HYDROGEN PEROXIDE FORMATION BY ZINC OXIDE IN OINTMENTS DISSERTATION

Transcription

1 HYDROGEN PEROXIDE FORMATION BY ZINC OXIDE IN OINTMENTS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philo sopty in the Graduate School of the Ohio State University Hy HECTOR ANTONIO LOZADA, B.3. in Phar., M.S. in Phar. The Ohio State University 195S Approved by Adviser College of Pharmacy

2 ii TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS... iv PURPOSE OF THE RESEARCH... v INTRODUCTION... 1 ZINC OXIDE... 5 ZINC OXIDE AS A PHOTOSENSITIZER ULTRAVIOLET L I G H T... IA INFRARED ABSORPTION S P E C T R A SCME PROPERTIES OF THE OINTMENT B A S E S WHITE O I N T M E N T HYDROPHILIC OINTMENT POLYETHYLENE GLYCOL OINTMENT HYDROUS WOOL F A T A Q U A P H O R CHOLESTEROL HYDROGEN PEROXIDE APPARATUS AND MATERIALS EXPERIMENTAL PREPARATION OF S A M P L E S IRRADIATION OF S A M P L E S METHOD OF ASSAY EXPERIMENTAL R E S U L T S DISCUSSION CONCLUSIONS... 84

3 iii TABLE OF CONTENTS (CONT»D. ) PAGE CONTEMPLATED FUTURE RESEARCH BIBLIOGRAPHY AUTOBIOGRAPHY... 91

4 iv ACKNOWLEDGEMENTS My deepest and sincere appreciation to Dr. Earl P. Guth, Professor, College of Pharmacy, for the guidance and assistance offered to me during the course of this research. His efforts have to a large extent contributed to the successful completion of this work. I also want to make grateful acknowledgement to Dean Luis Torres Diaz, University of Puerto Rico - College of Pharmacy, for his efforts in securing the financial assistance necessary for the continuation of my studies for the degree Doctor of Philosophy. Sincere appreciation is extended to Dr. Loyd E. Iferris and Dr. John W. Nelson, College of Pharmacy, for their invaluable assistance. I also wish to acknowledge all the graduate students in the College of Pharmacy for their cooperation, with special gratitude to V&lter Morozowich. Finally, all my love and devotion goes to ray dear wife, Lillian, for her moral support and understanding.

5 PURPOSE OF THE RESEARCH v Considerable work has been done in connection with the ability of photosensitized zinc oxide to produce hydrogen peroxide from water and oxygen. Blubaugh, Mathias, Minardi, Reese, and Young (6, 4» 5» 3» 7) contributed a good deal of knowledge in applying this phenomenon to pharmaceutical preparations containing zinc oxide or calamine. None of these investigators, however, worked with ointment bases containing zinc oxide. The main objectives of this investigation were - 1. To determine whether the official formula for Zinc Oxide Ointment would produce hydrogen peroxide under the conditions described in this investigation. 2. To compare the amount of hydrogen peroxide produced by the official formula with the amount produced by using hydrous wool fat, aquaphor, hydrophilic ointment, and polyethylene glycol ointment as bases. 3. To determine the effect of temperature on hydrogen peroxide formation. 4. To determine any relation between hydrogen peroxide production and time of exposure to ultraviolet irradiation. 5. To report any changes in hydrogen peroxide production when using sodium formate as additive. 6. To report other information disclosed during the course of the investigation.

6 HYDROGEN PEROXIDE FORMATION BY ZINC OXIDE IN OINTMENTS INTRODUCTION Ointments constitute one of the earliest types of medication employed by man. They were popular remedies in ancient Babylon and Egypt. Fats of animals mixed with such things as resins, waxes, and powdered herbs were very commonly used dermatological preparations in ancient times. The Papyrus Ebers, which dates from about the sixteenth century before Christ, shortly prior to the time of Moses, contained a formula for a greaseless ointment which was made from Hartshorn beaten up with incense and flour and mixed with sweet ale. Ointments are defined by the Pharmacopeia of the United States (l) as semi-solid preparations usually containing medicinal substances and intended for external application to the body. Zinc oxide is one of the most popular and frequently used ingredients in the treatment of dermatological conditions of varied nature. It is official in the Pharmacopeia of the United States (l) and is also an ingredient of such official preparations as Calamine, Calamine Lotion, Zinc Oxide Paste, Zinc Gelatin, Phenolated Calamine Lotion, Coal Tar Ointment, and Zinc Oxide Ointment. The National Formulary (2) includes it in its formulas for Calamine Ointment, Zinc Oxide Paste with Salicylic Acid, Compound Resorcinol Ointment, and Zinc Compounds and Eugenol Cement. Among all these formulas, one of the most frequently used is that of Zinc Oxide Ointment. It has been used for many years as an astringent, protective and mildly antiseptic external preparation. 1

7 Physicians all over the country continue to prescribe this kind of preparation for treatment of skin conditions, although sometimes they make sane changes in the official base by substituting for it other fats, glycols, or emulsion type bases. Reese, Mathias, Minardi, Blubaugh, and Young (3, 4* 5> 7) have studied the Formation of Hydrogen Peroxide in Calamine Lotions," "The Effect of Various Phenols on the Formation of hydrogen Peroxide by Zinc Oxide," "The Effect of Time on the Hydrogen Peroxide Production by Calamine Lotions," "A General Study of the Effects of lipids Upon the Quantity of Peroxides Produced by Fhotosensitized Zinc Oxide," and "The Effect of Selected Rienols on the Formation of Hydrogen Peroxide by Calamine liniment N. F. IX," respectively. All these researchers worked with water suspensions of zinc oxide or with emulsions containing zinc oxide as a major ingredient. None of these studies were concerned with ointments containing zinc oxide. This study deals with this type of preparation. Reese (3) found considerable variation in the production of hydrogen peroxide by different formulations of calamine lotion. This amount of peroxide was related to the kind of ingredients present in the formula. Seme substances were found to increase the peroxide. Among these were polyethylene glycols, sorbitan esters, propylene glycol alginates and the two already known, glycerine and phenol. They all showed a pronounced effect on the production of hydrogen peroxide, with phenol being the most efficient of those studied. large amounts of ferric oxide were reported to be detrimental to the production of hydrogen peroxide unless

8 there are additives present in the lotions. Fluorescent and incandescent lamps were found to stimulate peroxide production. Sunlight proved to be an excellent promoter of peroxide formation even in cloudy weather. The formation of hydrogen peroxide was not linear with respect to time, two hours yielding less than five times the amount formed in fifteen minutes. Reese also found that lotions which are bacteriostatic may be produced by adding 1 per cent of phenol and/or irradiation with ultraviolet light. The phenol and the peroxide so formed appeared to have complemental synergism rather than additive action. Mathias (4) reported significant difference in the amount of hydrogen peroxide produced by several commercial brands of calamine lotions upon irradiation with ultraviolet light. With one exception, he found no significant difference in the amount of peroxide produced by irradiation of a lotion prepared with native calamine and one made with prepared calamine. In his study of the effect of adding phenol, resorcinol, resorcinol monoacetate, and pyrogallol to the suspension, he concluded that the number of hydroxyl groups may have an effect upon the quantity of hydrogen peroxide produced by ultraviolet light. An increase in hydroxyl groups in the phenol brings an increase in ability to produce peroxide. Nevertheless, there appeared to be an optimum quantity of phenols beyond which there is no increase in hydrogen peroxide production. An increase of concentration beyond this limit showed a decrease in peroxide formation. Minardi (5) studied the effect of time on the hydrogen peroxide production of calamine lotions. He concluded that after a period of

9 fourteen weeks no significant increase is obtained upon irradiation with ultraviolet light. Another of his findings was the fact that sodium carboxymethylcellulose and/or dioctyl sodium sulfosuccinate retarded the photochemical reaction by which hydrogen peroxide is produced from zinc oxide and water. Blubaugh (6 ), working with lipids, found that the addition of fixed oils greatly reduced the amount of peroxides produced by photosensitized zinc oxide. Although he found a considerable variation in the reduction produced by different oils, no correlation could be made between this and the physical constants of the oils. Fixed oils also decreased the ability of sodium formate to act as an nadditiven in the reaction. Mineral oil did not markedly reduce the amount of peroxide produced by zinc oxide. Blubaugh observed a variation in peroxide production by using zinc oxide obtained from different sources, and he indicated that this might be connected with the method used in the manufacture of the zinc oxide. Squalene was shown to increase the peroxide production while addition of human blood plasma caused a reduction. In his work with selected phenols, Young (7) concluded that an olive oil-calcium hydroxide solution emulsion has a depressing effect on the catalytic activity of zinc oxide in the peroxide production. The amount of agitation was found to have a direct relationship on the amount of peroxide formed. The addition of various phenols to calamine liniment will alter its physical properties (i.e., viscosity) but increase the production of hydrogen peroxide. Contrary to the

10 observations of Mathias (4) regarding the effect of the number of hydroxyl ions in the phenol upon the formation of hydrogen peroxide by calamine lotion, Young found that the number of hydroxyl ions does not appear to influence directly the amount of peroxide formed by calamine liniment. The age of the calamine liniment containing various phenols influences the peroxide forming ability of the liniments by either increasing or decreasing it. The amount of peroxide formed varies also with the concentration of the phenols in calamine liniment. As pointed out before, none of these investigators worked with ointments, their main concern being with aqueous types of preparations and the emulsion types of preparations containing either zinc oxide or calamine. Also it was noticed that no information is given whatsoever about the possibility of the role of temperature on the peroxide formation by zinc oxide. It was thought that a study of these two new aspects of the problem was in order and the official formula (l) for Zinc Oxide Ointment was used as the basis for this study. ZINC OXIDE Zinc Oxide is described in the Pharmacopeia of the United States, XV Revision (l) as "very fine, odorless, amorphous, white or yellowish white powder, free from gritty particles. It gradually absorbs carbon dioxide from the air." When freshly ignited, it should contain not less than 99 per cent of zinc oxide. It is insoluble in water and alcohol but is soluble in dilute acids. Feitknecht and Haberli (8) report that the solubility product of

11 zinc cod.de varies with the method of preparation. They found that the solubility product of a sample prepared by precipitation was 1.6 x 10 ^ ; zinc oxide prepared by drying zinc hydroxide at 100 C was 8.3 x 1 0 ~ ^ t and zinc oxide prepared by decomposition of zinc carbonate at 1000 C was I r t 2.7$ x 10. This indicates that there are sane differences in the zinc oxide due to the different methods of preparation. Outcalt (9) reports that Le Clair and Sorel, in France, started the commercial production of zinc oxide during the latter part of the eighteenth century. Le Clair was a master painter and paint grinder and is reported to have been the first to use zinc oxide in combination with basic carbonate of white lead in paint. The zinc oxide used by Le Clair was made by burning slab zinc (spelter) with air, a process which is now known as the French process, the indirect process, or the two-step process for production of zinc oxide. The manufacture of zinc oxide in the United States was started from an observation made by a workman. A white fume was given off from sane zinc ore which he U3ed to repair temporarily a break in a boiler flue. Weatherill and Jones in 1885 perfected a furnace to produce zinc oxide by a direct or single step process, which has since been known as the American process (9)» Other furnaces have been developed and used in this connection since that time but seme of the original type Weatherill furnaces are still operated in this country. The French process involves burning of slab zinc metal (spelter) with air. In order to obtain a white pigment a very pure spelter must be used. If it contains lead it will burn to litharge giving a

12 yellowish tint. To render the lead harmless, a method has been developed in which the burning is done in air mixed with carbon dioxide, the lead is changed to white lead and thus the yellow tinge is avoided. The zinc is unaffected by the process (9)» The furnace used in this process is essentially a retort which is externally heated, usually by producer gas, but may also be heated by coal, oil or natural gas.. The zinc metal is heated in a retort to the boiling point when the zinc vapors come in contact with air and burn with a bright yellowish flame to produce zinc oxide fumes. The zinc oxide fumes are then conducted through suitable pipe lines to settling chambers. Although the physical properties of the zinc oxide may be controlled within certain limits by the rate of oxidation, the particle shape of the product is characteristically spherical. The Ralmerton process, which employs an air blast at the point of oxidation (9 ), produces a smaller particle size but the shape of the particle is still spherical. The American process uses the principle of vaporization of zinc directly from zinc ore rather than from the metal. The various types of American process furnaces have sufficiently different characteristics to warrant some brief description. (1) The original or Eastern type of Weatherill furnace U3es anthracite coal to heat the layer of zinc ore mixed with anthracite coal. This combined charge is allowed to burn until a temperature of about 1100 C is attained, at which temperature the carbon in the coal reduces the zinc from its ore, the rate being regulated by air blown through the grates. The particles obtained are predominantly nodular. The physical

13 properties of the zinc cocide are influenced by the temperature prevailing at the oxidizing zone, by the length of the time the fume is maintained at an elevated temperature, and by the amount of dilution of zinc oxide fume in the gases of combustion. Because of this and the large volume of gases of combustion that must be handled, the uniformity of the properties is very poor and little control can be achieved. (2) The Western Weatherill type of furnace uses bituminous coal. The process yields large acicular particles. Uniformity of the finished product is more difficult to be obtained than by using the previous method. (3) The mechanical furnace is an improvement upon the Eastern type furnace. This process uses a moving grate fed with coal and zinc ore. Good uniformity of the product is achieved. (4) The Electro-thermic furnace differs greatly in smelting design from other American processes. The charge to the furnace is purified and sintered zinc ore and coke. The heat necessary for reduction is supplied by an electric current. Combustion gases are not present and a wide range of control over properties is achieved. It combines in the zinc oxide the desirable properties of both the French and American processes. The zinc oxide thus obtained is of the high chemical purity associated with the French process and the particle sizes and shapes characteristic of both processes (9 )» (5) The Cornillat furnace is essentially of the French process type but uses semibituminous coal and internal heating. little control over properties is achieved.

14 (6) The Wet Process finds practically no commercial application at the present because of the high production cost. This process involves precipitation of the zinc from solutions in the form of zinc carbonate and subsequent calcination of the carbonate to obtain the oxide. Spherical particles are obtained and the particles are finer in size than those obtained by any other method (9). (7) A by-product zinc oxide is produced on a small scale by calcining zinc carbonate which is obtained as a by-product from industries where zinc dust is used as a reducing agent. The properties of this zinc oxide are of inferior grade. Zinc oxide has been an official product in the Pharmacopeia of the United States (10) since the first edition in The Pharmacopeia of the United States V, 1876 (11) and later the British Pharmacopeia of I885 (12), recognized zinc oxide prepared from zinc carbonate by the Wet Method. This process produces a zinc oxide the particles of which are finer in size than those produced by the fume processes (i.e., Weatherill and Electro-thermal Processes) (9). Zinc oxide has been widely used in medicine as well as in industry. The medicinal properties of zinc oxide are primarily associated with dermatology because of its effectiveness in the treatment of many skin disorders. It is a component of dermatological formulas such as powders, pastes, ointments, and lotions. Among other conditions of the skin it is included (13) in formulas for the treatment of seborrheic dermatitis, atopic dermatitis, eczematous dermatitis, lichen planus, and psoriasis. It is also used in formulas (13) as a drying agent in conditions like

15 sudorrhea* lichen planus; as an antipruritic in many types of dermatitis 10 and allergic urticaria. Although it has no well-defined pharmacologic action on the skin, its popularity may be attributed (14) to a combination of four qualities: it is protective, mildly astringent, possibly weakly antiseptic, and non-toxic. In dentistry, it has been used in treatment of Vincent s angina (14) or trench mouth, and also in combination with zinc acetate, zinc stearate and rosin as a dental protective (2), i.e., as a pulp capping or temporary filling. Internally it has been used as an antispasmodic in chorea, epilepsy, and whooping cough (14). It has also been employed a3 a protective in diarrhea. Nevertheless, it is seldom used internally in modern medicine. In industry zinc oxide has been employed in the production of paints and in the manufacture of synthetic rubber. ZINC OXIDE AS A PHOTOSENSITIZER The catalytic activity of zinc oxide in the photochemical formation of hydrogen peroxide was first reported by Baur and Neuweiler (15) in Ten years later Goodeve confirmed this action (16). All these investigations as well as that of Winter (17) have shown that hydrogen peroxide is formed at light activated zinc oxide surfaces in contact with oxygen water and miscellaneous compounds. Winter (17) stated that the zinc oxide prepared by the French process showed chalking in paint films, fluorescence with ultraviolet light, and photochemical activity leading to formation of hydrogen peroxide in aqueous solutions. Zinc

16 11 oxide prepared by the American process showed no chalking, a yellow fluorescence under ultraviolet light, and no photochemical activity. The proposed over-all reaction (17, IS) for peroxide formation is as follows: 2H * 2H202 This reaction involves a large increase in free energy. The light absorption region of zinc oxide limits the effective wave lengths for peroxide formation to those less than about 4000 A. Of the total energy of the sun which is incident at the earth*s surface about 4 per cent can be effective in promoting this reaction (19). Chari and Qureshi (20) studied the reaction in some detail and found that sunlight, artificial ultraviolet, and visible light up to 4700 A. are effective in causing the reaction to proceed. The zinc oxides were prepared from zinc hydroxide precipitated from zinc sulfate and zinc nitrate, from the ignition of the carbonate, and from ignition of the nitrate. The oxide prepared from the carbonate was the most effective and the one prepared from ignition of the nitrate the least active. This finding is in accordance with previous reports of Smith and Hawk (21) and Schleede, Ritcher, and Smith (22) in which they conclude that the oxide produced from zinc carbonate is more active than zinc oxide prepared by combustion of zinc or by thermal decomposition of the nitrate. Huttig and Feher (23)» in 1937, from their study of zinc oxides prepared from a variety of starting materials, concluded that the catalytic activity of zinc oxide depends upon the nature of the material

17 12 from which it was obtained presumably because of the nature of the original crystals. G a m (24)» in his dissertation in 1952» concluded that the causes of the differences in catalytic activity of zinc oxide appeared to be most likely because of impurities, lattice defects or metallic zinc. The principal cause is the different general methods of preparation producing differences in the surface characteristics of zinc oxide. It has been shown that the presence of a small concentration of one of many water soluble, easily oxidized, organic compounds (often called "stabilizers," "promoters" or "additives") such as sodium formate, potassium oxalate, phenol, etc., in the zinc oxide-water-oxygen mixture increases the rate of peroxide formation. Chari and Qureshi (20) tested nine compounds and phenol was the most efficient followed by glycerine and acetanilid. In systems containing additive, zinc oxide is a photosensitizer for peroxide formation (16). This is evidenced by the facts that hydrogen peroxide is formed only in the region of light absorption of zinc oxide, i.e,, 2500 A., and that no apparent change is observed in zinc oxide since it can be re-used again without change in the efficiency of peroxide formation (19). Oxygen is necessary in the system for hydrogen peroxide formation. Although the rate of peroxide formation is greatly increased when the additives are present in the irradiated system, peroxide decomposition is also very rapid when the oxygen supply is low. Yamahuzi, Nisioeda, and Imagawa (25), and Yamahuzi, Nisioeda and

18 13 Ifcrusi (26) studied the photochemical formation of hydrogen peroxide in the presence of biological sensitizers. The latter authors suggest that the formation proceeds by three reactions: 21^0 = H2O2 / 2H 2H / Os = H202 A / H20 / 02 = AO / H2O2 ( I) ( ID (III) where A" in reaction (III) is the organic sensitizer. While the energy of light is sufficient to promote these reactions, the heat liberated in (II) and (III) would accelerate reaction (I). These substances which are called stabilizers 1 or promoters are actually reactants and undergo oxidation simultaneously with the hydrogen peroxide formation. Experiments showed that oxalate and formate ions are oxidized to carbonate; phenol is oxidized in part to catechol (19) Example: H20 / 02 / HC00- ^ H202 / HCO3

19 V* ULTRAVIOLET LIGHT In 1800, Sir William Herschel discovered that the solar spectrum extended out beyond the portion which was visible to the human eye. One year later, J. W. Ritter investigated the portion of the spectrum beyond the violet and showed that chemical action was caused by some kind of energy in this region, the region which is now called the ultraviolet. Thomas Young, in 1804, showed that the invisible chemically active radiations beyond the violet end of the spectrum (subsequently called actinic rays) corresponded to some radiations of shorter wavelength than visible light (27). There is really no sharp dividing line between these forms of radiant energy. They are all manifestations of the same kind of electromagnetic radiation differing from each other only in the frequency. From the viewpoint of biological and therapeutic effects these ultraviolet radiations have received much more attention than the visible and infrared energy. They produce fluorescence, photographic action and many known biological effects. Ultraviolet light is conveniently divided into three parts in reference to the visible spectrum (28): 1. Extreme ultraviolet region (wavelength shorter than 2000 A.) is the least penetrating of all radiant energy and is absorbed by most substances including air. It must be produced and studied in a vacuum by means of special photographic plates, gelatin emulsions being opaque to it. 2. Middle ultraviolet energy (between 2000 A. and 3000 A. ) is a very important spectral region from biological and therapeutic viewpoints.

20 15 Ordinary glass will not transmit it, but quartz is transparent through^ out this range. The solar spectrum barely extends into this region for its short-wave limit is near 2900 A. The solar energy between 29OO A. and 3100 A., however, is known to be very valuable because of its antirachitic and germicidal action, in the production of vitamin D, in the production of erythema, and in the production of conjunctivitis in extreme cases. Ordinarily conjunctivitis is not produced by sunlight. The maximum of germicidal action is in the neighborhood of 2600 A. but it can extend to 3100 A. The maximum for production of conjunctivitis seems to be near 2500 A. and for erythemal effect 3000 A. The production of vitamin D by irradiation of ergosterol is apparently due to energy somewhere between 25OO to 3100 A. 3«Near ultraviolet energy (between 3000 and 3900 A.) produces fluorescence and photographic action. Ordinary glass is quite transparent between 3900 and 3500 A., but this transparency decreases until most glasses of ordinary thickness are fairly opaque at 3100 A. Most substances that are transparent to visible radiant energy, including ordinary glasses, become quite opaque in this region. Quartz, seme special glasses, and water are special exceptions. The source of ultraviolet light for this study was a DAZ0R Floating Fixture Ultraviolet Lamp, Model N. U-58, equipped with a General Electric UA~3» 360 Watt Quartz Photochemical "Uviarc lamp. The radiant energy output of this lamp is greatest at 2537 A., 3131 A., and 3564 A. (29).

21 16 INFRARED ABSORPTION SPECTRA As pointed out previously, Sir William Herschel in 1800, discovered that there was an extension of the solar spectrum beyond the visible region. He found a rise in temperature as he passed from the violet to the red end of the spectrum by passing a thermometer through the various colors obtained by a spectrum that he fomed. Still more astounding was the fact that the thermometer showed a higher temperature in the dark space beyond the red than it did in any part of the visible spectrum (27). This meant that there was some intense radiation beyond the visible region. Thi3 invisible region beyond the red end is now known as infrared radiation. The degree of molecular motion possessed by a molecule is dependent upon the amount of energy that it possesses. If energy is given in the form of radiant energy, motion begins and it can take any one of or a combination of four types of motion depending upon the amount of energy supplied to it. These four forms of motion are described (30) as follows, according to the energy requirements: 1. Translational - in which the molecule moves from one point in space to another. Radiant energy of a very low order (20 microns in wavelength) is sufficient to initiate this motion. 2. Rotational - in which the molecule rotates about seme central axis. Molecular motion of this type is initiated by radiation in the order of twenty microns. 3. Vibrational - in which atoms are displaced from their normal

22 positions and oscillate back and forth or move sidewise with swinging motion within the molecule. The rotations involving small energies are superimposed on atomic displacements giving rise to absorption bands in the near infrared region of the spectrum which extends from 2 to 16 microns. Electronic - in which an electron is displaced to a higher energy level within the molecule. Greater displacements and greater energies are involved in ultraviolet and visible absorption spectra. Infrared absorption spectra are concerned then with molecules which are capable of rotation and vibration. However, there is an additional requirement. A compound to be active in the infrared region must be sufficiently asymmetrical to possess a dipole moment. Vibrations of two similar atoms against each other, as for example in nitrogen and oxygen molecules, will not result in a change in the electrical symmetry of the molecules and these molecules will not absorb in the infrared region (31)» The most significant portion of the infrared region of the spectrum for structure determination is the region from 2 to 8 microns, because it is in this region that individual bands are more or less characteristic of specific pairs or groups of atoms. Above 8 microns, the bands are due to vibrations and rotations in which all the atoms in the molecule take part. The spectrographs of pure compounds are so highly specific that they have become accepted as the,tfingerprints of those compounds'* (31)* When a compound undergoes reaction the fingerprint changes to that characteristic of a new compound but the groups that are present in the parent

23 18 compound plus those groups in the compound with which the parent compound was reacted will quite often show their characteristic absorption bands. As an example of the above, reference is made to Figures 4 and 5> pages 75 and 76 respectively. In Figure 4* the infrared absorption spectrograph of cholesterol shows peaks of absorption between cm"-*-, cm ^, and cmt-*- due to the effect of the presence of a hydrcayl group in position 3 of the cholesterol molecule (32). However, in Figure 5 the infrared spectrograph of a mixture of cholesterol and,,axidized,, cholesterol is shown. In addition to those peaks of absorption due to hydroxyl group we can notice a new peak of absorption at cm ^* due to partial oxidation to a ketone. A further consideration in the use of infrared spectra for analytical work is the effect of the solvent used upon the spectrograph. The ideal situation is that of a liquid compound in which the absorption observed is due to molecules of the compound only. Since this is not usually the case, the bands due to the solvent used must be considered. These factors then place the determination of the structure of a new compound somewhat upon an empirical basis. There is some uncertainty present but when the information from the infrared spectrograph is combined with other data valid conclusions as to the structure may be withdrawn. In this investigation, the apparatus used was the Baird Associates Two-Beam Infrared Spectrophotometer, property of the Department of Chemistry, The Ohio State University. This is a self-recording instrument. Sodium chloride crystals were used and the samples were prepared in a Nujol mull. Nujol did not interfere in this particular case since

24 interest was only to show oxidation of cholesterol, and the bands of absorption of Nujol are fortunately distinguishable from those of cholesterol or "oxidized cholesterol. SOME PROPERTIES OF THE OINTMENT BASES White Ointment The official formula for White Ointment, Pharmacopeia of the United States XV (1), calls for 5 per cent of white wax in 95 per cent of white petrolatum. It is prepared by melting the wax in a suitable dish on a water bath, adding the white petrolatum until liquefied and letting it congeal. It is an ointment base for several official ointments like ammoniated mercury ointment, sulfur ointment, and zinc oxide ointment of the Fharmacopeia of the United States (1); boric acid ointment, ethyl aminobenzoate ointment, mild mercurial ointment, phenol ointment, and yellow mercuric oxide ointment of the National Formulary (2). Its characteristics are essentially those of white petrolatum, the white wax being added as a stiffening agent. Because of its good keeping properties and physical nature, it has been largely employed for many years as a base for ointments which are white or light in color. The white petrolatum used in its preparation is a colloidal dispersion of aliphatic liquid hydrocarbons (C^q to C24) in solid hydrocarbons (C25 to C^q) obtained from petroleum. This mixture is semisolid, and the disperser of the liquid phase in the solid phase is a non-crystalline, naturally occurring branched chain paraffinic type of hydrocarbon known as proto-substance. Without this, the oil will leak out or "sweat" (33)*

25 20 The white petrolatum differs from petrolatum only in color. Both contain a small amount of oc -tocopherol which is added as an antioxidant. The white ointment and the white petrolatum have been used as mild dressings for blistered or excoriated surfaces. There has been sane objection to their use in this respect and as bases for ointments because they seem to retard healing and also because of their greasy nature. Wool fat was formerly an ingredient of white ointment but it was emitted from the formula in the Pharmacopeia of the United States XIV because of the sensitization which it sometimes causes (14) Hydrophilic Ointment This is an official preparation in the Pharmacopeia of the United States (1). It is a rather new type of ointment ba3e. Its formula contains white petrolatum, stearyl alcohol, propylene glycol, polyoxyl 40 stearate, methyl and propylparaben, and purified water. This ointment is really an emulsion type of ointment base. The emulsion formed is of the oil in water type. It belongs to the general classification of hydrophilic or water washable ointment bases, most of which are 0/W type of emulsions. The advantages of this type of base are several: they are easily removed from the skin or clothing with water; they can be diluted with water and permit incorporation of aqueous ingredients; provide media to absorb serous discharges of wounds, and also allow the heat in inflamed areas to be more easily dissipated; they allow for a greater release of many medicaments incorporated in them, and they are more acceptable cosmetically.

26 21 hydrophilic ointment was first recognized in the Pharmacopeia of the United States XIII, the then official formula differing from the present in containing glycerine in place of propylene glycol and 1 per cent sodium lauryl sulfate instead of polyoxyl 40 stearate (34)» In the Pharmacopeia of the United States XIV (35) the glycerine which caused the ointment to soften when it was triturated or used as a levigating agent (14) was replaced by the propylene glycol which corrected this defect. In the present formula (1 ), sodium lauryl sulfate has been replaced by the nonionic surfactant polyoxyl 40 stearate (36) to avoid skin irritation produced by the Pharmacopeia of the United States XIV formula because the sodium lauryl sulfate is anionic and is recognized as being a primary irritant. The p-hydroxy-benzoic acid esters are used as preservatives. There is some evidence to indicate that the combination of both esters is more efficient in this respect than either one alone in equivalent concentrations. The stearyl alcohol stiffens the ointment and also acts as an adjuvant emulsifier. Propylene glycol serves as a humectant and also assists the parabens as preservatives (14 ) Polyethylene Glycol Ointment This ointment is official in the Pharmacopeia of the United States XV (1) and consists of a mixture of 60 per cent of polyethylene glycol 400 and 40 per cent of polyethylene glycol The mixture is prepared by heating the polyethylene glycols 400 and 4000 to 65 C on a water bath and then stirring the mixture until it congeals. It is an ointment base of the water soluble type. It is the official base for the Benzoic and

27 22 Salicylic Acid Ointment of the Pharmacopeia of the United States XV (1) and for the Compound Undecylenic Acid Ointment of the National Formulary X (2). Polyethylene glycols are polymers represented by the general formula HOCHg (CffjOCF^nCHjjOH. The nm ranges from 1 to a large number which produces solid, waxy materials having high molecular weights. The liquids polyethylene glycols cover the range of molecular weight from 150 to 700, while the solids vary from 1,000 to 10,000. The solid polyethylene glycols are sold under the trade name of Carbowaxes (Carbide & Carbon Company). They are commonly prepared by either polymerization of ethylene oxide, using a trace of water, heat and pressure or by dehydration of ethylene glycol (33)* With the advent of the liquid and solid polyethylene glycols and the development of synthetic emulsifiers, the preparation of a wide variety of water miscible and washable ointment bases was made possible. The idea of greasiness inseparably associated with ointments is not considered so today. Because these washable bases have seme advantages over oleaginous bases, there is an increasing tendency on the part of dermatologists to prescribe the water soluble bases as vehicles for cutaneous medicinal preparations. These series of polyethylene glycols possess certain physical properties combined with chemical inertness and heat stability (33) which favor their use as ingredients in washable ointment bases. The official Polyethylene Glycol Ointment is a homogenous, white, semisolid base having the consistency of petrolatum. The Pharmacopeia

28 23 of the United States XV (1) indicates in its monograph that "if a firmer preparation is desired, not more than 400 On. of polyethylene glycol 400 may be replaced by an equal amount of polyethylene glycol 4000." It is completely soluble in water and does not stain clothing or bed linens. However, because of its high solubility in water we cannot add more than 3 per cent of water to the ointment or otherwise the consistency of the base will be affected. This might be considered as a disadvantage in the use of thi3 base. Before incorporating zinc oxide, sulfur or other substance to the base, these should first be triturated with a small amount of glycerine, propylene glycol or polyethylene glycol 400. The formula for this ointment base was first prepared by Zopf and his associates at the State University of Iowa, College of Pharmacy, in 1950 (37) The ointment was made official in the Pharmacopeia of the United States XIV (35). A great deal of work has been done to determine any untoward effects of this type of ointment base, but the order of toxicity seems to be low and apparently no more capable of sensitizing the skin than many other materials commonly used in ointment bases (14). However, there have been reports of two cases of demonstrable sensitivity to polyethylene glycols in ointment bases (38). Also Sulzberger and Baer (39) expressed seme disappointment in polyethylene bases after longer usage, mentioning as disadvantage s, (1 ) the possible irritation to diseased or dry skin, (2 ) the possible allergic sensitization in a small percentage of cases and an increase of sensitizing potential of active ingredients, and

29 24 (3 ) less effectiveness in removing scales, particularly from the scalp, and reduced emollient and lubricating effect when compared with greasy bases. Hydrous Wool Fat This product is described in the Pharmacopeia of the United States XV (1) as "wool fat containing not less than 25 per cent and not more than 30 per cent of water. Wool fat is the purified, anhydrous, fatlike substance obtained from the wool of sheep. Wool fat is also known as anhydrous lanolin, while the hydrous wool fat is also called lanolin. Lanolin is included in the formulas of Compound Menthol Ointment and Mild Mercurous Chloride Ointment in the National Formulary X (2). The wool of sheep contains from 10 to 50 per cent of a grease which occurs as an external coating on the fiber. It is analogous in function and composition to the human sebum (14)«Lanolin contains the sterols cholesterol and oxycholesterol as well as triterpenes and aliphatic alcohols. About 7 per cent of the alcohols are found in the free state; the remainder occur as esters of the following fatty acids: carnaubic, cerotic, lanoceric, lanopalmitic, myristic, and palmitic acids. Some of them are found free (40). The content of free cholesterol has been reported as approximately 1 per cent, and the acetyl value is reported as 23*3 (4l)» The iodine value is between IS and 36 (1). Wool fat, although often related to the fats, is more accurately classified chemically as a wax. True fats consist mainly of esters of glycerol and fatty acids, while lanolin is composed largely of esters of high molecular weight alcohols, as cholesterol and oxycholesterol.

30 Common wool grease requires extensive purification to make it suitable for medicinal use (40). This is accomplished with the aid of solvents, bleaching agents and other suitable agents. After purification and addition of water, it appears as a brownish yellow, tenacious, unctuous mass. lanolin is insoluble in water but soluble in chloroform and ether with separation of water. Sulton and Gardiner reported that wool fat penetrated the human skin much less readily than did lard (14). However, Johnston and Lee (42), by using as a tracer radioactive sodium chloride dispersed as an aqueous solution in various ointment bases, found wool fat to produce better absorption through the skin than did a base of lard, petrolatum or of a washable type of ointment. The efficiency of wool fat, as that of any other base, in promoting release of the active medicament when applied locally, depends not only on the base itself but also on the nature of the medicament and the state of the skin to which the ointment is applied. All these things have to be taken into consideration before stating anything about the properties of a base in releasing a given drug. Lanolin has been much used in the treatment of rough, dry skin, and similar conditions. While it is satisfactory for this purpose it has been largely superseded by newer formulations of the water in oil emulsion type in which the emulsifying agents present in wool fat are used as emulsifiers. It is largely used as a vehicle for ointments, especially when a liquid is to be incorporated. It is found in sane ointments of the National Formulary X (2), since it gives a distinctive quality to the ointment, increasing its absorption on topical application

31 26 and maintaining a uniform consistency for the ointment under most climatic conditions. Reports of sensitization to wool fat led to the deletion in the Pharmacopeia of the United States XIV (35) of this substance from all ointments, in which it was formerly used, on the recommendation of dermatologists who had noticed this undesirable manifestation in seme of their patients using this animal wax. Sulzberger et al., (43) obtained results connecting the aliphatic alcohol fraction of wool fat with the development of sensitization. They also report that the hypersensitivity due to lanolin is of weak intensity when compared with other eczematous hypersensitivities. Aquaphor There is not too much to be said about aquaphor since it is a propietary ointment vehicle (Duke Laboratories) and its exact composition is not revealed. It is described in the Remington*s - Practice of Pharmacy (40) as na hydrophilic ointment base containing high molecular hydroxyl animal fats." It is said to contain about 3 per cent of the free alcohols of wool fat including cholesterol and oxycholesterol. Cholesterol This is an official product in the Pharmacopeia of the United States. It is also called cholesterin. Cholesterol occurs as white or faintly yellow, almost odorless, pearly leaflets or granules. It melts between 147 C and 150 C. Cholesterin is insoluble in water and sparingly soluble

32 in alcohol; it is soluble in hot alcohol, acetone, ether, chloroform, petroleum ether, and in vegetable oils. It is stable under normal storage conditions (33) The term cholesterol literally means bile solid alcohol, since it was first isolated, in 1770» from human gallstones, of which it is generally the chief component. In 1815 it was shown to be unsaponifiable and was called cholesterin. later, the alcoholic nature of the compound was established, and since then it has been designated cholesterol. It is a steroid alcohol which is widely distributed in the animal organism. In fact, it has been found in all animal tissues but does not occur in plant tissues. It is synthesized in the animal body. The amount of cholesterol in animal tissues varies widely but it is reported (44) as existing in the plasma of humans in a concentration of 152/24 milligrams per one hundred milliliters. In the blood it occurs free and also in the form of esters. An excessive amount of this compound in the blood may result in a precipitation from its colloidal solution and subsequent deposition in the arterial wall. This phenomenon leads to the condition known as atherosclerosis. The skin possesses the capacity to synthesize cholesterol at a rate comparable to that of the liver. Cholesterol is converted to 7**dehydrocholesterol in the body and upon irradiation with ultraviolet light (i.e., when skin is exposed to sun rays) it foras vitamin D3. Cholesterol is particularly abundant in brain and nerve tissue, adrenal glands, and egg yolk. Fish oils constitute one of the principal sources of cholesterol. Cue of the most important methods of commercial production

33 28 involves extracting the unsaponifiable matter in the spinal chord of cattle, with petroleum benzin. In pharmacy, cholesterol has extensive use as an emulsifying agent and absorption base for the emulsification and incorporation of medicinal products in oils or fats. The chemical formula for cholesterol is shown in Figure 3 page 74* HYDROGEN PEROXIDE Above its melting point (-1.7 C), 100 per cent hydrogen peroxide is a syrupy liquid which appears colorless when observed in thin layers and slightly blue in thick layers (45 ) The boiling point of absolute hydrogen peroxide is reported as 152 C (14)» It is a powerful oxidizing agent. The anhydrous hydrogen peroxide contains 47 per cent by weight of available oxygen. The pharmaceutical as well as the industrial applications of hydrogen peroxide solutions are related to its ability to yield oxygen. One of the chief advantages of its use in pharmacy and industry is the fact that it gives rise only to water and oxygen when it acts as an oxidizing agent. In commerce, hydrogen peroxide is available as an aqueous solution in concentrations varying from 3 per cent to 80 per cent by weight. The strength of hydrogen peroxide is frequently designated also by the volume of active oxygen that it yields under standard conditions. Every 3»3 volume units are equivalent to 1 per cent by weight (i.e., the official. (1) Hydrogen Peroxide Solution, which is 3 per cent by weight, will release approximately 10 times its volume of oxygen).

34 29 Hydrogen peroxide was first prepared by a French pharmacist, Thenard, in He obtained it by treating barium peroxide with hydrochloric acid. The greater portion of the hydrogen peroxide now available is prepared electrolytically (14, 40). Pure concentrated (30 per cent or stronger) hydrogen peroxide solutions are quite stable (40). The commercial products, however, rapidly deteriorate in the absence of a preservative or stabilizer. One of the most commonly employed stabilizers is acetanilid. Generally, a dosage of 1/5 grain of acetanilid per fluid ounce of the official solution is sufficient (45) Chari and Qureshi (20) tried seme organic compounds for their ability to preserve hydrogen peroxide solutions and listed them in the order of decreasing stabilizing properties as follows: phenol, glycerol, acetanilid, ethyl alcohol, ethyl oxide, acetone, benzidine, salicylic acid, and oxalic acid. Other factors that affect the stability of hydrogen peroxide solutions are temperature, purity, acidity, and effect of ultraviolet light. Stability decreases with an increase in temperature. Horkheimer (46) found that solutions of hydrogen peroxide retained their potency for six weeks at 16 to 18 C, but at 20 to 23 G they lose some strength over this same period. Kiss and Lederer (47) determined the rates of decomposition of hydrogen peroxide solution in the presence of various metallic ions and found that only Cu7^ and Fe7^ ions showed any considerable catalytic effect upon the decomposition. The presence of small quantities of mineral acids aids in the stabilization of hydrogen peroxide solutions, but with too much acid the stability is so great that it fails to liberate

35 "nascent oxygen and this impairs the use of the solution as an antiseptic. Alkalies, on the other hand, rapidly decompose hydrogen peroxide solutions with the liberation of oxygen (40)«It is also well known that ultraviolet rays of short wavelength ( millimicrons) decompose solutions of hydrogen peroxide at a measurable rate. Therefore, solutions must be protected from exposure to direct sunlight. Hydrogen peroxide is frequently used as a germicidal agent. Its ability to kill microorganisms depends upon the release of oxygen and will persist only as long as oxygen is being released. Although in relatively dilute solutions it will eventually destroy many of the pathogenic microorganisms, its action is extremely slow unless the solution is fairly concentrated. Heinemann (48) reached the conclusion from his experiments, that three teaspoonfuls of the official solution after six weeks of exposure will destroy 99 per cent of the bacteria present in a liter of drinking water. This is about 1:1000 solution of hydrogen peroxide. Kavanagh (49) studied several antibacterial substances against nine species of bacteria. The results he obtained are given below for hydrogen peroxide as compared with penicillin and streptomycin which were the most effective of the twenty two antibacterial substances that he used in his experiments.