POTENTIAL OF ENZYMES FOR TOPICAL APPLICATION 257 the hydrolyric reactions to take place. This is more likely to occur when the enzyme is applied to a wound which is actively discharging serum, etc. However, for most cosmetic applications, free tissue water will be absent and must therefore be supplied as part of the formulation. 2. Base Compatibility. This refers to the other side of the coin the stability of the emulsion in the presence of the enzymes. One cannot expect to obtain a stable emulsion with a lipid base in the presence of a lipolytic enzyme, nor can one expect a surfactant that is an ester to with- stand the hydrolytic activity of the esterases. So many commercial en- zyme products contain esterase activity present, either as nonspecific contamination or as part and parcel of proteolytic activity, that the selec- tion of the proper emulsifying agent may become a major problem to the formulatot. He must not only be certain that the surfactant is compatible with the enzyme but must be concerned with the reverse condition, as well. In some cases switching from an ester to an ether is all that may be neces- sary, whereas in others the search for the appropriate emulsifier could become a major research project. 3. In Fivo ./lctivity. An ingredient in an ointment can react only at the interface between the skin and the base. One might then expect in vivo enzyme activity to be a function of enzyme concentration up to the point where the interface becomes saturated. In some instances this could be modified by the proper selection of a hydrophilic or lipophilic base. A hydrophilic ointment should serve to attract moisture into a non- aqueous vehicle so that all of the enzyme at the interface would be reactive at any one time. A lipophilic base might be expected to keep at least part of the enzyme inactive throughout a period of contact by preventing the uptake of water. Both types of bases can serve useful purposes, if we assume that a given enzyme would be equally stable in both. If we wanted most rapid action it would appear that the hydrophilic ointment would be more suitable. On the other hand, the lipophilic base would offer a degree of "sustained release" by virtue of the enzyme's inability to hydrate all at once. 4. In Fitro ./lctivity. Usually the activity of a reagent in vitro is not considered if it can be shown to function properly in vivo. In the case of enzymes, however, a label claim can be made for a product only if the active ingredient can be assayed. It has been our experience that the dosage form can be a critical factor in determining whether an enzyme can be quantitatively recovered from a product. More often than not, if the enzyme cannot be recovered under the optimum conditions in our laboratory, it will not be available as an active agent in vivo. As might be expected, vehicles with high Hydrophilic-Lipophilic Balance (HLB) pose the least problem. On the other hand, mixtures that have to

258 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS be extracted with a fat so]vent in order to free the enzyme create the greatest concern. One example of such a problem is the inability to dis- tinguish between poor recovery due to poor stability, or due to the tech- nique used to separate the water soluble protein from the lipophilic base. The proper selection of the type of ointment, as well as the components with which it is made, can spell the difference between being able to demon- strate both in vitro and in vivo activity and having a preparation with the enzyme so bound as to make it virtually useless. And finally, it should also be recognized that an enzyme can be chem- ically, as well as physically, complexed to some components of cosmetic preparations, due to the large number of reactive groups present on all proteins. An example of this particular type of problem is the complexing of certain types of enzymes with carboxymethylcellulose, which appears to occur via an ion exchange-type of mechanism. Sometimes reactions of this nature can be minimized by adjusting pH or preferentially complexing the proteins with reagents that will not interfere with the activity, or the physical stability, of a cosmetic preparation. C. AS'eas of •uestionable Potential The cosmetic chemist most certainly will become better acquainted with the desirable features of enzymes that make them so suitable for cosmetic purposes. This does not mean that enzymes will become the panacea there are many areas in which nonenzymatic methods are, and will remain, superior. For example, it would take a most remarkable enzyme to function as a cuticle remover as effectively as do the inorganic alkalies currently in use. Also, as indicated above, an enzyme with the characteristics of the "ideal" depilatory more than likely does not exist. The areas of most questionable potential are those in which the demand is for an agent to react instantaneously on an insoluble substrate. The critical parameter as far as the enzymes are concerned is the insoluble substrate with which the cosmetic chemist is so often concerned. Under the proper conditions enzymes can perform in the desirable fashion, if the substrate is readily available. Unfortunately, many of the conditions with which the cosmetic chemist must deal are those that are least compatible with the requirements of the enzymes. However, by adjusting some of these, such as the extension of contact time, choice of suitable dosage form, etc., some of the drawbacks can be overcome. In those areas where nonenzymatic reagents may be more economical and function as well as or better than their enzymatic counterparts, it would be pointless to consider an enzyme as the active ingredient. How- ever, there are areas in which enzyme specificity would offer such ad- vantages (e.g., in cases of "oily skin" it may be possible to remove specific