Part IV:  Topical Cosmeceuticals, Formulations, and Indications

countries are in the process of harmonizing and adopting these legal safeguards. Any company that does not react ahead of the inevitable is going to find it an arduous and almost impossible task to implement in the time frames that are normally allowed for full compliance.

DEFINITION OF NATURAL

A natural is any material that is harvested, mined, or collected, and which may have subsequently been washed, decolorized, distilled, fractionated, ground, milled, separated, or concentrated, leaving a chemical or chemicals that would be available and detectable in the original source material. It is also the modification of natural material by the action of microorganisms, enzymes, or yeasts to modify or increase the yield of material by this process. Naturally derived materials are defined by the use of a natural raw material as the starting point in a chemical process that produces a new chemical or chemicals that in themselves may not be available in nature or in the starting material. Nature-identical materials are substances that have been synthetically produced, not usually from a natural starting material, in order to produce a material that is identical to that naturally occurring in nature.

SOURCES OF DATA FOR PRODUCTS THAT MAY HAVE TOPICAL COSMECEUTICAL BENEFIT

Many ethnopharmaceutical applications for topical application that have been used for countless generations have been tried and tested in those countries where their use is prevalent. In many cases, these are well described in the literature and have been identified from their phytochemical composition, preparation, part used, and dosage.

This search is no longer restricted to the European systems of herbal medicine but now extends to Russian, Chinese, Indian systems of Ayurvedic medicine and Unani, African, and more recently, Aboriginal traditional medicine. Less reliable sources are “grandmother’s recipes” or folklore, which pass from generation to generation without any real scientific basis. The works of the old herbalists such as Galen, Dio- scorides, Culpeper, Hildegarde von Bingen, and Paracelsus (Theophrastus Bombastus von Hohenheim) may give some insights to future investigation. In some cases, these remedies may be discounted after serious evaluation, but there are still many occasions when science substantiates their beneficial use.

PLANTS ARE COMPLEX CHEMICAL FACTORIES

A plant is a complex and ever-active chemical factory that produces a wide range of chemical moieties that it requires for protection against yeast and moulds, resistance to insect attack, even protection against UV and drought conditions in some special- ist plants. Other coastal region plants require very specific protection against salts and excess minerals. It is this complex environment that produces many dozens of chemical entities that may have benefit in human treatments for various skin condi- tions. The composition may vary according to the area of growth (country), the soil, the weather conditions, the time of harvest, the processing, and, of course, the part of the plant that is being extracted. The storage conditions of the plant, the time of extraction, and the solvents used in that extraction will all have a significant implica- tion on the final chemical composition, for example, the content of natural preserva- tives produced by plants to protect the fruit and the leaves will fall dramatically once

oil is reputed to contain vitamin A according to some references. A large body of evidence (mainly anecdotal clinical) suggests that this oil has exceptional cicatrizing properties and is an excellent oil for restoring skin elasticity especially for postsurgical conditions where tightness has become a problem for the patient. It was also shown to be effective for treating the hyperpigmentation of certain scar tissues.

Flavonoids

“Flava” means yellow in Greek and the collective name of flavonoids for this group of compounds was proposed by Geissman in 1952. This is a very large group of compounds showing extraordinary diversity and variation and as the Greek root for the word suggests, many of these compounds are yellow in color. They consist of a number of structurally related groups of products, which are often identified as polyphenols. Many have a basic skeleton (Fig. 2) that contains 15 carbon atoms, which are usually subdivided into one part made up from a phenolic (6C) moiety and another which has a cinnamic acid molecule (C13) as a building block. The group called the chalcones may be considered as the Friedel-Crafts reaction product of a (substituted) cinnamic acid and a phenol.

The flavonoids in red wine (Vitis vinifera) such as quercetin, kaempferol, and anthocyanidins account for the free radical–scavenging activity. In green tea (Camellia sinensis or Thea viridis), it is the catechins and catechin gallate esters that are shown to be effective antioxidants against free radicals. The dietary effectiveness of these materials has been known for generations and similar antioxidant effective- ness has been shown to occur when these materials are topically applied to protect skin cells.

Flavonoids also are a source of natural color, with yellows from the chalcones and flavonols, and reds, blues, and violets from the anthocyanidins. The flavones are colorless, but are still able to absorb UV strongly and so act as a beacon to pollinating insects. The exploitation of these molecules as a source of natural color in cosmet- ics and toiletries is just beginning, but their poor light stability is often a stumbling block. Flavonoids may be found as their glycosides. These are molecules that are substituted on one or more of the hydroxyl groups with a sugar such as galactose, glucose, mannose, or rhamnose, etc. The aglycons do not carry a sugar moiety.

Chalcones

Chalcones act as precursors for an enormous range of flavonoid derivatives. Re- ductive ring closure of chalcones results in the formation of a flavone. Naringenin chalcone (Fig. 3A) is converted to a flavanone, naringenin (Fig. 3B), from which api- genin (Fig. 3C) (4′,5,7-trihydroxyflavone) is formed. Flavones are generally found in herbaceous families such as Labiatae, Umbelliferae, and Compositae. Flavones have the skeleton 2-phenylchromen-4-one and include apigenin, baicalein, chrysin, dios- metin, diosmin, flavone, luteolin, tangeretin, techtochrysin, rhamnazin, nobiletin,

and natsudaidain. The most common flavones are apigenin found in celery (Apium graveolens) and parsley (Carum petroselinum); luteolin found in German chamomile (Matricaria recutita) and horsetail (Equisetum arvense), and diosmetin found in rosemary (Rosmarinus officinallis). All these materials are known for their soothing, calm- ing, and cicatrizing effects, although each has additional properties that are a result of other chemical moieties within their constituents, for example, the silicate content in horsetail gives it nail strengthening properties. The flavones often occur as glyco- sides. Flavones also occur in nature in association with tannins (polyesters of gallic acid; 3,4,5-trihydroxybenzoic acid). Gallic acid and its esters (e.g., propyl gallate, dodecyl gallate) are well-known powerful antioxidants, and it is probable that these products fulfill a similar role in higher plants.

Flavones

The simplest representative of the group of flavones is “flavone” (Fig. 4), which does not carry any hydroxy, methoxy, or glycosidic groups.

Flavanone Glycosides

The flavanone glycosides are very important, because they include naringin (Fig. 5A), which has been found specifically in grapefruit peel and seeds, and hesperidin (Fig.

5B) found in citrus peels and seeds. These materials have been shown to exhibit antimicrobial activity and have been used as preservatives (even though they are not listed in Annex VI of permitted preservatives).

O

Aurones

Some aurones occur naturally. In this group, the six-membered heterocyclic ring is replaced by a five-membered ring. An example of an aurone is sulfuretin, 6,3′,4′- trihydroxyaurone (Fig. 6) found in Rhus verniciflua and Dalbergia odorifera, which are being studied for their anti-inflammatory properties.

Flavonols

The most common flavonols (3-hydroxyflavones) are kaempferol, quercetin (Fig. 7), and rhamnetin. The name for quercetin comes from the genus in which the chemical was first identified, namely, Quercus spp., or oak, but the highest levels have been seen in evening primrose leaf (O. biennis) and the sap of mayapple (Podophyllum peltatum).

Kaempferol (Fig. 7B) is found in neem or Nimba flowers (Azadirachta indica) and pea (Pisum sativa). It is also widely found throughout the Brassica spp., such as cabbage, kohlrabi cauliflower, and kale. Interestingly, these plants have not been exploited for their anti-inflammatory activity. The flavonols also exist as flavonol glycosides such as rutin (Fig. 8), found in buckwheat (Fagopyrum esculentum) and rue (Ruta graveolens).

Isoflavones

The isoflavones (Fig. 9A) are mainly found within the Leguminosae (specifically in the sub-family Papilionoideae), although many other species (e.g., Compositae, Iridaceae, Myristicaceae, and Rosaceae) contain these chemical moieties (1). These

OH

OH

HO O

isoflavones can act as steroidal mimics by filling the stereochemical space that could be occupied by estrogenic compounds. This spatial chemistry helps explain the ef- fects of many nutritional herbal supplements and topical preparations (Fig. 9B,C).

Daidzein is a phytoestrogen (also called a phenolic estrogen, to distinguish it from a steroidal estrogen such as 17β-estradiol). The activity of phytoestrogen is much weaker than steroidal estrogens, varying from 0.005% to 2% (2). The estro- genic properties are insufficient in strength to replace steroidal estrogens, but they do have significant value in reducing the effects of aging and improving the quality of the skin. Phytoestrogens may also be viewed in relation to the phytochemical division of terpenoids, which comprise the largest group of natural plant products. All terpenoids are biogenetically derived from isoprene. The largest group of terpenoids are the triterpenoids, which include, among other divisions, the triterpenoid and steroid saponins and the phytosterols.

Nature has a rich portfolio of phytosterols, and it is easy to understand why compounds such as stigmasterol and β-sitosterol (Fig. 10A,B) have anti-inflammatory effects and are capable of reducing swelling and erythema when their structure is compared to corticosterone (Fig. 10C) and hydrocortisone (Fig. 10D).

The most commonly occurring isoflavones are: ▪ Biochanin A  5,7-Dihydroxy-4′-methoxyisoflavone

▪Daidzein  4′,7-Dihydroxyisoflavone

▪(±)-Equol  4′,7-Isoflavandiol

▪Formonometin  7-Hydroxy-4′-methoxyisoflavone

▪Glycitein  4′,7-Dihydroxy-6-methoxyisoflavone

▪Genistein  4′,5,7-Trihydroxyisoflavone

▪Genistein-4′,7-dimethylether  5-Hydroxy-4′,7-dimethoxyisoflavone

▪Prunetin  4′,5-Dihydroxy-7-methoxyisoflavone

will naturally alleviate symptoms occurring as a result of the aging process and a deficiency in estrogen levels. Aging signs and symptoms will, to a certain extent, be reversed. The rich source of sterols and phytohormones also indicates the plant for the topical treatment of wrinkles and aging skin conditions.

Red Clover (Trifolium pratense L.) (Leguminosae)

Red clover flower heads contain the following isoflavones: biochanin A, daidzein, formononetin, genistein, pratensein, and trifoside. The plant has alterative, antispasmodic, expectorant properties and is a sedative dermatological agent. Its main use is for skin complaints such as psoriasis and eczema, as well as an expectorant in coughs and bronchial conditions (5). Biochanin A (Fig. 13A) and formononetin (Fig. 13B) are two isoflavones from red clover. They are very similar to genistein and daidzein, ex- cept that the hydroxyl groups have been methylated. These two isoflavones are con- siderably less estrogenic in their original forms; the stereochemistry of the methoxy groups means they are not able to efficiently bind to estrogen receptors. However, once ingested, they are demethylated by bacteria in the colon; biochanin A becomes genistein (Fig. 13C) and formononetin becomes daidzein. Daidzein can be further metabolized to equol (Fig. 13D). Internally, therefore, biochanin A and formononetin are a source of considerable estrogenic activity. It may well be that these mechanisms give red clover its reputation as an alterative remedy, cleansing the system yet mild enough for many children’s skin problems, even eczema. A lotion of red clover can be used externally to give relief from itching and other skin disorders (6). Red clover is recommended for athlete’s foot, sores, burns, and ulcers (7), and has been used in the herbal treatment of cancer, especially of the breast or ovaries (8). It is also a very popular alternative remedy for hormone replacement therapy.

Sweet Yellow Melilot (Melilotus officinalis)

Melilot is soothing, astringent, and anti-irritant possessing similar properties to red clover. It may also be anti-inflammatory, antiedema, and anesthetic (9). However, it is perhaps not the isoflavones at force here, but possibly the β-sitosterol or coumarin contained in the roots. M. officinalis L. extract, containing 0.25% coumarin (Fig. 14),

potential in skin care and body care, being anti-inflammatory and anti-rheumatic. It is also cited for dysmenorrhea, and ovarian and uterine pain (14,15).

It is interesting to note that Vitex agnus-castus is a source of natural progesterone, and many documented studies have investigated the use of these products to treat various gynecological disorders (16). The fruit of Vitex contains essential oils (including limonene, 1,8-cineole, and sabinene), iridoid glycosides (agnuside and aucubin), and flavonoids (including castican, orientin, and isovitexin). The active constituents have been determined as 17-α-hydroxyprogesterone (leaf), 17hydroxyprogesterone (leaf), androstenedione (leaf), δ-3-ketosteroids (leaf), epites- tosterone (flower), progesterone (leaf), testosterone (flower and leaf) (17,18).

It is highly unlikely that diosgenin in the plant could ever be metabolized to a corticosteroid or hormonal derivative on application to the skin. However, it does seem likely that this material (being the precursor to these estrogenic molecules) will, to some extent, mimic the function of those pharmaceutical active materials and benefit the skin (19). However, the production of wild yam was unable to sus- tain the demand for diosgenin as the starting precursor, for the production of birth control materials, which was dominated by estrone (Fig. 16D).

Fenugreek (Trigonella foenum graecum)

The world turned its attention to fenugreek (T. foenum graecum) for its source of diosgenin. Fenugreek seeds are emollient and accelerate the healing of suppuration and inflammation. Seeds are cooked with water into a porridge and used as hot compresses on boils and abscesses in a similar manner to the usage of linseed (20). A cataplasm obtained by boiling the flour of the seeds with vinegar and saltpeter is used for swelling of the spleen (21). Extracts of the seeds are incorporated into several cos- metics claimed to have effect on premature hair loss and as a skin cleanser (22), and it is also present in hair tonics and claimed to cure baldness (23). Many of the herbal ma- terials found to have an effect on hair growth have a hormonal or hormonal-mimetic basis. Likewise, there are a number of references to fenugreek having galactagogue (increase milk in nursing mothers) activity (8,24,25), which again is indicative of an estrogen-like activity. Fenugreek is reputed to be oxytocic and uterine stimulant ac- tivity has been documented in vitro (16), so its use during pregnancy and lactation is not advisable.

Pomegranate (Punica granatum)

The seeds of pomegranate, an ancient symbol of fertility, contain an estrone identical to the genuine hormone, and they are the best source of plant estrone (26). Pomegranate fermented juice and seed oil showed strong antioxidant activity (determined by measuring the coupled oxidation of carotene and linoleic acid), close to that of butylated hydroxyanisole (Fig. 17) and green tea, and significantly greater than that of red wine (27). This is clearly a fruit worthy of further exploration. The rind is used as an astringent (28) and the leaf has antibacterial properties (29).

Date Palm (Phoenix dactylifera)

A decrease of normal body hormones plays a role in skin aging, reduced skin thickness, and the disturbance of normal collagen turnover, which in turn results in a decrease in collagen I and III synthesis. Date palm kernel has seven compounds with regenerative, anti-oxidizing, firming, and soothing properties, extracted: phytosterols, phytosteroids, ursolic acid, isoflavones, policosonols, provitamin A, and vitamin E. Some studies suggest that dehydroepiandrosterone (DHEA) (Fig. 18), known for its

OH

C(CH3)3

capacity to promote keratinization of the epidermis, would have a beneficial effect against signs of aging. The effects of date palm kernel extract were compared with those of DHEA using ex vivo skin. There was a decrease of wrinkles within five weeks of date palm kernel extract application and the skin structure was also improved in a way superior to that of DHEA (30). The seed and the pollen have both been shown to contain estrone and this may further explain the reasons for this activity (31,32).

Hops (Humulus lupulus)

The hop contains β-sitosterol, estradiol, stigmasterol, and estrone, together with many other materials that are known for their sedative attributes. Regular doses of the herb can help regulate the menstrual cycle (33). It had long been known that menstrual periods came early in young girls hop picking. Considerable amounts of estrogen (30,000–300,000 IU per 100 g) have been found in hops. The presence of antiandrogens may explain why hops will suppress sexual excitement in men (26). Hop extract recovered the proliferation of hair follicle derived keratinocyte suppressed by androgen, stimulated the proliferation of hair follicle keratonicytes and demonstrated a potent acceleration of hair growth (34).

Sarsaparilla (Smilax ornate)

Sarsaparilla is used in concoctions with other plants as a tonic or aphrodisiac (35). It was formerly used in the treatment of syphilis (36), gonorrhea (37), rheumatism, and certain skin diseases. Used in soft drinks, the genins are also used in the partial synthesis of cortisone and other steroids (6). It is especially useful as part of a wider treatment for chronic rheumatism. It has also been shown that sarsaparilla contains chemicals with properties that aid testosterone activity in the body (15). Sarsaparilla contains saponins, sarsaponin, and parallin, which yield isomeric sapogenins, sarsapogenin, and smilogenin. It also contains sitosterol and stigmasterol in the free form and as glucosides. It is antirheumatic, antiseptic, antipruritic, and indicated for psoriasis and other cutaneous conditions. It is specifically used in cases of pso- riasis especially where there is desquamation (14).

Sugars, Polysaccharides, and Mucopolysaccharides

The skin appears to have an affinity for sugars, and there are many examples where they have been shown to have a significant cutaneous effect. In Third World countries

and poorer communities, honey, the first choice as a natural source of these sugars, has been shown to be of great benefit in the treatment of burns, scalds, and wounds, especially because it has antibacterial properties when used undiluted. Reepithelialization is accelerated, granulation is even, and there is less necrotic tissue formed. Honey absorbs exudates and makes noninvasive cleaning simple and painless.

Mucopolysaccharides are present in numerous plant materials, including the ribwort and greater plantains (Plantago lanceolata and Plantago ovata). Mucilages are found in numerous species of seaweeds, for example, bladderwrack (Fucus vesiculosus), sea lettuce (Ulva lactuca), and oarweed, tangleweed, or kombu (Laminaria digitata). These plants have similar effects and they are used for dry, desquamatous, pruritic skin conditions.

SPECIALIST PLANTS

There are a number of plants that have unique chemical entities and are specific for treating skin diseases and damage.

Gotu Kola (Centella asiatica)

C. asiatica (Indian pennywort or cotyle) is an important medicinal herb in India. Its Sanskrit name is Brahmi and in Tamil it is known as Mandukaparni. An infusion of the leaves and stems has long been used in India for leprosy and other skin diseases.

Asiaticoside (Fig. 19) was found to be active against leprosy (by dissolving the waxy coating of Mycobacterium leprae) and an oxidized form, oxyasiaticoside, inhibited growth of tubercle bacillus in vitro and in vivo (38,39).

C. asiatica extracts are used for the treatment of skin ailments, particularly ulcers, wounds, and for prevention of keloid and hypertrophic scars. Centella extracts have been found to accelerate wound healing, particularly in cases of chronic, post- surgical, and posttraumatic wounds. Extracts have also been successfully used as a therapy in the treatment of secondand third-degree burns. The pharmacological activity of C. asiatica is thought to be attributable to several saponin constituents, including the asiatic acid and madecassic acid, and both compounds stimulate the production of human collagen I, a protein involved in wound healing. Asiaticoside

is effective in accelerating the healing of superficial postsurgical wounds and ulcers by an acceleration of cicatricial action.

Topical application of asiaticoside has been shown to promote wound healing in rats and significantly increased the tensile strength of the newly formed skin. Ex- tracts of C. asiatica, and in particular asiaticoside, have also been shown to be valuable in the treatment of hypertropic scars and keloids. The mechanism is because of the effect of the plant on the synthesis of collagen and acidic mucopolysaccharides, and by the inhibition of the inflammatory phase of hypertropic scars and keloids. Asiaticoside is likely to interfere with scar formation by acting on myofibroblasts and immature collagen (40).

Licorice (Glycyrrhiza glabra)

Theophrastus recommended licorice for quenching thirst, to combat cramps caused by stomach ulcers and asthma. Napoleon chewed licorice root regularly and eventually blackened his teeth (41). Licorice is one of the most commonly used plant materials in TCM, and various species, including Glycyrrhiza glabra, Glycyrrhiza uralensis, and Glycyrrhiza inflata, are used. The roots are most often used with other herbs to mediate their effects (41). It was introduced into Britain by the Black Friars in the 16th century and was later cultivated extensively in the Pontefract district of Yorkshire

(43). Glycyrrhizin, a triterpenoid saponin found in the roots, has anti inflammatory and antiallergic activity, the former attributed to the corticosteroid-like activity of glycyrrhetinic acid or enoxolone (Fig. 20A) and glycyrrhizin (Fig. 20B) (40).

Comfrey (Symphitum officinale)

Comfrey’s medieval reputation for knitting broken bones is reflected in its name of knitbone, which comes from the Latin conferre, meaning to bring together (45). The plant contains allantoin (Fig. 21), which acts as a vulnerary because of its cell prolifer- ant effect. The demulcent action is because of the high mucilage content. Significant anti-inflammatory activity has been demonstrated in vivo (46). The plant also contains

mucilage, tannins, starch, and two alkaloids, consolidine and symphytocynglossine. Large amounts of potassium, phosphorus, and vitamins A and C are also present (47).

Aloe Vera (Aloe barbadensis)

There are many studies on the healing power of aloe vera. Aloe-treated wounds in dogs had smaller unhealed areas than untreated controls and wounds treated with antibiotics (48). Mannose-6-phosphate is the major sugar in aloe, and mice receiving 300 mg/kg of mannose-6-phosphate had improved wound healing over saline controls (49).

After full-face dermabrasion, the abraded face was divided in half. One side was treated with a standard polyethylene oxide gel wound dressing, and the other was treated with a similar gel dressing saturated with stabilized aloe vera. By 24 to 48 hours, there was dramatic vasoconstriction and accompanying reduction in edema on the aloe-treated side. By the third to fourth day, there was less exudate and crust- ing at the aloe site, and by the fifth to sixth day the reepithelialization at the aloe site was complete. Overall, wound healing was approximately 72 hours faster at the aloe site. This acceleration in wound healing was important to reduce bacterial contamination, subsequent keloid formation, and pigmentary changes (50).

The influence of oral and topical aloe vera on wound healing (induced using a biopsy punch) in mice was studied. In the oral study, test animals received aloe vera (100 mg/kg/day) in drinking water for 2 months, and the controls received water only. In the topical study, test animals were given 25% aloe vera cream and the controls received cream only. There was a 62.5% reduction in wound diameter (oral aloe vera group) and a 50.8% reduction (topical aloe vera group). These data suggest that aloe vera is effective by both oral and topical routes of administration (51).

Eight topical agents in current use were studied for their effects on wound con- traction and rate of reepithelialization of full thickness skin excisions in pigs. The fol- lowing were applied daily for 27 days: scarlet red ointment, benzoyl peroxide lotion, bacitracin ointment, silver sulfadiazine cream, aloe vera gel, tretinoin cream, capsa- icin cream, and mupirocin ointment. Reepithelialization was significantly enhanced by capsaicin, bacitracin, silver sulfadiazine, and scarlet red, and was markedly re- tarded by tretinoin. Wound contraction was significantly retarded by mupirocin, bacitracin, and silver sulfadizine. Knowledge of the effects of topical agents on vari- ous aspects of healing allows the clinician to choose the most appropriate material to use in a given clinical situation to optimize the healing process (52).

Aloe vera (100 and 300 mg/kg daily for 4 days) blocked the wound healing suppression of hydrocortisone acetate by up to 100% using the wound tensile strength assay. The growth factors present in aloe vera mask wound healing inhibitors such as sterols and certain amino acids. The sterols showed good anti inflammatory ac- tivity in reducing the croton oil induced ear swelling (53,54). The use of aloe in treating leg ulcers has been described (55). Kligman writes in his conclusions, “It is our opinion that the Aloe vera materials tested did not interfere with the nor- mal rate of superficial dermal wound reepithelialization nor did they enhance the process any faster than the covered nontreated control wounds at the end of three

weeks. It can be stated that the wounds treated with Aloe vera healed better than uncovered wounds and were more cosmetically gratifying” (56). A review of the literature showed that the healing, soothing, and cooling claims routinely made for aloe are justified (57). Aloe vera also has a prophylactic effect in the protection of the skin against UV radiation (58) and also protects the skin against the radiation produced in radiotherapy treatment (59–61).

Witch Hazel (Hamamelis virginiana)

Known as hamamelis, snapping hazel, winter bloom, spotted alder, tobacco wood, and hamamelis water, witch hazel is a well-known plant with a long history of use in the Americas. Uses include the treatment of hemorrhoids, burns, and eye irritation. Preparations have been used topically for symptomatic treatment of itching and other skin inflammation, and its drying and astringent effects help treat skin inflammation (62) and also protects against infection. The astringent action is be- cause of the hamamelitannin (Fig. 22A) and hamamelose (Fig. 22B). Inflammation of mucous membranes including mouth, throat, and gums may also be treated with a witch hazel gargle. It is also used for swollen or tired eyes, to relieve the pain of sunburn, as a face splash for oily skin, and to control minor pimple formation. It reduces the pain of insect bites and can be used cold or with ice to reduce the pain of sprains or athletic injuries including bruises. It is used in some hospital recovery rooms to reduce the swelling from intravenous feeding (63).

CONCLUSIONS

A plant is a complex chemical factory, where each chemical component delivers spe- cific properties that in concert are often synergistic in their performance. In addition to some of the simple categories discussed herein, there are numerous plant species that have chemicals that are unique to them.

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forming. The efficacy of these cosmeceutical products was simply not enough. Cos- metic marketers became unhappy and started asking for more efficacious products while at the same time, regulators of cosmetic products started to ask for clinical evidence for the claims that were made. Cosmetic claim substantiation became a cosmetic discipline. Customers of cosmetic products wanted to see a real consumerperceivable benefit, cosmetic marketers wanted to deliver this benefit, and cosmetic regulators wanted to see the evidence. In the meantime, cosmetic formulators were the ones who had to deliver the clinical effect via the delivery of the active ingre- dient to the site of action in the skin at sufficiently high concentrations for a suf- ficiently long period of time. They tried new active ingredients with promising intrinsic activity profiles but were often not able to create efficacious products when using their standard cosmetic formulations. Suppliers of the new active ingredients were blamed. Some suppliers were smart and realized that the lack of skin delivery was the root cause of this and started to provide cosmetic delivery systems to the cosmetic industry: liposomes, nanosomes, microcapsules, and the like emerged into the market, and these were popular for some time and continue to be so in selected applications where they have demonstrated their benefit. But skin delivery sys- tems cannot be used generically. Something that is good for one active ingredient, drug, or cosmeceutical, is not necessarily good for another. Moreover, not everyone wanted to include costly gadgets into their topical preparations that would hopefully give them enhanced delivery. There was need for a more generic approach that would work with every active ingredient, under every condition (concentration) at no extra cost.

In 2003, I presented, together with my colleagues, a paper at the International Federation of Societies of Cosmetic Chemists Conference in Seoul, South Korea, that went a long way in providing exactly that. It was not set up to meet this objective, but started as a means to explain some seemingly contradictory requirements for skin delivery to my colleagues. When I was finally able to explain this to my col- leagues, the formulating for efficacy concept was born. Although very simple in principle, its consequences are far-reaching from both a marketing perspective as well as a consumer perspective. The use of this concept allows the formulator to cre- ate a more efficacious product at lower concentrations of active ingredient, a target that would delight people both within as well as outside the cosmetic or pharmaceutical industry.

In this chapter, I will first discuss the various factors influencing skin delivery from topical formulations, such as formulation type, structure, and composition.

I will explain the theoretical background of the formulating for efficacy concept, show how this has been used to enhance the skin penetration of active ingredients into the skin, and show clinical results from formulations that were and that were not optimized via this concept. This will be followed by new clinical results indi- cating that the dose of active ingredient can be lowered without a loss of efficacy.

The chapter will end with some further perspectives of possible improvements that could be made to the formulating for efficacy concept.

FACTORS INFLUENCING SKIN DELIVERY FROM TOPICAL FORMULATIONS

Various factors influence skin penetration. In short, they are the condition of the skin (whether it is healthy or diseased), the physicochemical properties of the pen- etrating molecule, the formulation in which the penetrating molecule is applied, and finally, the dosing conditions (1). The condition of skin, which includes such

aspects as skin lipid structure and organization, and how this may be influenced by various disease states, is discussed elsewhere in this book. It has been shown in patients suffering from atopic dermatitis, for instance, that the barrier function of the skin is reduced because of changes in the packing rigidity of skin lipids (2). The physicochemical properties that favor skin penetration in short are the following:

(1) a 10log octanol/water partition coefficient of 1 to 2, that is, the molecule should be 10to 100-fold more soluble in octanol than in water; (2) a low molecular weight (ideally, lower than 500, but when higher, generally the lower the better); (3) un- charged over the physiological pH range encountered during skin penetration; (4) a high dipole moment (1). A molecule such as dimethyl sulfoxide fulfills all these re- quirements and it penetrates human skin within seconds. The influence of formula- tion design on skin penetration is the subject of this chapter. The dosing conditions are generally of relatively minor importance, although in individual cases, they may have a big impact. This encompasses issues such as application under occlusion, which was shown to be very relevant for the topical application of corticosteroids, the number of daily applications, and the skin temperature. In the following, it is assumed that a formulation is applied onto healthy skin and contains a drug, an active ingredient, or a cosmeceutical, which has the right chemical characteristics to penetrate the skin.

Formulation Type

There are three fundamental levels on which a formulation can influence topical delivery. On the first level, there is the influence of formulation type, that is, whether the drug or active ingredient is dosed in a gel, an oil-in-water (O/W) emulsion or a water-in-oil (W/O) emulsion, a microemulsion, or an oil. In a systematic study, four model drugs of differing polarities (the polar 5-fluorouracil, the intermediate polarity hydrocortisone, the lipophilic testosterone, and the very lipophilic keto- conazole) were dosed at 1% w/w from the five formulation types mentioned earlier.

Both transdermal and dermal delivery were measured and results are discussed accordingly.

For transdermal delivery, the microemulsions were almost always superior to the other formulations tested (Fig. 1). In the table attached to Figure 1, a score of 1 means that that particular formulation type delivered the greatest amount of drug and a score of 5 means that it delivered the least amount. Please note that this is not an equidistant scale; 2 and 3 may be close together, whereas 1 and 2 may be numeri- cally far apart. The graph in Figure 1 clearly indicates that 5-fluorouracil penetration from a microemulsion is significantly higher than from any of the other formulation types tested. It must be said that considerably higher levels of surfactants were used to create this microemulsion and the microemulsions containing the other polarity drugs. It is therefore assumed that the higher transdermal penetration can be explained from the high levels of surfactant in the formulation because these are known to act as skin penetration enhancers. Transdermal delivery from these mi- croemulsions was consistently high but the dermal delivery was variable (results not shown).

Although this was not very surprising, the next finding was. When compar- ing the transdermal delivery from O/W and W/O emulsions, it was noted that this was always roughly the same with average scores ranging from 2 to 4. This suggested that transdermal delivery from an O/W emulsion is roughly the same as that from a W/O emulsion. However, when comparing the dermal delivery,

the formulation type study described earlier). This droplet size study was the first systematic investigation that used exactly the same formulation components and only differed in the way the formulations were manufactured (4).

Results from the Total Surfactant and the Free Surfactant experiments are shown in Figure 3. Dermal delivery, expressed as microgram of tetracaine per mil- ligram of skin, is plotted on the left-hand Y axis (open symbols), whereas transder- mal delivery, expressed as the percentage of the dose that penetrated skin within 24 hours, is plotted on the right-hand Y axis (closed symbols). Droplet size is plot- ted logarithmically on the X axis. Although the lines are not perfectly straight, the observed differences are far from statistically significant and it can therefore be con- cluded that there is no influence of emulsion droplet size on both transdermal and dermal delivery.

Again, as described earlier for the influence of formulation type on skin pen- etration, care should be taken not to generalize this finding. After all, we evaluated only one active ingredient (tetracaine) and one surfactant system (C12–15 Pareth-7, commercial name Synperonic A7). However, feedback has been received from other cosmetic scientists who claim to have found the same results using another emollient as the active ingredient and another surfactant system, but they never pub- lished their findings because they go very much against normally accepted rules.

r (nm)

Figure 3  Dermal (open squares) and transdermal (filled in circles) delivery of tetracaine from macroemulsion and nanoemulsion either at a constant total free surfactant concentration or a constant free surfactant concentration. Both experiments show that there is no influence of droplet size on either dermal or transdermal delivery because observed differences are not statistically significant (P > 0.05). These findings contradict the general belief that skin penetration from smaller droplet sizes is enhanced, a finding that is based on studies in which the formulations vary not only in droplet size but also in surfactant concentrations required to stabilize emulsions with smaller droplet sizes, which is probably the reason for this enhancement. The formulations used in this study only differed in the way they were mechanically processed and not in chemical composition.

The conclusion of all these studies is that there is some influence of formula- tion type but not of formulation structure on dermal and transdermal delivery. Most of the claimed differences of droplet size seem to be due to the presence of (excess) surfactant in the formulation, that is, the composition of the formulation seems to be influencing skin delivery of a given active ingredient more than anything else.

As a consequence, formulation design could very well become a trial-and-error pro- cess before one will find the best composition to optimize the skin delivery of a particular active. Therefore, a more systematic approach, based on understanding rather than on a trial-and-error method, is needed. Formulating for efficacy is such a systematic approach that was developed on a fundamental understanding of skin delivery processes. In the following, the theoretical background of formulating for efficacy will be explained as well as the use of this concept in formulation design. Clinical results will be shown for formulations that were and were not optimized by this concept to show the validity of this concept.

THEORETICAL BACKGROUND OF FORMULATING FOR EFFICACY

Both dermal and transdermal delivery aim to achieve one goal summarized in the four R’s of delivery: to deliver the right chemical at the right concentration to the right site in (dermal delivery) or beyond (transdermal delivery) the skin for the cor- rect period of time. If one of these four R’s is not achieved, there is no delivery and as a result no clinical efficacy can be obtained. The active ingredient or drug needs to be stable in the formulation as well as during its passage through the stratum corneum and viable epidermis and dermis, although in certain cases, prodrugs that penetrate the skin better and rely on skin metabolism to transform the prodrug to the parent drug that subsequently has the right intrinsic activity, are used. The right site is often achieved in topical delivery by applying the drug to the relevant surface area of the body but this site definition goes much further. One may want to target a specific cell type within the viable epidermis and the common way to achieve this is to sim- ply dose enough on the skin surface in an attempt to deliver sufficient quantities of active ingredient to the site of action. The correct length of time is seldom an issue in topical delivery; topical formulations are applied indefinitely and the only issue could be a too-short application time because of accidental removal of the formulation by the mechanical movement of clothes or our body movements. This leaves only the right concentration. Efficacy is only obtained if we deliver more than the MEC and less than the toxic concentration, two concentrations that can be obtained from in vitro experiments using the drug, cosmeceutical, or active ingredient.

Two separate issues need to be ensured if you want to deliver enough material to reach levels beyond the MEC. On the one hand, one needs to ensure that there is enough active ingredient in the formulation that the MEC can be reached. This requires a high absolute solubility of the cosmeceutical in the formulation. This can be achieved by matching the polarity of the formulation to the polarity of the drug or active ingredient. If the two are similar, the solubility of the active ingredient in the formulation will be high but its driving force for delivery will be low. One therefore also needs to optimize the driving force to ensure that the cosmeceutical will leave the formulation and penetrate into the skin in order to reach its site of action. For this, it needs to have a low solubility in the formulation relative to its solubility in the stratum corneum. The two requirements seem contradictory, but what one is really looking for is for the active ingredient to be very soluble in the formulation but even more soluble in the stratum corneum. Our work revealed that it is possible

to calculate the optimal polarity of the phase in which the active ingredient is incor- porated that will give you the maximally achievable skin delivery possible for that specific molecule (5). The formula for the polarity of the formulation from which you will achieve 50% skin penetration is:

polarity of formulation = polarity of penetrant ± penetrant polarity gap

The penetrant polarity gap is the polarity difference between the active ingredient and the stratum corneum. Polarity of penetrants, stratum corneum, and formula- tions can be calculated via polarity index (PI) values, which are a mixture of many different molecular physicochemical characteristics that influence solubility and skin penetration. The polarity of the stratum corneum was assumed to be slightly more polar than n-butanol based on literature reports (6). In formulating for efficacy terminology, the relative polarity index (RPI) is the difference between the PI values of active ingredient and the formulation component under study, whereas the pen- etrant polarity gap is the difference between the PI values of active ingredient and the stratum corneum (5). The optimal polarity of a formulation can now be realized by mixing a primary emollient with a low RPI (hence a high solubility of the active ingredient in that emollient) with a secondary emollient with a high RPI (hence a low solubility of the active ingredient in that emollient but therefore with high driving force to leave the emollient) in the right proportions to achieve the best of both worlds, that is, a high solubility and a high driving force. This is schematically illustrated in Figure 4.

Experimentally, one uses a primary emollient with a small RPI (i.e., with a polarity that is very similar to the active ingredient that one wants to formulate) to dissolve the desired amount of active ingredient (green arrow in Fig. 4) and then adds a secondary emollient with a large RPI (i.e., with a polarity that is very differ- ent from the active ingredient that one wants to formulate) to maximize the driving force for diffusion (red arrow). The resulting mixture, indicated by the blue arrow, has the optimal polarity to ensure a maximum driving force for the amount of active ingredient incorporated into the formulation.

Figure 4  A schematic overview of formulating for efficacy. The active ingredient is dissolved in the primary emollient with a low relative polarity index (RPI) in which it is very soluble (light grey arrow). Subsequently, a secondary emollient with a high RPI ensuring a high driving force for diffusion (medium grey arrow) is added. The proportions of primary and secondary emollient relative to the concentration of the active ingredient will ensure that the optimal polarity for the formulation is achieved (dark grey arrow) where skin delivery is maximal.

SKIN DELIVERY AND CLINICAL EVIDENCE VALIDATING THE FORMULATING FOR EFFICACY CONCEPT

We have used the formulating for efficacy concept to make skin delivery–optimized formulations containing octadecenedioic acid, a novel skin whitener. Formulation

A was made without using the Formulation for Efficacy concept and contained 2% active ingredient. Formulation B was made using the formulating for efficacy concept and also contained 2% octadecenedioic acid. The exact composition of the formulations is provided in Table 1. The only difference between the formulations is the choice of the emollients. Propylene glycol isostearate is used as the primary emollient in formulation B, whereas triethylhexanoin was used as the secondary emollient. Skin delivery experiments using the two formulations on full-thickness pig skin were performed and showed a statistically significant, 3.5-fold increase in dermal delivery (Fig. 5) (5).

The delivery result (Fig. 5) illustrates that the formulating for efficacy concept delivers significantly more octadecenedioic acid into the skin. The fact that this in- crease was 3.5-fold is accidental because this depends on the extent of skin delivery from the formulation that was not optimized for skin delivery. Therefore, it cannot in general be stated by what factor skin delivery will increase using the formulating for efficacy concept. But both formulations contained 2% active, yet the delivery was much more effective from the second optimized formulation.

This enhanced skin delivery suggests that the clinical efficacy should also increase. Octadecenedioic acid is a new skin-whitening molecule that interacts with the γ isoform of the peroxisome proliferator–activated receptor, PPARγ. This interaction results in reduced tyrosinase mRNA production, which subsequently results in reduced tyrosinase production (7). As tyrosinase is the rate-limiting enzyme in skin melanogenesis, its reduced production means that skin should become whiter. Clinical studies in which skin color was measured using a chro- mameter were carried out. In both studies, 20 subjects applied either formulation

A or B for a period of eight weeks twice a day. The chromameter measures the L, a, and b values that characterize the color of any object. The L scale is the luminosity

TABLE 1 Composition of the Nonoptimized (A) and Two Skin Delivery Optimized Formulations (B and C)

=non skiin delivery--optimizedformulationn

=skin delivery--optimizedformulation

Figure 8  The three possible benefits of formulating for efficacy. When one does not have sufficient delivery to reach the minimum effective concentration, use of the formulating for efficacy concept can make your formulation clinically effective (situation A). When one has insufficient skin delivery to have maximal clinical efficacy, use of the formulating for efficacy concept can enhance the clinical efficacy of your formulation (situation B). Finally, when you have a formulation with maximal clinical efficacy, use of the formulating for efficacy concept will allow you to reduce the concentration of your active in your formulation without losing clinical efficacy (situation C). Mixed forms of benefit where you reduce the concentration yet achieve more clinical efficacy are also possible.

illustrates that you can reduce the concentration without losing clinical efficacy. This was shown in the comparison between formulations B and C in Figure 7.

CONCLUDING REMARKS

In order for cosmetic products to exert clinical efficacy, the active ingredient or cos- meceutical will need to penetrate the skin to reach the site of action at a sufficiently high concentration for a sufficiently long period of time. The most important de- terminant for skin penetration is the physicochemical nature of the molecule, in particular its octanol-water partition coefficient and molecular weight and volume.

If a molecule does not have the right physicochemical properties to penetrate, nothing can be done via formulation design to enable the chemical to penetrate the skin apart from special delivery systems such as transfersomes, etc.

Assuming the chemical of interest does have the right physicochemical properties, the formulation type in which the active ingredient is incorporated does have an influence on skin delivery, but the influence depends on the polarity of the cos- meceutical. Information as provided in Figure 2 may give some guidance towards the influence of formulation type although it should be realized that more than 20 formulations are needed to conclusively state the influence of formulation type on skin delivery.

Reducing the droplet size in emulsions is often claimed to enhance the skin delivery of cosmeceuticals but we were not able to confirm this when we kept the composition of the formulations exactly the same and only changed the way in which the emulsions were mechanically processed: neither dermal nor transdermal delivery showed a dependence on droplet size. Formulation composition, on the other hand, had a pronounced influence on skin delivery.

Our research indicated that the emollients determine how much active ingre- dient penetrates into the skin, whereas the emulsifiers determine where it penetrates into the skin. The formulating for efficacy concept allows calculation of the required polarity of the formulation that will maximize the skin delivery. This should not only result in more skin delivery but also in increased efficacy of the topically ap- plied formulation as was shown for formulations containing octadecenedioic acid.

In essence, use of formulating for efficacy should allow three different scenarios:

(1) to create a clinically effective formulation from a previously noneffective formu- lation at the same dosage of active ingredient; (2) to enhance the clinical efficacy of a suboptimal delivering formulation, again at the same dosage of active ingredient; and (3) to reduce the level of the active ingredient of an already optimized formula- tion without losing any clinical efficacy. Of course, mixed possibilities such as ob- taining greater efficacy at a lower dose level of active ingredient are also possible.

Cosmetic and pharmaceutical product developers should realize that topical formulation design is no longer a matter of trial and error but that it can be opti- mized by concepts such as formulating for efficacy. This, in turn, should result in more efficacious products so that there is no longer an excuse for lack of clinical efficacy from a formulation that contains sufficient active material.

REFERENCES

1.Wiechers JW. The barrier function of the skin in relation to percutaneous absorption of drugs. Pharm Weekbl Sci Ed 1989; 11:185–198.

2.Pilgram GSK, Vissers DCJ, van der Meulen H, et al. Aberrant lipid organization in stratum corneum of patients with atopic dermatitis and lamellar ichthyosis. J Invest

Dermatol 2001; 117:710–717.

3.Potts RO, Guy RH. Predicting skin permeability. Pharm Res 1992; 9:663–669.

4.Izquierdo P, Wiechers JW, Escribano E, et al. A study on the influence of emulsion droplet size on the skin penetration of tetracaine. Skin Pharmacol Physiol, accepted for publication, 2007; 20:263–270.

5.Wiechers JW, Kelly CL, Blease TG, et al. Formulating for efficacy. Int J Cosmet Sci 2004; 26:173–182.

6.Scheuplein RJ, Blank IH. Mechanism of percutaneous absorption: IV. Penetration of nonelectrolytes (alcohols) from aqueous solutions and from pure liquids. J Invest Dermatol 1973; 60:286–296.

7.Wiechers JW, Rawlings AV, Garcia C, et al. A new mechanism of action for skin whitening agents: binding to the peroxisome proliferator-activated receptor. Int J Cosmet Sci 2005; 27:123–132.

Figure 2  Typical structure of the epidermis and critical steps in formation of the SC. Source: From Ref. 1 and 9.

Figure 3  (A) Water profile averaged over a single rectan­ gular region of a cryosection con­ tained from an individual with good skin, grade 0.5. The hori­ zontal axis is distance across the SC with the SC/granulosum junction indicated by a vertical line. (B) Water profile averaged over a single rectangular region of a cryosection obtained from an individual with dry skin, grade 4. Source: From Ref. 7.

Figure 4  High-magnification cryoscanning elec­ tron microscopy images of two sheets of stratum corneum (SC) hydrated to 90% w/w. Both sheets show an increased hydration level in their central regions and a low hydration in the superficial and lowest part of the SC. Source: From Ref. 13.

for electron microscopy methods and visualized an SC that appeared more compact with smaller intercellular spaces and, hence, tighter cell-cell interactions. More con- troversial, however, was the lack of keratohyalin granules in the epidermis. Norlen (11) has also developed novel cryotransmission electron microscopy techniques to image vitreous sections of skin without the use of cryoprotectants and, again, a more densely packed SC was apparent compared with conventional images and new organelles or tubular structures were observed in the epidermis. Norlen (12) has further proposed a cubic rod-packing model for SC keratin structures. How- ever, even with a more compacted SC, several swelling regions have been estab- lished by Bouwstra et al. (13) and Richter et al. (14) upon skin hydration that appear to be related to loss of barrier function and loss of NMF in the outer layers of the SC, hydrolysis of filaggrin to NMF and lysis of nonperipheral corneodesmosomes, allowing greater intercorneocyte freedom and transglutaminase-mediated matura- tion of corneocytes toward the surface layers of the SC (Fig. 4). All these events become aberrant in dry skin.

STRATUM CORNEUM LIPID CHEMISTRY AND BIOPHYSICS

All SC lipids are important for barrier function of the skin, but because of their unique properties and structure, the ceramides have been of most interest in re- cent years. Ceramides constitute (on a weight basis) approximately 47% of the SC lipids (15). A new nomenclature based on structure, rather than the original chro- matographic migration characteristics, was proposed by Motta et al. (16). In this system, ceramides are classified in general as CER FB, where F is the type of fatty acid and B indicates the type of base. When an ester-linked fatty acid is present, a prefix E is used. Normal fatty acids (saturated or unsaturated), α-hydroxy fatty acids, and ω-hydroxy fatty acids are N, A, and O, respectively, whereas sphingo- sines, phytosphingosines, and 6-hydroxysphingosine are indicated by S, P, and H, respectively. Sphinganine (not previously classified) is proposed to be SP in this nomenclature system. A novel long-chain ceramide containing branched-chain fatty acids is also found in vernix caseosa (17). Typical structures of human ceramides are given in Figure 5. Newly identified ceramides have also been found attached to the corneocyte envelope. In addition to ceramide A (sphingosine) and ceramide B (6-hydroxysphingosine), Chopart et al. (18) recently identified covalently bound ω-hydroxyl fatty acid containing sphinganine and phytosphingosine ceramides. These covalently bound ceramides should now be named CER OS, CER OH, CER OSP, and CER OP.

Figure 8  Organization of SC lipids in tape strippings of individuals with clinically nor­ mal skin. Transmission electron micrographs of tape strippings. Ultrastructural changes in lipid organization toward the sur­ face of the SC: (A) first strip, absence of bilayers and pres­ ence of amorphous lipidic material; (B) second strip, dis­ ruption of lipid lamellae; (C) third strip, normal lipid lamel­ lae (×200,000). Source: From Ref. 24.

CORNEODESMOSOMES AND CORNEODESMOLYSIS

Corneodesmosomes (27) are macromolecular glycoprotein complexes incorporated into the corneocyte envelope (CE) that consist of the cadherin family of transmem- brane glycoproteins, desmoglein 1 (Dsg 1), and desmocollin 1 (Dsc 1). These gly- coproteins span the cornified envelope into the lipid enriched intercellular spaces between the corneocytes and provide cohesion by binding homeophilically with proteins on adjacent cells. Within the corneocytes, Dsg 1 and Dsc 1 are linked to keratin filaments via corneodesmosomal plaque proteins such as plakoglobin, desmoplakins and plakophilins. The corneodesmosomal protein, corneodesmosin (Cdsn), after secretion by the lamellar bodies together with the intercellular lipids and certain proteases, becomes associated with the desmosomal proteins just before transformation of desmosomes into corneodesmosomes. - these proteins are crosslinked into the complex by transglutaminase, their controlled disruption must oc- cur by proteolysis to allow desquamation to proceed. Indeed, Rawlings et al. (24) and Fig. 9 demonstrated degradation of the corneodesmosomes toward the surface of the SC in humans.

Corneodesmolysis, and ultimately desquamation, is facilitated by the action of specific hydrolytic enzymes in the SC that degrade the corneodesmosomal link- ages. Currently, several serine, cysteine, and aspartic enzymes are believed to be involved in this process, namely, SC chymotryptic enzyme (SCCE), SC tryptic en- zymes (SCTE), SC thiol protease (SCTP now known as cathepsin L-2), cathepsin E, and the aspartic protease cathepsin D. SCCE and SCTE are alkaline-optimal en-

Figure 9  Electron micrographs of tape strippings of normal skin (grade 1). Degradation of corneo­ desmosomes (CD) toward the sur­ face of the SC: (A) First strip, CD fully degraded; (B) second strip; CD partially degraded and encap­ sulated by lipid lamellae; (C) third strip; CD partially degraded, vacu­ lation of structure. (D) Third strip, normal CD in contact with lamellar lipids. Source: From Ref. 24.

zymes, whereas the latter ones are acidic-optimum enzymes (28–32). Cathepsin L has also recently been implicated in Cdsn hydrolysis (33). Only SCTE and not SCCE, however, was capable of degrading Dsg 1 (34). This enzyme was also reported to be involved in the processing of pro-SCCE. Bernard et al. (35) have also identified an endoglycosidase, heparanase 1, within the SC, thought to play a role in the preproteolytic processing of the protecting sugar moieties on corneodesmosomal pro- teins. Cdsn undergoes several proteolytic steps. Cleavage of the N-terminal glycine loop domain occurs first at the compactum-disjunctum interface (48–46- to 36–30-kDa transition), followed by cleavage of the C-terminal glycine loop domain in exfoliated corneocytes (36–30- to 15-kDa transition) (36). The last step appears to be inhibited by calcium resulting in residual intercorneocyte cohesion. Nevertheless, the presence of oligosaccharides did not protect Cdsn against proteolysis by SCCE (34). A complete list of the putative desquamatory enzymes is given in Table 1.

These enzymes are secreted with the lamellar bodies and have been immu- nolocalized to the intercorneocyte lipid lamellae. Sondell et al. (37) used antibodies that immunoreact precisely with pro-SCCE to confirm that this enzyme was trans- ported to the SC extracellular space via lamellar bodies. In later studies, using an- tibodies to both pro-SCCE and SCCE, Watkinson et al. (38) demonstrated that the processed enzyme was more associated with the corneodesmosomal plaque. More recently, Igarashi et al. (39) have immunolocalized cathepsin D to the intercellular space, whereas cathepsin E was localized within the corneocytes. Finally, KLK8 has also been reported to be localized to the intercellular spaces of the SC (40).

Naturally, as the desquamatory enzymes are present in the intercellular space, the physical properties of the SC lipids, together with the water activity in this mi- croenvironment, will influence their activity. However, SCCE appears to have a