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The evaluation of various bases for sulfonamide ointments*
Scient$c Edit iolz
JOURNAL OF THE AMERICAN PHARMACEUTICAL .ASSOC I A T I ON JUSTIN
VOLUMEXXXV
L. POWERS, EDITOR, WASHINGTON, D. C.
MARCH, I946
NUMBER 3 CONSECUTIVE No. 6
The Evaluation of Various Bases for Sulfonamide Ointments* By F. J. BANDELIN and C.R. KEMPt A method for the estimation of the amopnt of sulfonarnidesreleased from various ointments upon contact with saline or serum fdr varying lengths of time is described.
the advent of sulfonamide therapy there has been an increasing interest in the direct application of these compounds to infected or potentially infected areas. The local use of sulfonamides in any type of accessiblewound or infection,or as a prophylactic in surgery, is so well established that a review of the literature is unnecessary. The use of sulfonamides in ointment bases has likewise become common practice since in this form they are not only easily applied and h d d in place but, as Strakosch and Olsen (1) have pointed out, the advantage of such topical application is twofold as compared with oral administration. First, they state, it can be assumed that the local tissue concentration of the drug can be reached INCE
*
Received Aug 15 1945 from the Research Laboratories, Flint, Eaton & cdmpany 6ecatur Ill. t Praent address: De&. of Int&nal Medicine. State University of Iowa, University Hospitals. Iowa City, Iowa.
with smaller amounts of the drug when thus applied, and second, the possibility of untoward toxic reactions is minimized. Furthermore, the external use of sulfonamide powders has the disadvantage of caking or forming crusts in or about the wound. For these reasons, and because of convenience of use, a variety of sulfonamide pastes and ointments have been used or suggested. Some sulfonamide ointments have been suspensions of the drug in bases such as petrolatum and lanolin, or in mixtures of petrolatum, lanolin, oils, fats, and waxes. Others have been suspensions in emulsified bases containing, in addition to these constituents, varying quantities of water. Preliminary work has indicated that the emulsion type bases seem to be superior to the hydrocarbon bases and that, of the emulsified bases, the oil-in-water type is more effective than the water-in-oil type. The disadvantage of the hydrocarbon bases and, to some extent, of the water-in-oil emulsions seems to be due to the fact that the oils and the grease form a barrier, allowing little or no 65
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contact between the drug and the tissues. Although the work of some investigators (27) indicates that the oil-in-water emulsion bases are superior, other workers (8,9) claim better results with the water-in-oil emulsions. In a recent editorial (10) it has been pointed out that many apparent therapeutic failures of sulfonanlides are due to improper topical application of the drug with the resulting low local concentrations of the drug in the tissues of the area applied. Although the amount of sulfonamide applied b a y be sufficient, the form and manner in prhich it is applied may be such that absorption is insufficient and therefore bactericidal concentrations of the drug in the infected area are not obtained. On examination it would seem that the hydrophilic oil-in-water bases offer several physical advantages over the water-in-oil bases. In the former, much of the sulfonamide is dissolved in the aqueous or continuous phase of the emulsion and this presents to the tissues a readily available source of the drug. In the latter type, the water is the dispersed phase, the aqueous part of the emulsion existing as minute droplets or globules each surrounded by the continuous phase of oil. This oily material forms a barrier between the drug, either in solution or as powder particles, and the tissues. Furthermore, the hydrophilic bases, because of their low surface tension, more readily form a continuous surface on the tissues and because of their water and serum miscibility, aid the disseminati6n of the drug in the wound. It has been pointed out (5,ll) that because of their serum miscibility, these ointments do not interfere with drainage of the wound, nor do they interfere with the normal function of the skin nearly as much as do the greasy ointments which inevitably introduce interfacial tension at the apposition of the mutually immiscible oil and exudate. It has been illustrated by Gershenfeld and Miller (12)) Foley and Lee (13)) and Gibson, et al. (14)) among others, that antiseptics and germicides are generally more active in oilin-water bases than in water-in-oil bases. Fantus (15) states that greasy ointments are
contraindicated in acute inflammatory conditions of the skin because of their “heating quality.” He shows that cream ointments or emulsions do not have this objection and further states that the success or failure of the treatment depends upon the proper choice of the base. In order to evaluate further various sulfonamide ointment bases we have attempted : ( a ) to determine the general availability of the sulfonamide and the rapidity of its release from the different bases when a static surface is exposed to a fluid such as saline or serum under conditions simulating those existing in the wound; ( b ) to correlate the results, if possible, with the .bacteriological data obtained in testing these ointments against Staphylococcus aureus using the F. D. A. agar cup plate method. EXPERIMENTAL I n the investigation of the various ointment bases we have attempted t o choose representative ointments of the various types, and in the case of the emulsion bases, with varying quantities of water. We have divided them into three classes: the water-free hydrocarbon bases, the water-in-oil emulsions, and the oil-in-water emulsions. Some of these formulas are taken directly from the literature where in some cases they have been recommended as vehicles for sulfonamides, others are official bases such as the b.S. P. Ointment and Hydrous Lanolin, and still others are either extemporaneous preparations or formulas taken from the literature and modified. T h e following various formulas are designated by numbers which will be used throughout the paper to identify t h e bases instead of the entke formulas which are space consuming. Hydrocarbon Base-Water Free No. 1. Lanolin, anhydrous.. ........... 5% White Wax.. .................. 570 White Petrolatum.. .............90% This is the U. S. P. XI1 White Ointment (16) generally used as a base in official ointments. Water-in-Oil Emulsion Bases No. 2. Hydrous Lanolin This also is an official preparation (17) containing 30y0 water emulsified in lanolin. No. 3. Arlacel C1 ................... 5.00% Lanolin, anhydrous. . . . . . . . . . 5.00% Petrolatum, white.. . . . . . . . . . .32.50% Mineral Oil, heavy. . . . . . . . . . . 7.50% Glycerin.. . . . . . . . . . . . . . . . . . . 5.00% Water.. . . . . . . . . . . . . . . . . . . . .45.00’% 1 Sorbitan Monooleate, Atlas Powder Company, Wilmington, Del.
SCIENTIFIC EDITION
67
This also is a water-in-oil emulsion containing more water, however, than No. 2.
Sulfonamide ointments were prepared in 5% and 10% strengths by warming the base t o 70' on a
No. 4. Absorption Base. . . . . . . . . . . . . . . .22% ............
water bath, adding the sulfonamide, then removing from the water bath and stirring continuously until cooled to room temperature. In attempting to estimate the rate and amount of sulfonamide released &om the ointment, the following procedure was adopted. Four grams of the ointment was applied evenly to the inside surface of a 6-x 1-inch test tube using a glass rod to spread the ointment in a uniform layer with a circular motion. Twenty-five cubic centimeters of saline or serum was then pipetted into t h e tube (capacity 60 cc.) and the tubes then placed in an incubator at 37.5' for varying lengths of time. Five tubes were thus prepared with each ointment and one taken for analysis after two, four, six, eight, and twenty-four hours' incubation. The contents of the tube taken for analysis were poured carefully into another tube and agitated by inverting three times. An aliquot of this taken for analysis was used directly or after appropriate dilution depending upon t h e relative concentration of the drug in the liquid. The amount of sulfonamide in t h e aliquot was determined colorimetrically by the method of Bratton and Marshall (20) using an electrophotometers and a filter with a maximum transmission at 525 mp. Although this method is merely an estimation of the amount of sulfonamide absorbed from the ointment, we have obtained, in preliminary work, fairly uniform and reproducible results by carrying out determinations on a number of series of tubes of the same ointment. For instance, 6 tubes of the same sulfonamide ointment were found to release in twenty-four hours 61,65,64,71,62,and 57 mg. of the drug in 25 cc. of saline. On this basis we have assumed that the method as given above might be used as a criterion of the ability of various ointment bases t o liberate sulfonamides incorporated in them. The following tables and graphs indicate both the rate and amount of sulfonamide released in the various periods of time designated.
Petrolatum, white.. . . . . . . . . . . . . . Glycerin. . . . . . . . . . . . . . . . . Water. . . . . . . . . . . . . . . . . . . This is an extemporaneous absorption base cream containing more water than either of the previous two emulsions. Oil-in-Water Emulsion Bases No. 5. Cetyl Alcohol.. . . . . . . . . . . Propylene Glycol. . . . . . . . . White Wax. . . . . . . . . . . . . . . . . . . . Sodium Lauryl Sulfate.. ... Water. . . . . . . . . . . . . . . . . . . . This hydrophilic, washable ointment base has been recommended (18,19) as a base for sulfonamides.
No. 6. Cetyl Alcohol.. . . . . . . . . . . . . . . . . 6% Lanolin ........................ 6% Sodium Lauryl Sulfate.. . . . . . . . . . 2% Petrolatum, white.. . . . . . . . . . . . . .14% Mineral Oil, heavy.. . . . . . . . . Water. .......................
This formula contains the same emulsifying agent as No. 5 but contains considerably more oil.
No. 7. Stearic Acid.. . . . . . . . . . . . . . . . . . .25% Triethanolamine.. ... Carbitol . . . . . . . . . . . . Lanolin. ...... Water. .......
This base is a triethanolamine stearate emulsion of the vanishing cream type.
No. 8. Stearic Acid.. ..................15% Lanolin. . . . . . . . . . . . . . . 4% Mineral Oil, heavy. . . . . . . . . . . . . .25% Triethanolamine.. . . . . . . . . . . . . . . 2y0 Glycerin. . . . . . . . . . . . Water . . . . . . . . . . . .. : . . . . . . . . . . This base, like No. 7, is also a triethanolamine stearate emulsion but containing more oil. No. 9. Glycerylmonostearate. . . . . . . . . . .18% Carbowax 15002.. . . . . . . . . . . . . . . 2% Glycerin. ............. Water. ....................... This is a simple glycerylmonostearate cream with
a high water content.
. . . .12% Lanolin. ...... . . . .lo% Cetyl Alcohol.. . . . . . . . . . . . . . . . 2Y0 Mineral Oil, heavy. . . . . . . . . . . . .25% Carbowax 1500a.. . . . . . . . . . . . . . 2%
No.10. Glycerylmonos
Glycerin. . . . . . . . . . . . . . . . . . . . . . Water.. .....................
5%
.#yo
This has the same emulsifying agents as No. 9 but contains more oil.
* Carbide & Chemicals Corp.. New York, N. Y.
TABLE I.-TOTAL SULFATHIAZOLE (IN MILLIGRAMS) RELEASEDINTO 25 CC. OF SALINE BY 570 SULFATHIAZOLE OINTMENTS~ Base No.
1 2 3 4 5 6 7 8 9 10 a
Hours of Exposure of Ointments to Saline 2 4 6 8 24
0.2 1.0 0.3
0.0 12.0 4.0 11.5 13.0 11.0 8.0
0.2 1.0 0.4 0.2 20.0 14.5 17.5 17.0 15.0 11.0
0.3 1.2 0.4 0.4 27.5 26.0 20.0 23.0 20.0 16.0
0.3 1.5
0.4 1.7
1.4
1.9 ~-
0.7 31.0 31.0 24.0 25.0 25.0 19.0
2.0 40.0 34.0 35.0 27.5 27.5 22.5
-.
~
See Fig. 1.
Fisher Electrophotometer, Fisher Scientific Co.. Pittsburgh, Pa. J
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(IN MILLITABLEII.-ToTAL SULFATHIAZOLE (IN MILLI- TABLE IV.-TOTAL SULFONAWDE GRAMS) RELEASED INTO 25 cc. OF SALINE B Y 10% GRAMS) RELEASED INTO 25 cc. SERUM BY SULFONAMIDE OINTMENT^ SULFATHIAZOLE OINTMENTP
Hours of Exposure of Ointments to Saline 4 6 8 24
Base No.
2
1 2 3 4 5 6 7 8 9 10
0.2 0.9 0.7 0.1 19.0 6.0 16.0 13.5 12.0 8.0
0.2 1.2 0.8 0.4 35.0 11.5 31.0 33.0 16.0 12.5
0.3 . 1.6 r.2 0.8 41.0 32.5 35.0 37.0 23.5 19.0
0.5 1.8 1.4 1.0 43.2 48.5 37.0 36.0 32.0 24.0
0.5 1.8 3.2 3.1 52.0 50.5 43.7 50.5 42.5 37.0
See Fig. 2.
TABLE111.-TOTAL SULFANILAMIDE (IN MILLIGRAMS) RELEASED INTO 25 cc. OF SALINEBY
Base No.
2
10% Sulfathiazole Ointments 0.2 0.2 0.4 0.7 0.5 1.1 2.0 2.6 17.0 39.0 51.0 77.0 9.5 26.5 33.7 34.0 75.0 50.0 55.0 19.0 10% Sulfanilamide Ointments 0.2 0.3 0.4 0.6 1.0 4.0 9.0 15.0 62.5 110.0 142.0 187.0 58.0 93.0 137.0 156.0 75.0 120.0 142.0 162.0
1 3 5 8
9 1 3 5 8 9 a
Hours of Exposure of Ointments to Serum 4 6 8 24
0.7 2.6 92.0 58.7 97.0 0.9 19.0 205.0 190.0 192.5
See Figs. 5 and 6.
SULFANILAMIDE OINTMENTS4
Hours of Exposure of Ointments to Saline 2 4 6 8 24
Base No.
5% Suuanilamide Ointments 0.3 0.4 0.4 0.3 7.0 1.2 4.3 7.0 17.0 35.0 37.5 50.2 45.0 77.5 18.5 40.0 95.0 95.0 25.0 ,40.0 25.0 10.0 21.0 6.25 10% Sulfanilamide Ointments 0.5 0.5 0.6 0.7 2.0 7.7 12.0 17.0 23.0 55.0 75.0 90.5 40.0 60.0 90.0 150.0 80.0 125.0 160.0 160.0 31.0 45.0 10.0 20.0
1 3 5 7 8 9
1 3 5
7 8 9 6
0.4 18.5 60.0 82.5 95.0
42.0 0.7 40.0 107.5 195.0 185.0 60.0
See Figs. 3 and 4.
d
60
I-
F.
2
4
6
8
Time in Hours Fig. 2.-Total Sulfathiazole Released into 25 cc. of Saline by 10% Sulfathiazole Ointments.
I
20
' "
M
0
A
1 1 6 Q Time in Hours
Fig. 1.-Total Sulfathiazole Released into 25 cc. of Saline by 5% Sulfathiazole Ointments.
All ointments were tested bacteriologically against Staphylococcus aureus using the F. D. A agar cup plate method (21). Tubes containing 20 cc. of sterile nutrient agar were inoculated with 0.1 cc. of a twenty-four hour culture of Staphylococcu: aureus at 42" and immediately poured into sterile Petri dishes. After the agar had solidsed, 1.5 cm. cups were cut in the center of the plates and 2.0 Gm. of the various ointments introduced. A drop of sterile broth was dropped into the cup t o assure uniform contact of the ointment with the agar media. Thc Petri dishes prepared in this manner were incubated a t 37.5' for twenty-four hours. A t th: end of this period they were removed from the
SCIENTIFIC EDITION
i
69
i
P
F9
20 9
3
I
0
2 4 d B
k
Time in Hours Fig. 3.-Total Sulfanilamide Released into 25 cc. of Saline by 5% Sulfanilamide Ointments.
Time in Hours Fig. 5.-Total Sulfathiazole Released into 25 cc. of Serum by 10% Sulfathiazole Ointments.
d
z
Time in Hours Fig. 4.-Total Sulfanilamide Released into 25 cc. of Saline by 10% SulfanilamideOintments.
Time in Hours Fig. 6.-Total Sulfanilamide Released into 25 cc. of Serum by 10% SulfanilamideOintments.
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incubator and the zone of inhibition measured and recorded in millimeters. The following results were obtained with the various ointment bases. TABLE V
Base No.
5% Sulfathiazole Ointment
Zone of Inhibition, Mm.10% 5% 10% SulfaSulfanilSulfanilthiazole amide amide Ointment Ointment Ointment
0 3
0 3
0 1
0
2
3
1 0
1 0 15 10 10 6 15 6
0 0 15
0
1 4
5 6 7 8 9 10
14 10 10 6 14 6
8
1
0 15 10 12
11 5 18
7 18
12
12
DISCUSSION Analysis of saline and serum in contact with the ointments for varying lengths of time indicated that the greasy hydrocarbon base, i.e., U. S. P. Ointment, released relatively little of the sulfonamide incorporated in it. Release of the sulfonamides from water-in-oil emulsions was only slightly better while release from the oil-in-water hydrophilic bases was much more rapid and far greater in every case. This is undoubtedly due t o the low surface tension and water or serum miscibility of these bases. It, was observed that these bases diffused into the saline or serum forming a uniform suspension. The hydrocarbon and water-in-oil emulsions formed a distinct interface with the fluid in contact with it so that only the sulfonamide at the interface was in contact with the fluid. In some instances, the drug available from the U. S. P. base or the water-in-oil emulsions was so little that only the results obtained from one of these was plotted on the graphs to show the contrast. It is an interesting fact that base No. 3, which is a water-in-oil emulsion containing 50% aqueous phase, and base No. 6, a n oil-in-water emulsion containing 50% water, exhibit marked differences in their abilities to give up their sulfonamide content. The hydrophilic bases used in this study were developed in pairs, each pair having the same emulsifier but with one member containing considerably more petrolatum or grease than the other. This was done to determine whether or not a high concentration of
the oil phase interfered with the release of the sulfonamides from ointments of this type. Bases 5 and 6 have as an emulsifier sodium lauryl sulfate with base No. 6 having a rather high petrolatum content. Bases Nos. 7 and 8 contain triethanolamine as an emulsifier, No. 8 having in it lanolin and petrolatum in addition to the triethanolamine stearate. Bases Nos. 9 and 10 are emulsified with glyceryl monostearate and Carbowax 1500, base No. 10 containing a considerable amount of lanolin and petrolatum not present in No. 9. I n all cases where lanolin and petrolatum were added they largely replaced water in the formula so that by increasing the oily phase of the emulsion, the aqueous phase was reduced. In most cases, increasing the oily phase by the addition of lanolin and petrolatum seemed t o interfere only slightly, if a t all, with the ability of the bases to give up their sulfonamide content. This is apparently due to the concehtration of the sulfonamide in the aqueous phase in which case the oil globules do not interfere with the solubility of the sulfonamides . As indicated in the graphs and tables, considerably more sulfanilamide than sulfathiazole was available from the ointments. This may be expected because of the higher solubility of sulfanilamide. It. also seemed generally true that the ointments released more sulfonamide into serum than into saline. This is undoubtedly due t o the higher solubility of these compounds in serum as pointed out by Clark, e t a l . (22). The oil-in-water bases seemed t o give up most of their sulfonamide content quite rapidly in the first six to eight hours and liberated only slightly more in the period up to twenty-four hours. The water-in-oil emulsions gave up only a fraction of their sulfonamide content very slowly and continued to release small increasing amounts up to and beyond the twenty-four hour period. Ointments tested by the F. D. A. agar cup plate method against Staphylococcus aureus gave inhibition zones which generally paralleled the liberation of the drug from the various ointments by the method described. Ointments which liberated a large amount of sulfonamide gave the largest inhibition zones while those liberating small amounts of sulfonamide gave little or no zone of inhibition. Here, again, it seemed that the oil-in-water emulsions were the most satisfactory bascs.
SUMMARY
A method for the estimation of the amount of sulfonamides released from various sulfonamide ointments upon contact with saline or serum for varying lengths of time is described. By this method, the availability of the sulfonamides incorporated in various ointment bases is estimated.
The types of bases studied were a hydrocarbon water free base (U. S. P. Ointment), water-in-oil emulsion bases and oil-in-water emulsion bases. Ointments were prepared by incorporating 5 per cent and 10 per cent of sulfanilamide or sulfathiazole in the various bases. Of these ointments the oil-
SCIENTIFIC EDITION in-water emulsion type was most efficient, liberating its sulfonamide content more rapidly and more completely than the others. The hydrocarbon base wks the poorest in this respect and the water-in-oil emulsions only slightly better. Increasing .the oil phase of the oil-in-water emulsion bases did not seem to materially affect the liberation of the sulfonamides. All ointments were tested against Stuphyl-
71
ococcusaureususing the F. D. A. agar cup plate method. Largest zones of growth inhibition of the organisms were obtained with the oil-in-water emulsions with other bases showing little or no inhibition. From these results it seems logical to conclude that because of their low surface tension and water miscibility, the oil-in-water type emulsions are superior bases for sulfonamide ointments.
REFERENCES (13) Foley, E., and Lee, C. 0..THISJOURNAL., 31, 105
“%]’ Gibson, A. J., Parker, H.E.,and Almus, A., ibid., 30, 196(1941).
1 (1942). (5) Gurd, F. B., Ackman, D., and Smith, F., Ann. Surg., 118, 921(1943). (6) Kern R. A. U.S. Naval Med. Bull. 41,1654(1943). (7).Pillsdury, D. M., Wammock, V. S.: Twingood, C. S., and Nichols A. C. A m . J . Mcd. Scr 202, 808(1941). (8) Wimmer, i. H.. and Strako&h, E. A., J. A m . Med. Assoc., 118, 221(1942). (9) Kuney E. L. Pembroke R. H., Chatard. F. E., and Ziegler, J. M.,’ibid., i17, 1415(1$41). (10) Editorial, U.S.Naval Mcd. Bull., 44,861(1944). (11) Ackman D. Gerrie J. W Pritchard, J. E., and Miller, E. s.,Ann. ‘ s ~ & ,119, i61U.944). (12) Gershenfeld, L., and Mlller. R. E., A m . J . Pharm., 105, 194(1933).
(15) Fantus, Bernard, J . A m . Mcd. Assoc., 107, 861-63 (1936). (16) “Pharmacopceia of the United States,” Twelth revision Mack Printing Co,, Easton, Pa.,1942,p. 533. i7 Ibid. p. 35. 1181 Eva& C. H..THISJOURNAL, Piact. Pharm. Ed., 3, 235(1942). (19) Baker W. B. Calco Technical Bulletin N o . 686, Calco
Chemical Diiision, American Cyanamid Company, Bound Brook N. J. (20)’ Bratton, A. C., and Marshall, C. M., J . B i d . Chcm..
128, 537(1939). (21) Ruehle,. George D.,, and Brewer, C. M., U.S. Food and Drus AdmrnrstrafronCrrcular No. 198, U . S. Dept. Agr., 1931. (22) Clark, G. W., Strakosch, E. A,, and Levitan, N. I., J . Lob. C l m . Med.. 28, 188(1942).
The Separation of Acetylsalicylic Acid, Acetophenetidin, and Caffeine* By K E N N E T H E. HOLTf which are freely soluble in ether. The results obtained are comparable to those ration of mixtures containing acetylsalicylic obtained with the A. 0. A. C. method acid, acetophenetidin, and caffeine. Al- (1, 3) while the time required for the comthough the present A. 0. A. C. method (1) plete analysis by an experienced analyst is produces satisfactory results the hydrolysis reduced to less than three hours. of acetophenetidin is time-consuming and EXPERIMENTAL affords considerable chance of error. Acetophenetidin.-Accurately weigh a sample The method outlined here eliminates the containing about 0.3 Gm. of acetophenetidin and hydrolysis of acetophenetidin by utilizing transfer to a separatory funnel. Add 25 cc. of the relative insolubility of caffeine in ether HCl (1 + 10) and extract with purified ether using to effect the separation of caffeine from one 40-cc. portion and five 20-cc. portions. Combine ether extracts in another separatory funnel and acetylsalicylic acid and acetophenetidin, wash with one 10-cc. portion HC1 (1 + 10) and one FREQUENT
operation of the pharma-
A ceutical control laboratory is the sepa-
*Received May 23, 1945, from the Chemical Laboratories, Cole Chemical Company, St. Louis, Mo. t Acknowledgment is made to Wendell L. Holt and Margaret Miller for their suggestions,
10-cc. portion ‘of water, washing these in turn through two 25-cc. portions of ether and adding to the original acid solution, (Save the acid solution
JOURNAL OF THE AMERICAN PHARMACEUTICAL .ASSOC I A T I ON JUSTIN
VOLUMEXXXV
L. POWERS, EDITOR, WASHINGTON, D. C.
MARCH, I946
NUMBER 3 CONSECUTIVE No. 6
The Evaluation of Various Bases for Sulfonamide Ointments* By F. J. BANDELIN and C.R. KEMPt A method for the estimation of the amopnt of sulfonarnidesreleased from various ointments upon contact with saline or serum fdr varying lengths of time is described.
the advent of sulfonamide therapy there has been an increasing interest in the direct application of these compounds to infected or potentially infected areas. The local use of sulfonamides in any type of accessiblewound or infection,or as a prophylactic in surgery, is so well established that a review of the literature is unnecessary. The use of sulfonamides in ointment bases has likewise become common practice since in this form they are not only easily applied and h d d in place but, as Strakosch and Olsen (1) have pointed out, the advantage of such topical application is twofold as compared with oral administration. First, they state, it can be assumed that the local tissue concentration of the drug can be reached INCE
*
Received Aug 15 1945 from the Research Laboratories, Flint, Eaton & cdmpany 6ecatur Ill. t Praent address: De&. of Int&nal Medicine. State University of Iowa, University Hospitals. Iowa City, Iowa.
with smaller amounts of the drug when thus applied, and second, the possibility of untoward toxic reactions is minimized. Furthermore, the external use of sulfonamide powders has the disadvantage of caking or forming crusts in or about the wound. For these reasons, and because of convenience of use, a variety of sulfonamide pastes and ointments have been used or suggested. Some sulfonamide ointments have been suspensions of the drug in bases such as petrolatum and lanolin, or in mixtures of petrolatum, lanolin, oils, fats, and waxes. Others have been suspensions in emulsified bases containing, in addition to these constituents, varying quantities of water. Preliminary work has indicated that the emulsion type bases seem to be superior to the hydrocarbon bases and that, of the emulsified bases, the oil-in-water type is more effective than the water-in-oil type. The disadvantage of the hydrocarbon bases and, to some extent, of the water-in-oil emulsions seems to be due to the fact that the oils and the grease form a barrier, allowing little or no 65
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AMERICAN ’PHARMACEUTICAL ASSOCIATION
contact between the drug and the tissues. Although the work of some investigators (27) indicates that the oil-in-water emulsion bases are superior, other workers (8,9) claim better results with the water-in-oil emulsions. In a recent editorial (10) it has been pointed out that many apparent therapeutic failures of sulfonanlides are due to improper topical application of the drug with the resulting low local concentrations of the drug in the tissues of the area applied. Although the amount of sulfonamide applied b a y be sufficient, the form and manner in prhich it is applied may be such that absorption is insufficient and therefore bactericidal concentrations of the drug in the infected area are not obtained. On examination it would seem that the hydrophilic oil-in-water bases offer several physical advantages over the water-in-oil bases. In the former, much of the sulfonamide is dissolved in the aqueous or continuous phase of the emulsion and this presents to the tissues a readily available source of the drug. In the latter type, the water is the dispersed phase, the aqueous part of the emulsion existing as minute droplets or globules each surrounded by the continuous phase of oil. This oily material forms a barrier between the drug, either in solution or as powder particles, and the tissues. Furthermore, the hydrophilic bases, because of their low surface tension, more readily form a continuous surface on the tissues and because of their water and serum miscibility, aid the disseminati6n of the drug in the wound. It has been pointed out (5,ll) that because of their serum miscibility, these ointments do not interfere with drainage of the wound, nor do they interfere with the normal function of the skin nearly as much as do the greasy ointments which inevitably introduce interfacial tension at the apposition of the mutually immiscible oil and exudate. It has been illustrated by Gershenfeld and Miller (12)) Foley and Lee (13)) and Gibson, et al. (14)) among others, that antiseptics and germicides are generally more active in oilin-water bases than in water-in-oil bases. Fantus (15) states that greasy ointments are
contraindicated in acute inflammatory conditions of the skin because of their “heating quality.” He shows that cream ointments or emulsions do not have this objection and further states that the success or failure of the treatment depends upon the proper choice of the base. In order to evaluate further various sulfonamide ointment bases we have attempted : ( a ) to determine the general availability of the sulfonamide and the rapidity of its release from the different bases when a static surface is exposed to a fluid such as saline or serum under conditions simulating those existing in the wound; ( b ) to correlate the results, if possible, with the .bacteriological data obtained in testing these ointments against Staphylococcus aureus using the F. D. A. agar cup plate method. EXPERIMENTAL I n the investigation of the various ointment bases we have attempted t o choose representative ointments of the various types, and in the case of the emulsion bases, with varying quantities of water. We have divided them into three classes: the water-free hydrocarbon bases, the water-in-oil emulsions, and the oil-in-water emulsions. Some of these formulas are taken directly from the literature where in some cases they have been recommended as vehicles for sulfonamides, others are official bases such as the b.S. P. Ointment and Hydrous Lanolin, and still others are either extemporaneous preparations or formulas taken from the literature and modified. T h e following various formulas are designated by numbers which will be used throughout the paper to identify t h e bases instead of the entke formulas which are space consuming. Hydrocarbon Base-Water Free No. 1. Lanolin, anhydrous.. ........... 5% White Wax.. .................. 570 White Petrolatum.. .............90% This is the U. S. P. XI1 White Ointment (16) generally used as a base in official ointments. Water-in-Oil Emulsion Bases No. 2. Hydrous Lanolin This also is an official preparation (17) containing 30y0 water emulsified in lanolin. No. 3. Arlacel C1 ................... 5.00% Lanolin, anhydrous. . . . . . . . . . 5.00% Petrolatum, white.. . . . . . . . . . .32.50% Mineral Oil, heavy. . . . . . . . . . . 7.50% Glycerin.. . . . . . . . . . . . . . . . . . . 5.00% Water.. . . . . . . . . . . . . . . . . . . . .45.00’% 1 Sorbitan Monooleate, Atlas Powder Company, Wilmington, Del.
SCIENTIFIC EDITION
67
This also is a water-in-oil emulsion containing more water, however, than No. 2.
Sulfonamide ointments were prepared in 5% and 10% strengths by warming the base t o 70' on a
No. 4. Absorption Base. . . . . . . . . . . . . . . .22% ............
water bath, adding the sulfonamide, then removing from the water bath and stirring continuously until cooled to room temperature. In attempting to estimate the rate and amount of sulfonamide released &om the ointment, the following procedure was adopted. Four grams of the ointment was applied evenly to the inside surface of a 6-x 1-inch test tube using a glass rod to spread the ointment in a uniform layer with a circular motion. Twenty-five cubic centimeters of saline or serum was then pipetted into t h e tube (capacity 60 cc.) and the tubes then placed in an incubator at 37.5' for varying lengths of time. Five tubes were thus prepared with each ointment and one taken for analysis after two, four, six, eight, and twenty-four hours' incubation. The contents of the tube taken for analysis were poured carefully into another tube and agitated by inverting three times. An aliquot of this taken for analysis was used directly or after appropriate dilution depending upon t h e relative concentration of the drug in the liquid. The amount of sulfonamide in t h e aliquot was determined colorimetrically by the method of Bratton and Marshall (20) using an electrophotometers and a filter with a maximum transmission at 525 mp. Although this method is merely an estimation of the amount of sulfonamide absorbed from the ointment, we have obtained, in preliminary work, fairly uniform and reproducible results by carrying out determinations on a number of series of tubes of the same ointment. For instance, 6 tubes of the same sulfonamide ointment were found to release in twenty-four hours 61,65,64,71,62,and 57 mg. of the drug in 25 cc. of saline. On this basis we have assumed that the method as given above might be used as a criterion of the ability of various ointment bases t o liberate sulfonamides incorporated in them. The following tables and graphs indicate both the rate and amount of sulfonamide released in the various periods of time designated.
Petrolatum, white.. . . . . . . . . . . . . . Glycerin. . . . . . . . . . . . . . . . . Water. . . . . . . . . . . . . . . . . . . This is an extemporaneous absorption base cream containing more water than either of the previous two emulsions. Oil-in-Water Emulsion Bases No. 5. Cetyl Alcohol.. . . . . . . . . . . Propylene Glycol. . . . . . . . . White Wax. . . . . . . . . . . . . . . . . . . . Sodium Lauryl Sulfate.. ... Water. . . . . . . . . . . . . . . . . . . . This hydrophilic, washable ointment base has been recommended (18,19) as a base for sulfonamides.
No. 6. Cetyl Alcohol.. . . . . . . . . . . . . . . . . 6% Lanolin ........................ 6% Sodium Lauryl Sulfate.. . . . . . . . . . 2% Petrolatum, white.. . . . . . . . . . . . . .14% Mineral Oil, heavy.. . . . . . . . . Water. .......................
This formula contains the same emulsifying agent as No. 5 but contains considerably more oil.
No. 7. Stearic Acid.. . . . . . . . . . . . . . . . . . .25% Triethanolamine.. ... Carbitol . . . . . . . . . . . . Lanolin. ...... Water. .......
This base is a triethanolamine stearate emulsion of the vanishing cream type.
No. 8. Stearic Acid.. ..................15% Lanolin. . . . . . . . . . . . . . . 4% Mineral Oil, heavy. . . . . . . . . . . . . .25% Triethanolamine.. . . . . . . . . . . . . . . 2y0 Glycerin. . . . . . . . . . . . Water . . . . . . . . . . . .. : . . . . . . . . . . This base, like No. 7, is also a triethanolamine stearate emulsion but containing more oil. No. 9. Glycerylmonostearate. . . . . . . . . . .18% Carbowax 15002.. . . . . . . . . . . . . . . 2% Glycerin. ............. Water. ....................... This is a simple glycerylmonostearate cream with
a high water content.
. . . .12% Lanolin. ...... . . . .lo% Cetyl Alcohol.. . . . . . . . . . . . . . . . 2Y0 Mineral Oil, heavy. . . . . . . . . . . . .25% Carbowax 1500a.. . . . . . . . . . . . . . 2%
No.10. Glycerylmonos
Glycerin. . . . . . . . . . . . . . . . . . . . . . Water.. .....................
5%
.#yo
This has the same emulsifying agents as No. 9 but contains more oil.
* Carbide & Chemicals Corp.. New York, N. Y.
TABLE I.-TOTAL SULFATHIAZOLE (IN MILLIGRAMS) RELEASEDINTO 25 CC. OF SALINE BY 570 SULFATHIAZOLE OINTMENTS~ Base No.
1 2 3 4 5 6 7 8 9 10 a
Hours of Exposure of Ointments to Saline 2 4 6 8 24
0.2 1.0 0.3
0.0 12.0 4.0 11.5 13.0 11.0 8.0
0.2 1.0 0.4 0.2 20.0 14.5 17.5 17.0 15.0 11.0
0.3 1.2 0.4 0.4 27.5 26.0 20.0 23.0 20.0 16.0
0.3 1.5
0.4 1.7
1.4
1.9 ~-
0.7 31.0 31.0 24.0 25.0 25.0 19.0
2.0 40.0 34.0 35.0 27.5 27.5 22.5
-.
~
See Fig. 1.
Fisher Electrophotometer, Fisher Scientific Co.. Pittsburgh, Pa. J
68
JOURNAL
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(IN MILLITABLEII.-ToTAL SULFATHIAZOLE (IN MILLI- TABLE IV.-TOTAL SULFONAWDE GRAMS) RELEASED INTO 25 cc. OF SALINE B Y 10% GRAMS) RELEASED INTO 25 cc. SERUM BY SULFONAMIDE OINTMENT^ SULFATHIAZOLE OINTMENTP
Hours of Exposure of Ointments to Saline 4 6 8 24
Base No.
2
1 2 3 4 5 6 7 8 9 10
0.2 0.9 0.7 0.1 19.0 6.0 16.0 13.5 12.0 8.0
0.2 1.2 0.8 0.4 35.0 11.5 31.0 33.0 16.0 12.5
0.3 . 1.6 r.2 0.8 41.0 32.5 35.0 37.0 23.5 19.0
0.5 1.8 1.4 1.0 43.2 48.5 37.0 36.0 32.0 24.0
0.5 1.8 3.2 3.1 52.0 50.5 43.7 50.5 42.5 37.0
See Fig. 2.
TABLE111.-TOTAL SULFANILAMIDE (IN MILLIGRAMS) RELEASED INTO 25 cc. OF SALINEBY
Base No.
2
10% Sulfathiazole Ointments 0.2 0.2 0.4 0.7 0.5 1.1 2.0 2.6 17.0 39.0 51.0 77.0 9.5 26.5 33.7 34.0 75.0 50.0 55.0 19.0 10% Sulfanilamide Ointments 0.2 0.3 0.4 0.6 1.0 4.0 9.0 15.0 62.5 110.0 142.0 187.0 58.0 93.0 137.0 156.0 75.0 120.0 142.0 162.0
1 3 5 8
9 1 3 5 8 9 a
Hours of Exposure of Ointments to Serum 4 6 8 24
0.7 2.6 92.0 58.7 97.0 0.9 19.0 205.0 190.0 192.5
See Figs. 5 and 6.
SULFANILAMIDE OINTMENTS4
Hours of Exposure of Ointments to Saline 2 4 6 8 24
Base No.
5% Suuanilamide Ointments 0.3 0.4 0.4 0.3 7.0 1.2 4.3 7.0 17.0 35.0 37.5 50.2 45.0 77.5 18.5 40.0 95.0 95.0 25.0 ,40.0 25.0 10.0 21.0 6.25 10% Sulfanilamide Ointments 0.5 0.5 0.6 0.7 2.0 7.7 12.0 17.0 23.0 55.0 75.0 90.5 40.0 60.0 90.0 150.0 80.0 125.0 160.0 160.0 31.0 45.0 10.0 20.0
1 3 5 7 8 9
1 3 5
7 8 9 6
0.4 18.5 60.0 82.5 95.0
42.0 0.7 40.0 107.5 195.0 185.0 60.0
See Figs. 3 and 4.
d
60
I-
F.
2
4
6
8
Time in Hours Fig. 2.-Total Sulfathiazole Released into 25 cc. of Saline by 10% Sulfathiazole Ointments.
I
20
' "
M
0
A
1 1 6 Q Time in Hours
Fig. 1.-Total Sulfathiazole Released into 25 cc. of Saline by 5% Sulfathiazole Ointments.
All ointments were tested bacteriologically against Staphylococcus aureus using the F. D. A agar cup plate method (21). Tubes containing 20 cc. of sterile nutrient agar were inoculated with 0.1 cc. of a twenty-four hour culture of Staphylococcu: aureus at 42" and immediately poured into sterile Petri dishes. After the agar had solidsed, 1.5 cm. cups were cut in the center of the plates and 2.0 Gm. of the various ointments introduced. A drop of sterile broth was dropped into the cup t o assure uniform contact of the ointment with the agar media. Thc Petri dishes prepared in this manner were incubated a t 37.5' for twenty-four hours. A t th: end of this period they were removed from the
SCIENTIFIC EDITION
i
69
i
P
F9
20 9
3
I
0
2 4 d B
k
Time in Hours Fig. 3.-Total Sulfanilamide Released into 25 cc. of Saline by 5% Sulfanilamide Ointments.
Time in Hours Fig. 5.-Total Sulfathiazole Released into 25 cc. of Serum by 10% Sulfathiazole Ointments.
d
z
Time in Hours Fig. 4.-Total Sulfanilamide Released into 25 cc. of Saline by 10% SulfanilamideOintments.
Time in Hours Fig. 6.-Total Sulfanilamide Released into 25 cc. of Serum by 10% SulfanilamideOintments.
70
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incubator and the zone of inhibition measured and recorded in millimeters. The following results were obtained with the various ointment bases. TABLE V
Base No.
5% Sulfathiazole Ointment
Zone of Inhibition, Mm.10% 5% 10% SulfaSulfanilSulfanilthiazole amide amide Ointment Ointment Ointment
0 3
0 3
0 1
0
2
3
1 0
1 0 15 10 10 6 15 6
0 0 15
0
1 4
5 6 7 8 9 10
14 10 10 6 14 6
8
1
0 15 10 12
11 5 18
7 18
12
12
DISCUSSION Analysis of saline and serum in contact with the ointments for varying lengths of time indicated that the greasy hydrocarbon base, i.e., U. S. P. Ointment, released relatively little of the sulfonamide incorporated in it. Release of the sulfonamides from water-in-oil emulsions was only slightly better while release from the oil-in-water hydrophilic bases was much more rapid and far greater in every case. This is undoubtedly due t o the low surface tension and water or serum miscibility of these bases. It, was observed that these bases diffused into the saline or serum forming a uniform suspension. The hydrocarbon and water-in-oil emulsions formed a distinct interface with the fluid in contact with it so that only the sulfonamide at the interface was in contact with the fluid. In some instances, the drug available from the U. S. P. base or the water-in-oil emulsions was so little that only the results obtained from one of these was plotted on the graphs to show the contrast. It is an interesting fact that base No. 3, which is a water-in-oil emulsion containing 50% aqueous phase, and base No. 6, a n oil-in-water emulsion containing 50% water, exhibit marked differences in their abilities to give up their sulfonamide content. The hydrophilic bases used in this study were developed in pairs, each pair having the same emulsifier but with one member containing considerably more petrolatum or grease than the other. This was done to determine whether or not a high concentration of
the oil phase interfered with the release of the sulfonamides from ointments of this type. Bases 5 and 6 have as an emulsifier sodium lauryl sulfate with base No. 6 having a rather high petrolatum content. Bases Nos. 7 and 8 contain triethanolamine as an emulsifier, No. 8 having in it lanolin and petrolatum in addition to the triethanolamine stearate. Bases Nos. 9 and 10 are emulsified with glyceryl monostearate and Carbowax 1500, base No. 10 containing a considerable amount of lanolin and petrolatum not present in No. 9. I n all cases where lanolin and petrolatum were added they largely replaced water in the formula so that by increasing the oily phase of the emulsion, the aqueous phase was reduced. In most cases, increasing the oily phase by the addition of lanolin and petrolatum seemed t o interfere only slightly, if a t all, with the ability of the bases to give up their sulfonamide content. This is apparently due to the concehtration of the sulfonamide in the aqueous phase in which case the oil globules do not interfere with the solubility of the sulfonamides . As indicated in the graphs and tables, considerably more sulfanilamide than sulfathiazole was available from the ointments. This may be expected because of the higher solubility of sulfanilamide. It. also seemed generally true that the ointments released more sulfonamide into serum than into saline. This is undoubtedly due t o the higher solubility of these compounds in serum as pointed out by Clark, e t a l . (22). The oil-in-water bases seemed t o give up most of their sulfonamide content quite rapidly in the first six to eight hours and liberated only slightly more in the period up to twenty-four hours. The water-in-oil emulsions gave up only a fraction of their sulfonamide content very slowly and continued to release small increasing amounts up to and beyond the twenty-four hour period. Ointments tested by the F. D. A. agar cup plate method against Staphylococcus aureus gave inhibition zones which generally paralleled the liberation of the drug from the various ointments by the method described. Ointments which liberated a large amount of sulfonamide gave the largest inhibition zones while those liberating small amounts of sulfonamide gave little or no zone of inhibition. Here, again, it seemed that the oil-in-water emulsions were the most satisfactory bascs.
SUMMARY
A method for the estimation of the amount of sulfonamides released from various sulfonamide ointments upon contact with saline or serum for varying lengths of time is described. By this method, the availability of the sulfonamides incorporated in various ointment bases is estimated.
The types of bases studied were a hydrocarbon water free base (U. S. P. Ointment), water-in-oil emulsion bases and oil-in-water emulsion bases. Ointments were prepared by incorporating 5 per cent and 10 per cent of sulfanilamide or sulfathiazole in the various bases. Of these ointments the oil-
SCIENTIFIC EDITION in-water emulsion type was most efficient, liberating its sulfonamide content more rapidly and more completely than the others. The hydrocarbon base wks the poorest in this respect and the water-in-oil emulsions only slightly better. Increasing .the oil phase of the oil-in-water emulsion bases did not seem to materially affect the liberation of the sulfonamides. All ointments were tested against Stuphyl-
71
ococcusaureususing the F. D. A. agar cup plate method. Largest zones of growth inhibition of the organisms were obtained with the oil-in-water emulsions with other bases showing little or no inhibition. From these results it seems logical to conclude that because of their low surface tension and water miscibility, the oil-in-water type emulsions are superior bases for sulfonamide ointments.
REFERENCES (13) Foley, E., and Lee, C. 0..THISJOURNAL., 31, 105
“%]’ Gibson, A. J., Parker, H.E.,and Almus, A., ibid., 30, 196(1941).
1 (1942). (5) Gurd, F. B., Ackman, D., and Smith, F., Ann. Surg., 118, 921(1943). (6) Kern R. A. U.S. Naval Med. Bull. 41,1654(1943). (7).Pillsdury, D. M., Wammock, V. S.: Twingood, C. S., and Nichols A. C. A m . J . Mcd. Scr 202, 808(1941). (8) Wimmer, i. H.. and Strako&h, E. A., J. A m . Med. Assoc., 118, 221(1942). (9) Kuney E. L. Pembroke R. H., Chatard. F. E., and Ziegler, J. M.,’ibid., i17, 1415(1$41). (10) Editorial, U.S.Naval Mcd. Bull., 44,861(1944). (11) Ackman D. Gerrie J. W Pritchard, J. E., and Miller, E. s.,Ann. ‘ s ~ & ,119, i61U.944). (12) Gershenfeld, L., and Mlller. R. E., A m . J . Pharm., 105, 194(1933).
(15) Fantus, Bernard, J . A m . Mcd. Assoc., 107, 861-63 (1936). (16) “Pharmacopceia of the United States,” Twelth revision Mack Printing Co,, Easton, Pa.,1942,p. 533. i7 Ibid. p. 35. 1181 Eva& C. H..THISJOURNAL, Piact. Pharm. Ed., 3, 235(1942). (19) Baker W. B. Calco Technical Bulletin N o . 686, Calco
Chemical Diiision, American Cyanamid Company, Bound Brook N. J. (20)’ Bratton, A. C., and Marshall, C. M., J . B i d . Chcm..
128, 537(1939). (21) Ruehle,. George D.,, and Brewer, C. M., U.S. Food and Drus AdmrnrstrafronCrrcular No. 198, U . S. Dept. Agr., 1931. (22) Clark, G. W., Strakosch, E. A,, and Levitan, N. I., J . Lob. C l m . Med.. 28, 188(1942).
The Separation of Acetylsalicylic Acid, Acetophenetidin, and Caffeine* By K E N N E T H E. HOLTf which are freely soluble in ether. The results obtained are comparable to those ration of mixtures containing acetylsalicylic obtained with the A. 0. A. C. method acid, acetophenetidin, and caffeine. Al- (1, 3) while the time required for the comthough the present A. 0. A. C. method (1) plete analysis by an experienced analyst is produces satisfactory results the hydrolysis reduced to less than three hours. of acetophenetidin is time-consuming and EXPERIMENTAL affords considerable chance of error. Acetophenetidin.-Accurately weigh a sample The method outlined here eliminates the containing about 0.3 Gm. of acetophenetidin and hydrolysis of acetophenetidin by utilizing transfer to a separatory funnel. Add 25 cc. of the relative insolubility of caffeine in ether HCl (1 + 10) and extract with purified ether using to effect the separation of caffeine from one 40-cc. portion and five 20-cc. portions. Combine ether extracts in another separatory funnel and acetylsalicylic acid and acetophenetidin, wash with one 10-cc. portion HC1 (1 + 10) and one FREQUENT
operation of the pharma-
A ceutical control laboratory is the sepa-
*Received May 23, 1945, from the Chemical Laboratories, Cole Chemical Company, St. Louis, Mo. t Acknowledgment is made to Wendell L. Holt and Margaret Miller for their suggestions,
10-cc. portion ‘of water, washing these in turn through two 25-cc. portions of ether and adding to the original acid solution, (Save the acid solution