of tetraResults: Table II gives the average mininial hydrofurfuryl as a diagnostic test. concentration producing erythema on topical

cases. Approximately 70% of those with acute rheumatic fever give the same abnormal reaction as the atopic (12). Non-reactivity in uncontrolled diabetes was the only abnormal response en-

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PERCUTANEOUS ABSORPTION OF NICOTINIC ACID

TABLE I

considered greater than 50% soluble when 1 ml. or 1 gin, of a liquid or solid substance respectively would dissolve in 1 ml. of ether or water.

Intradermal injection in normal saline (0.05 ml.)

Nicotinic Acid Nicotinic Acid UC1 Methyl Nicotinate

Ethyl Nicotinate Butyl Nicotinate Hexyl Nicotinate Octyl Nicotinate Tetrahydrofurfuryl tinate * Minimal

Nico-

10—'

1O—°.

++ ++ ++ ++ ++ ++

+ + + + + + +

lO—

++ ++ +

0 0 0 0 0 0 0 0

concentration giving erythema.

Results: The absolute solubiities in water and ether are shown in Table IV.

TABLE II Topical application in 95% alcohol (0.01 ml.) End Points Penetration

Nicotinic Acid Nicotinic Acid HC1 Methyl Nicotinate

1:150 1:150 1:20000 1:18000 1:2700

Ethyl Nicotinate

application. Erythema always appeared first Butyl Nicotinate in the follicular and perifollicular areas at the Hexyl Nicotinate

site of application. When higher concentrations Octyl Nicotinate were employed erythema would become diffuse Tetrahydrofurfuryl tinate but was always confined to the area of applica-

1:300

1 1 133 120 18 27 2

1:4300

29

1:4.000

Nico-

tion. At the dilutions given in the table as the * Minimal concentrations giving erythema. end-point, only follicular and perifollicular t Relative concentrations giving erythema. erythema were noted. For ease in comparing activity, a "penetration index" was derived for TABLE III each substance by division of the end-point Water ether partition coefficients dilution of each nicotinate by the end-point dilution of nicotinic acid which was arbitrarily Nicotinic Acid 9.25 designated as 1.

3. Water/Ether Partition (Joefflcient8 Procedure: A known amount of nicotinate was

Nicotinic Acid HCI Methyl Nicotinate

Ethyl Nicotinate Butyl Nicotinate added to a mixture of equal parts of ether and Hexyl Nicotinate water. After shaking, aliquots of ether and water Octyl Nicotinate were taken for determination of nicotinic acid Tetrahydrofurfuryl Nicotinate after hydrolysis in excess 0.1 N NaOH. Titration with a standard acid (.1 N HC1) was carried out TABLE IV to determine the amount of alkali remaining following hydrolysis. From this the number of Absolute solubilities milliequivalents of nicotinate dissolved in 25 ml. Ab. Sol. H,0 samples of water and ether was calculated and the partition coefficient derived. Nicotinic Acid 24.7 mg/ml Re8ults: The water/ether partition coefficients Nicotinic Acid HC1. 304.2 mg/mi are given in Table III. Methyl Nicotinate.. >50% Ethyl Nicotinate.... 45.9 mg/mi 4. Absolute Solubilities in Water an4 Ether Butyl Nicotinate.... 10.7 mg/mi 4.9 mg/mI Procedure: Water and ether were saturated with Hexyl Nicotinate. .. 2.1 mg/nil excess amounts of the various nicotinates. Each Octyl Nicotinate.... solution was placed on a magnet mixer for two Tetrahydrofurfuryl Nicotinate days to ensure saturation. Care was taken to avoid >50% evaporation. One ml. samples were taken of each * Absolute solubility in water. saturated solution and titrated according to the procedure described in section 3. A compound was t Absolute solubility in ether.

45.4 .373 .136 .028 .012 .032 .394

Ab. SoL Ethert

1.1 mg/mI .4 mg/mI >50% >50% >50% >50% >50%

>50%

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TABLE V Summary of Findings

Nicotinic Acid Nicotinic Acid HC1 Methyl Nicotinate

Ethyl Nicotinate Butyl Nicotinate Hexyi Nicotinate Octyl Nlcotinate Tetrahydrofurfuryi Nicotinate

Penetration

W/EPart.

1 1 133 120 18

9.25 45.4

27

2 29

.373 .136

.028 .012 .032 .394

Ab. Sol. H,O

24.7 mg/mi 304.2 mg/mi

>50% 45.9 mg/mI 10.7 mg/mi 4.9 mg/mi 2.1 mg/mi >50%

Ab. Sol. Ethers

1.1 mg/mi .4 mg/mi >50% >50% >50% >50% >50% >50%

* Relative rates of penetration. f Water/ether partition coefficient.

Absolute solubility in water. in ether.

* Absolute solubility

DISCUSSION

The lipid theory of Meyer and Overton regard-

ing percutaneous absorption was proposed 35 years ago (15). Subsequent modifications such as

the mosaic theory and the importance of the

water/ether partition co-efficient have not drastically altered the original postulate that it is

disproof to the Meyer-Overton concept. It was felt that a related series of biologically active substances with varying water/ether solubilities would be useful in this endeavor. Accordingly, nicotinic acid and 7 derivatives were obtained and studied by the methods outlined. All of the derivatives proved active at equal concentrations on intradermal injection (Table I). Topical applications (Table II) show a wide

the ratio of the solubiities in water and lipid that is important in enhancing percutaneous variation in percutaneous absorption and produce absorption. measurable, biologic response as Although this theory and its modifications are consistent, evidenced by vasodilation (erythema). The

widely accepted there has been relatively little correlation of ability to penetrate with relatively direct experimental evidence to support it. good solubiities in both water and ether (Table Technical difficulties are formidable. Most V) is of sufficient degree to support our present workers since the introduction of radioactive or concept of the importance of this factor in the tagged compounds have employed electrolytes. regulation of percutaneous absorption. Their studies have confirmed the impression that the epidermis is relatively impermeable to these

SUMMARY

substances. One study by Treherne (16), who

Nicotinic acid and 7 derivatives are compared used an in vitro system and C'4 and 535 labelled regarding percutaneous absorption and solubility non-electrolytes, does support the concept of the

characteristics. Ability of these compounds to

importance of the water/ether partition co- penetrate into the coriuni (percutaneous absorp-

efficient. Another by Valette (17) indicates that tion) seems related to their relative and absolute viscosity as well as liposolubiity is of importance. solubilities in water and ether. Hence, for a series of related non-electrolytes with

similar partition coefficients, penetration will be

greatest when a certain optimum chain length exists. In his series this ranged between 6 and 9 carbon atoms.

Our work was undertaken for two reasons. First, to develop the biologic method of studying

percutaneous absorption to a practical level; second, in the hope of adding further proof or

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DISCUSSION Do you know anything about the mechanism of action of these chemicals on the blood vessels? there are regional differences in the skin's What is the chemical basis of their effect? response to nicotirdc acid. The "blush" areas of DR. WILLIAM CLENDENNING (in closing): I the ears, face, neck and upper chest showed the would like to thank the discussers. most pronounced erythema from this compound. In answer to Dr. Malkinson, we feel that it Because there seem to be anatomic variations in will be interesting to investigate different areas the skin's threshold for response I wonder if of the body with regard to variation in absorpthe authors have found more rapid vasodilation tion and we are planning to do this in the imin these sites than in others. mediate future. As a further comment I would like to add that In answer to Dr. Fitzpatrick, we have not there have been no extensive studies of changes done this ourselves, but Fulton, Faber and in percutaneous absorption in specific skin disMoreci (1) have studied the action of nicotinates orders. Illig showed some time ago that in patients Da. FREDERICK D. MALKINSON (Chicago, Ill.):

I think it was an old observation of Spies that

with atopic dermatitis the skin responds para- and found there is both a direct action on the doxically to the application of iiicotinic acid and

blood vessel wall and also a local nerve conduction

its esters with vasoconstriction rather than mechanism. vasodilatation. The work that Dr. Stoughton and his colleagues have carried out could be logically extended to study the extent of increased permeability of the skin -if any -in such patients. PRESIDENT FITZPATRICK (Boston, Mass.):

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