CZ20003302A3 - Protease inhibitor absorbent composition and method of use - Google Patents

Abstract

The invention describes an absorbent article, at least one portion of which comprises a protease inhibitor that reduces the activity of fecal proteases that could otherwise cause or contribute to inflammation of the skin of the wearer of the article, resulting in diaper rash or dermatitis. The article further comprises a delivery system that comprises and delivers the protease inhibitor to at least a portion of the wearer's skin. More preferably, the delivery system comprises a dermatological component, a portion of which, including the protease inhibitor, is automatically delivered from the article to the wearer's skin during normal use of the article, thereby providing protection against fecal proteases at the skin-stool interface. Repeated application of similarly formulated articles to the wearer's skin provides an available source from which the protease inhibitor can be continuously delivered to the skin and provide active protection against fecal proteases in the treatment and/or prevention of diaper dermatitis caused by proteolytic enzymes.

Description

Absorbent composition containing a protease inhibitor and method of use thereof

Technical field

The invention relates to absorbent articles such as diapers, training pants, incontinence pads, feminine hygiene products, etc. These absorbent articles contain fecal protease inhibitors and are useful in the prevention and treatment of diaper rash.

State of the art

Diaper rash is a common form of irritation and inflammation of the areas of a child's body that are normally covered by a diaper. This condition is also called diaper dermatitis. Although it is certainly most common in children, it is not unique to them. Anyone who suffers from incontinence to the extent that they require the use of absorbent materials can develop this condition. Susceptible individuals include newborns, the elderly, the seriously ill, or those who are bedridden.

There are many types of disposable absorbent articles available, such as diapers, training pants, incontinence products, sanitary napkins, etc., which have a high absorbent capacity for urine and other bodily exudates. Disposable articles of this type typically comprise some type of liquid-permeable topsheet, an absorbent core, and a liquid-impermeable backsheet. Although these types of absorbent structures can be highly effective in absorbing fluids, it is known that prolonged wear of such absorbent articles can damage the skin by hyperhydration or by exposing the skin to irritants commonly found in bodily exudates. Part 21, Chapter 333 503 C.F.R. defines diaper rash as “an inflammatory skin condition of the diaper area (perineum, buttocks, lower abdomen, and inner thighs) caused by one or more of the following factors: moisture, occlusion, friction, prolonged contact with urine or feces or both, or mechanical or chemical irritation.” Doctors generally consider diaper rash or dermatitis to be a condition that is, in its mildest stage, an irritant contact dermatitis resulting from extensive skin contact with urine or feces or both. Among the factors most commonly cited in connection with diaper rash are ammonia, fecal enzymes, bacteria, bacterial products, urine pH, and Candida albicans.

There is evidence that proteolytic and lipolytic enzymes from feces, which originate from the intestine and/or pancreas, play a key role in the skin irritation and inflammation of diaper dermatitis, see U.S. Patent 4,556,560, U.S. Patent 4,657,537, U.S. Patent 4,685,909, U.S. Patent 4,842,593, Andersen et al. (Contact Dermatitis 30:152158, 1994), Mac Farlane et al. (J. 4,842,593), Andersen et al. (Contact Dermatitis 30:152-158, 1994), MacFarlane et al. (J. Appl. Bacteriol. 64:37-46, 1988) and Buckingham and Berg, (Pediatrics

Dermatology 3:107-112, 1986). Studies with various inhibitors designed to inhibit the enzymatic activity of different classes of proteases have shown that serine proteases, cysteine proteases, and metalloproteases are most commonly responsible for the overall proteolytic activity of feces. It is known that serine proteases,

<· · · · · · · · · · · · · · · · · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · «· primarily trypsin and chymotrypsin, are almost always present in large quantities in the stools of healthy young children, and smaller, but detectable, amounts are present in the stools of healthy adults.

The irritant effects of faecal enzymatic activity on the skin are usually enhanced when urine is present and/or the skin is covered. The formation of ammonium hydroxide by the action of the bacterial enzyme urease on the urine results in an increase in pH, e.g. to values of 7.0 and above, at which the activity of proteases and other enzymes such as lipases present in the stool increases. For example, the optimal pH range for urease is 6.4-6.9, for trypsin 7.8-8.2 and for lipases 7.5-9.5. If the pH exceeds 7.0, free ammonia begins to be released from the urine, which is another irritating toxic compound acting on the skin. Urine itself can also contribute to the development of diaper rash by moistening the environment. Water, especially water in the form of urine, is particularly effective in reducing the skin barrier, thereby increasing the susceptibility of the skin to irritation by faecal enzymes. Since urine and feces are commonly present in the absorbent material at the same time, and skin exposure for several hours is not uncommon, there is ample time and suitable conditions for these interactions to occur and subsequent skin damage. Another factor that contributes to the enhancement of enzymatic activity of feces is its alkaline pH. For example, it is well known that although the feces of breastfed infants are usually acidic, the feces of formula-fed infants are usually alkaline, with a pH ranging from slightly alkaline (pH

7.2 - 7.5) to strongly alkaline (pH 8.7 and higher). So, especially formula-fed babies are prone to developing diaper dermatitis caused by increased enzyme activity in the stool due to higher pH.

• ·

In view of these established causes of diaper dermatitis, a number of attempts have been made to reduce or prevent the occurrence of diaper dermatitis. Many of these best practices attempt to address multiple causes or important cofactors. Most commonly, these include reducing skin hydration by frequent diaper changes, the use of moisture-absorbing powders, the use of superabsorbents, and improvements in

airflow in diapers. The use of artificial barriers is also common. It is common to apply a topical cream, ointment, solution, or paste that creates some degree of physical barrier to protect the skin from irritants from stool or urine. However, such a barrier approach, while reducing the access of irritants to the skin, may be aesthetically undesirable.

In view of the effect of alkaline pH on increased enzyme activity in feces, several attempts have been made to maintain skin pH by using pH-adjusting agents, such as buffering agents or acidic ammonia-neutralizing agents, in absorbent compositions or as additives in topical dermatological compositions. It is believed that effectively maintaining skin pH at its natural low pH (i.e., 3.0 to 5.5) may counteract the irritant effect of ammonia and reduce the activity of fecal enzymes. Reducing skin enzyme activity in this way, however, is quite difficult in situations where alkaline feces are directly on the skin after bowel movements.

Certain antienzyme components have been added to topically applied preparations for the treatment and prevention of diaper rash caused by prolonged contact of human skin with body secretions.

• ·

For example, U.S. Patent No. 4,556,560 describes compositions comprising water-soluble lipase inhibitors, preferably metal salts such as zinc chloride, in a barrier carrier such as polyethylene glycol. U.S. Patent No. 5,091,193 describes compositions for application to the skin during diaper changes which contain a chelating agent such as folic acid, ethylenediaminetetraacetic acid (EDTA) and the like, which limit the availability of metals which urease and proteases require as cofactors for activity. The composition may further comprise a lipase inhibitor such as a fatty alcohol ester or an added antienzyme, saturated or unsaturated, linear or branched, zinc salt of a fatty acid having 2 to 22 carbon atoms or an aminoacyl acid such as propionylcysteine, propionylhydroxyproline or caproylcysteine. Cleansing wipes for removing body secretions from the skin that contain protease inhibitors have also been described.

Although many factors are involved in the development of diaper dermatitis, it seems likely that there are some common mechanisms underlying the physiological response of the skin to irritants such as fecal enzymes, ammonia, etc. For example, it is known that the production of cytokines by skin cells is a common response to the presence of irritants and disruption of the outer layer of the skin (stratum corneum). The main cell type involved in the production of cytokines is the keratinocyte, which is located directly beneath the stratum corneum and is most likely to encounter the irritant first. It has been shown that the keratinocyte produces a wide variety of different cytokines, including the pro-inflammatory cytokine interleukin 1-alpha (IL-10), in response to irritants. This cytokine and other cytokines induce a cascade of events that can ultimately lead to the development of redness, pimples, scaling, and ulcers, collectively described as diaper dermatitis.

Compositions for the treatment or prevention of diaper rash have been described which contain certain urease, lipase and/or protease inhibitors, but it was not previously known that fecal proteases play an important role in inducing the initial cytokine response of keratinocytes, which leads to a cascade of inflammatory responses and protease inhibition, and therefore it is now possible to more specifically prevent and treat diaper dermatitis. No treatment method has previously been described which would reduce or prevent the occurrence of diaper dermatitis, in which protease inhibitors would be directly incorporated into an absorbent article, e.g. diapers, or the like, or where effective amounts of the inhibitors would be automatically transferred to the skin of the user of the modified article, without the aid of hands. Furthermore, it has not been described that use, especially repeated use, of the modified absorbent article could automatically transfer sufficient amounts of protease inhibitors to selected areas of the user's skin, thereby providing proactive protection against protease penetration and activity.

The essence of the invention

The invention describes an absorbent article, at least a portion of which contains a protease inhibitor that reduces the activity of fecal proteases that would otherwise trigger or contribute to inflammation of the skin of the wearer of the article with the subsequent development of diaper dermatitis. As used herein, the term protease inhibitor refers to any substance that inhibits the activity of proteases in one or more of the seven assays described below with (i) an IC50 _of 30 μM or less, typically from 0.00001 μM to 30 μM, more preferably from 0.0001 μM to 20 μM, more preferably from 0.001 μM to 10 μM, and most preferably from 0.01 μM to 5 μM, when measured by the purified protease method described below, with (ii) an IC50 _of 90 μM or less, typically from 0.00001 μM to 90 μM, more preferably from 0.0001 μM to 30 μM, more preferably from 0.001 μM to 10 μM, and most preferably from 0.01 μM to 5 μM, when measured by the specific fecal protease method described below or (iii) an IC50 of less than 500 μΜ, more preferably less than 300 μΜ and most preferably less than 100 μΜ, when measured by the general fecal protease method described below.

The protease inhibitor included in the composition inactivates one or more of the major types of proteases in feces, i.e., serine proteases, metalloproteases, cysteine proteases, and aspartyl proteases. Although any protease inhibitor or mixture thereof having the desired IC ₅₀ as set forth above may be used in the absorbent composition, it has been found that inhibition of fecal serine proteases with a serine protease inhibitor, such as soybean trypsin inhibitor and hexamidine, significantly reduces the initial cytokine response of skin cells to feces. Suitable protease inhibitors for use in the absorbent compositions of the present invention include soybean trypsin inhibitor and hexamidine, as well as aprotinin, p-aminobenzamidine, leupeptin, pepstatin A, chymostatin, and the like.

The composition should contain from 0.0001% to 30%, more preferably from 0.0001% to 10% by weight of the protease inhibitor. The inhibitor may be present in pure form, such as a powder, flakes, granules, etc., or may be in a carrier vehicle, such as a solution, suspension, dispersion, emulsion, etc. In addition, the inhibitor may be contained in free form in microcapsules, an absorbent, a chamber, an adhesive, a dermatological component, a solid support, a nanophase structure, etc. The inhibitor in the absorbent composition reduces protease activity, as measured by the absorbent composition test method (described below), by at least 10%, more preferably by at least 20%, more preferably by at least 50%, and most preferably by at least 80%. The inhibitor in the absorbent reduces protease activity by 10% to 99%, more preferably by 20% to 99%, even more preferably by 50% to 99%, and most preferably by 80% to 99%.

The absorbent device further comprises a delivery system for delivering the protease inhibitor to at least a portion of the skin of the wearer of the device. The delivery system may have, among others, any of the following configurations, e.g., a configuration in which the protease inhibitor is in powder, particulate, or flake form, or in a solution, suspension, emulsion, or the like. The delivery system may comprise a structure such as a microcapsule, an absorbent, a nanophase structure, a chamber, an adhesive, a solid support, or the like, or a component such as a dermatological component. The delivery system delivers, during wear of the device, the protease inhibitor to the vicinity of the skin, and preferably to at least a portion of the skin of the wearer of the device, such that the inhibitor can arrest proteases in the stool at the skin/stool interface before they can penetrate the stratum corneum of the skin, thereby reducing or preventing the activation of an inflammatory response.

In a preferred embodiment of the invention, the delivery system comprises a dermatological component comprising from 0.01% to 50%, more preferably from 0.05% to 25%, and most preferably from 0.1% to 10% by weight of the protease inhibitor. At least a portion of the surface of the absorbent article that contacts the user should comprise the dermatological component with the enzyme inhibitor, such that a portion of the dermatological component with • · λ · · · ····

Μ · ··· ····· • · · · · · « · ♦ ·· ·· ··· ··· ··· «· inhibitor is automatically transferred, during normal wear, from the device to the user's skin, where it then provides protection against fecal proteases. Repeated application of similarly formulated devices to the user's skin creates an available source from which the protease inhibitor can continuously transfer to the skin, where it accumulates and provides proactive protection against fecal proteases, which reduces or prevents the development of diaper dermatitis caused by proteolytic enzymes.

The advantage of an absorbent article containing a protease inhibitor is that the inhibition of fecal proteases and thus the reduction of skin irritation caused by contact with feces is a direct consequence of the inhibitor-enzyme interaction, rather than an indirect effect such as a change in pH, inactivation of a cofactor necessary for enzyme activity, or other dermatological agents. This method of treating and/or preventing diaper dermatitis is based on the judicious selection of inhibitors that inactivate the main types of proteases present in feces. In addition, the inhibitor-enzyme interaction occurs at high pH values, which are common in soiled diapers and other absorbent articles.

Overview of images in drawings

Figure 1 is a schematic representation of an absorbent article, in the form of a diaper, according to the present invention.

Figure 2 is a side view showing the placement of a skin analog used in a dermatological ingredient transfer test and/or a protease inhibitor transfer test.

♦ · · · · 9 9

9 · ······ • 9 9 9 9 9 9

9999999 99 99

Figure 3 is a plan view showing the placement of a skin analog used in a dermatological ingredient transfer test and/or a protease inhibitor transfer test.

Examples of embodiments of the invention

I. Definition

The term "comprising", as used herein, means that various components, ingredients or steps may be used together in the practice of the present invention. Accordingly, the term "comprising" also includes the narrower terms "consisting primarily of" and "consisting of".

The term "IC ₅₀ " refers to the inhibitory concentration (e.g., micromolar concentration, μΜ) of a substance (inhibitor) that will reduce the level of substrate cleavage by the enzyme by 50%, when measuring enzyme activity using standard in vitro assays, see below. IC ₅₀ is calculated according to the equation IC ₅₀ = (I)/((v/vj)-l), where (I) is the inhibitory concentration tested, v is the level of substrate cleavage in the absence of the inhibitor, and V; is the level of substrate cleavage in the presence of the inhibitor. As will be described below, the IC50 of a protease inhibitor can be measured using a purified enzyme method, a specific fecal protease method, or a general fecal protease method.

Other terms are defined here on their first use.

All percentages, coefficients and ratios used herein are weighted unless otherwise stated.

II. Invention ez • ·« · ·· 99 99

The invention provides an absorbent composition comprising a protease inhibitor that inhibits one or more proteases present in stool and meets the requirement for an IC ₅₀ against trypsin, chymotrypsin and/or leucine aminopeptidase in the purified protease assay, the stool specific protease assay or the stool general protease assay, as described below. The invention provides an absorbent composition comprising a protease inhibitor having an IC ₅₀ of 30 μΜ or less, typically 0.00001 μΜ to 30 μΜ, more preferably 0.0001 μΜ to 20 μΜ, more preferably 0.001 μΜ to 10 μΜ, and most preferably 0.01 μΜ to 5 μΜ, as measured by the Purified Protease Method. The invention separately provides an absorbent composition comprising a protease inhibitor having an IC50 _of 90 μM or less, typically 0.00001 μM to 90 μM, preferably 0.0001 μM to 30 μM, even more preferably 0.001 μM to 10 μM and most preferably 0.01 μM to 5 μM, as measured by the stool specific protease method. The invention further provides an absorbent composition comprising a protease inhibitor or a mixture of protease inhibitors having an IC50 of less than 500 μM, preferably less than 300 μM and most preferably less than 100 μM, as measured by the stool general protease method. In the context of the present invention, the term adapted composition refers to an absorbent composition comprising a protease inhibitor. In one embodiment of the present invention, the protease inhibitor is readily available, or is capable of being carried, in a portion of the absorbent article that may come into contact with stool, particularly watery stool, so as to directly inhibit the activity of proteases. As will be discussed below, the inhibitor may be in an active form or may be inactive but activatable, e.g., by an external source such as moisture from urine or stool.

·«··

In another embodiment of the invention, the absorbent article comprises a delivery system comprising a protease inhibitor that automatically delivers an effective amount of the inhibitor to at least a portion of the wearer's skin during wear of the article. Upon use or repeated use of similar articles having a delivery system comprising a protease inhibitor, a sufficient amount of the protease inhibitor is automatically delivered to selected areas of the wearer's skin prior to contact with feces, thereby providing proactive protection against penetration and activity of fecal proteases.

It is believed that fecal proteases cleave proteins present in the stratum corneum (outer barrier layer) of the skin, resulting in cytokine production by keratinocytes and activation of a cascade of inflammatory responses that produces the symptoms of diaper rash. As demonstrated herein, the protease inhibitor agents used in the absorbent articles of the present invention have the surprising ability to inhibit the induction of cytokine production by keratinocytes in the presence of feces, which is due to a direct inhibitor-enzyme interaction. It is believed that the presence of a protease inhibitor that meets the IC ₅₀ requirement described above, which is in contact with feces within the article or at the skin/feces interface, reduces or prevents the initial attack of the fecal protease on the stratum corneum. Therefore, an absorbent article containing a protease inhibitor or a delivery system that delivers an effective amount of the protease inhibitor directly to the skin is useful in the treatment and/or prevention of diaper dermatitis.

It is well known that one of the most important functions of the skin is to act as a barrier against the leakage of physiological fluids, electrolytes and other components, as well as to act as a barrier against the penetration of

4444 • · 4 444 · « 4 4 4

4 4 4 4 4 4 ·

B· 44 4 · · 4 4 · 4

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444 44 444 ···· 44 44 microbes, toxins and other inflammatory or harmful substances. In light of the finding that faecal proteases contribute significantly to the development of diaper dermatitis, it is likely that in addition to skin irritation by digesting the stratum corneum, damage to the barrier allows other components of urine and faeces, ammonia, fatty acids, etc., which would not themselves be irritants, to penetrate, thereby causing further irritation. Candida albicans, which produces aspartic protease, is, for example, often found as a major component of the stool of people treated with antibiotics. This yeast thrives in the moist environment of dirty diapers and, if the skin is broken, aspartic protease can contribute to further skin damage or even the development of a serious candidal infection. Therefore, the addition of protease inhibitors in absorbent products helps maintain the integrity of the stratum corneum barrier and effectively prevents the occurrence of secondary irritation and/or infection that contributes to diaper dermatitis.

III. Protease inhibitors

Protease is a common term that represents a group of proteolytic enzymes that are capable of splitting proteins and peptides into fragments by cleavage or hydrolysis of peptide bonds. Proteases can be further divided into proteinases (endopeptidases) and peptidases (exopeptidases). Peptidases act on a peptide bond near a free amino or carboxy group at the end of a protein, thereby cleaving the protein. The main types of peptidases are carboxypeptidases, dipeptidases, and aminopeptidases. Proteinases act on specific internal peptide bonds of proteins and can be divided into four groups, namely serine proteases, metalloproteases, cysteine proteases, and aspartic proteases. The main proteinases include trypsin and chymotrypsin. Since proteases are widely present in plants, • 444 ·· 44 » 4 · « » » · 4 » · · 4 » · · 4 • 4 »4 soil, bacteria, milk, dairy products and almost all animal tissues, as well as the digestive juices of the gastrointestinal tract, they are almost always present in the area of a diaper that has been soiled with body exudates. Each of the protease inhibitors present in the absorbent articles of the invention is a chemical that meets at least one of the seven criteria for IC ₅ o described above and reversibly or irreversibly inhibits the hydrolytic action of one or more proteases represented in the preceding subclasses of proteases commonly found in human feces, as well as proteases whose substrate specificity has not yet been determined.

Protease inhibitors that can be used in embodiments of the present invention include all naturally occurring inhibitors of plant, microbial and/or animal (including human) origin and synthetically prepared chemical inhibitors that meet the IC50 criteria described above. Examples of protease inhibitors that meet the IC50 criteria and inhibit the type of protease indicated in parentheses include, but are not limited to, soybean trypsin inhibitor and other plant-derived trypsin inhibitors, such as lima bean (Phaseolus limensis) protease inhibitor, corn protease inhibitor, and the like, Bowman-Birk inhibitor (serine and trypsin-like protease inhibitor), pancreatic trypsin inhibitor, such as bovine pancreatic alkaline trypsin inhibitor and other animal-derived pancreatic trypsin inhibitors, egg white trypsin inhibitor (serine and trypsin-like protease inhibitor), ovomucoids containing ovoinhibitors, such as those from chicken or turkey egg white (trypsin and chymotrypsin inhibitors), chymostatin (serine and chymotrypsin-like protease inhibitor), aprotinin (serine protease inhibitor); leupeptin and its analogues, such as propionyl-leupeptin, Ν-α-t-BOC-deacetylleupeptin (inhibitor • ···· · ·· *· *·

4 · · ·4 4 · * 9 · · • 4 4 4 4 4 4 4 ΐς 4 4 4 · 4 4 4 4 4 4

J 4 4 4 4 4 4 4 4 4 4 4

444 44 444 9444 44 44 serine and cysteine proteases), bestatin and its analogues, such as epibestatin and nitrobestatin (aminopeptidase and metalloprotease inhibitors), amastatin and its analogues, such as epiamastatin (aminopeptidase inhibitor), antipain (trypsin inhibitor), antithrombin III (serine protease inhibitor), hirudin (thrombin-like serine protease inhibitor), cystatin (egg white cysteine protease inhibitor), E-64 (trans-epoxysuccinyl- _L leucylamido-(4-guanidino)-butane) and its analogues (cysteine protease inhibitor), and ₂ -macroglobulin (universal endoprotease inhibitor), αι-antitrypsin (trypsin inhibitor), pepstatin and its analogues, such as acetylpepstatin, pepstatin A, Nle-Sta-Ala-Sta (aspartyl protease inhibitor), apstatin (aminopeptidase P inhibitor), (2R)-2-mercaptomethyl-4-methylpentanoyl-b-(2-naphthyl)-Ala-Ala amide (matrix metalloprotease inhibitor), (2R)-2-mercaptomethyl-4methyl-pentanoyl-Phe-Ala amide (matrix metalloprotease inhibitor), N-acetyl-Leu-Leu-methioninal (calpain inhibitor), N-acetyl-LeuLeu-norleucinal (calpain inhibitor), p-aminobenzoyl-Gly-Pro- _D Leu-o-Ala hydroxamic acid (matrix metalloprotease inhibitor); 2(R)-(N-(4-methoxyphenylsulfonyl)-N-(3-pyridylmethyl)amino)-3methyl-butano-hydroxamic acid (metalloprotease inhibitor), 4(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (broad spectrum inhibitor/general protease inhibitor) and mixtures of the above.

For use in the absorbent articles of the present invention, we prefer protease inhibitors that have an inhibitory effect that is not limited to one class of proteases. Such compounds include, but are not limited to, hexamidine and its salts, pentamidine and its salts, benzamidine and its salts and derivatives, p-aminobenzamidine and its salts and derivatives, and guanidinebenzoic acid and its salts and derivatives, such as those described in U.S. Patent 5,376,655 to Imaki.

et al., Dec. 27, 1994, which is incorporated herein by reference. Other proposed protease inhibitors are polymeric derivatives of guanidine benzoic acid, described in copending U.S. Patent Application Serial No. 09/041,196 (T. L. Underiner et al.), which was filed concurrently with this application.

Protease inhibitors can be used in the absorbent article alone or in mixtures, such as inhibitor cocktails. Furthermore, different protease inhibitors can be used in different parts of the absorbent article.

Due to the wide variety of enzymes present in feces, it is expected that agents such as those described above that inhibit fecal proteases will also inhibit enzymes that cleave substrates other than proteins and peptides. Therefore, protease inhibitors that inhibit lipases and other esterases, amylases, and/or ureases are also within the scope of the present invention, provided that these inhibitors meet the IC 50 criteria described above.

Protease inhibitors preferred in this invention are soybean trypsin inhibitor, Bowman-Birk inhibitor, aprotinin, hexamidine (e.g., hexamidine diisethionate), p-aminobenzamidine, leupeptin, pepstatin A, chymostatin, and polymeric derivatives of guanidinebenzoic acid (described in U.S. application Ser. No. 09/041,196).

The most suitable protease inhibitors are soybean trypsin inhibitor, hexamidine, p-aminobenzamidine and polymeric derivatives of guanidinebenzoic acid.

IV. Absorbent materials • 4 • 4

4

The term “absorbent article” is used herein to refer to a product that absorbs and contains bodily exudates. The term “disposable” is used herein to describe an absorbent article that is not intended to be washed or otherwise reprocessed or reused. Examples of disposable absorbent articles include feminine hygiene products such as sanitary napkins, sanitary pads and tampons, diapers, incontinence pads and pads, exercise pants, etc.

Protease inhibitors may be included in any portion or portions of any of the absorbent devices described herein. Protease inhibitor delivery systems are part of the absorbent devices and will be described separately.

A typical disposable absorbent article consists of a top layer that is liquid-permeable, a bottom layer that is impermeable, and an absorbent core disposed between the top and bottom layers. Disposable absorbent articles and their components, including the top layer, the bottom layer, the absorbent core, and any individual layers of these components, have a body-facing surface and a garment-facing surface. As used herein, the term "body-facing surface" means that this surface of the article or a portion thereof is intended to be worn toward the wearer's body, while the "garment-facing surface" is on the opposite side and should be worn toward the garment.

The following section generally describes absorbent core, topsheet, and backsheet materials that are suitable for disposable absorbent articles. This general description applies to portions of the specific absorbent article shown in Figure 1 and ^described below, in addition to other absorbent articles that are generally described herein.

The absorbent core is typically capable of absorbing or retaining fluids (e.g., blood, urine, and/or other body exudates). The absorbent core is typically compressible, conformable, and non-irritating to the wearer's skin. The absorbent core can be manufactured in a variety of sizes and shapes (e.g., rectangular, oval, hourglass, "T" shape, dog bone, asymmetrical, etc.). In addition, the absorbent core may also contain, in addition to the absorbent components, any of a wide variety of fluid-absorbent materials, such as cellulose. Examples of other suitable absorbent materials used for the absorbent core are cellulose wadding, blended polymers such as coform, chemically strengthened, modified or cross-linked cellulose fibers, synthetic fibers such as corrugated polyester fibers, peat, laminated paper, absorbent foams, absorbent sponge materials, superabsorbent polymers including composites, absorbent gelling materials or any similar material, combination of materials or mixtures thereof.

The arrangement and construction of the absorbent core may vary (e.g. the absorbent core may have different straddle zones and/or a profile that is thicker in the center, hydrophilic gradients, absorbent component gradients, superabsorbent gradients or areas of lower average density or lower average base weight, e.g. acquisition areas, or may be composed of one or more layers or structures). However, the overall absorbent capacity of the absorbent core should be appropriate for the proposed load and intended use of the absorbent article. Furthermore, the size and absorbent capacity of the absorbent core must be adequate for different uses, such as « · · · · ·

diapers, incontinence pads, menstrual pads, sanitary napkins and must be adapted to a range of users from children to adults. The absorbent core may also contain other absorbent components that are often used in absorbent articles, e.g. a backer layer, an wicking layer to retain urine or an additional top layer to increase the user's comfort.

The topsheet is typically pliable, soft, and non-irritating to the wearer's skin. Furthermore, the topsheet is liquid permeable, allowing liquids (e.g., blood and/or urine) to pass through it easily. Suitable topsheets can be made from a variety of materials, such as fleece and nonwoven materials (e.g., nonwoven webs of fibers), including nonwoven materials with apertures; polymers such as porous thermoplastic film, porous plastic film, and hydroformable thermoplastic films; porous foams; reticulated foams; reticulated thermoplastic films, and thermoplastic scrims. Suitable fleece and nonwoven materials include natural fibers (e.g., wood or cotton fibers), synthetic fibers (e.g., polymer fibers such as polyester, polypropylene, or polyethylene fibers), or materials formed by a combination of natural and synthetic fibers. If the topsheet is made from a nonwoven material, the material can be processed by a variety of known techniques. The net may be made, for example, by spinning, carding, hot-blown, wet-knitting, wet-forming, a combination of the above or the like. The top layer should contain a dermatological component with an enzyme inhibitor, regardless of whether it is made of wool or non-wool material, see below.

The backsheet is impermeable to liquids (e.g., blood and/or urine) and typically comprises a thin plastic layer, although other impermeable flexible materials may be used. The term "flexible" is used to refer to materials that are pliable and readily conform to the shape and contours of the human body. The backsheet prevents exudates absorbed and retained in the absorbent core from wetting articles in contact with the absorbent, such as sheets, underpants, pajamas, and undergarments. The backsheet may therefore comprise a fleece or nonwoven material, a polymer coating, such as a thermoplastic layer of polyethylene or polypropylene, or composite materials, such as a coated nonwoven material. A suitable backsheet is a polyethylene film having a thickness of from 0.012 mm to about 0.051 mm. Suitable polyethylene films are manufactured by Clopay Corporation of Cincinnati, Ohio, under the designation P18-1401, and by Tredegar Film Products of Terre Haute, Indiana, under the designation XP-39385. The backsheet is typically embossed and/or matted to give a textile appearance. Furthermore, the backsheet should be permeable to vapors from the absorbent core (i.e., the backsheet is breathable) while being impermeable. The size of the backsheet is determined by the size of the absorbent core and its chosen shape.

The backsheet and the topsheet are positioned, respectively, adjacent the garment and body-facing surfaces of the absorbent core. The absorbent core is typically attached to the topsheet, the backsheet, or both layers by one of the fastening elements (not shown in Figure 1) commonly used in the art. However, embodiments of the absorbent device may be apparent if parts or all of the absorbent core is not attached to either the topsheet or the backsheet, or both layers.

• · • · · ·

The backsheet and/or topsheet may be attached to the absorbent core or to each other by a continuous layer of adhesive, a patterned layer of adhesive, or a series of individual lines, spirals, or dots of adhesive. Suitable adhesives are manufactured by H.B. Fuller Company of St. Paul, Minnesota under the designation HL-1258 or H2031. The bonding means preferably comprises a network of adhesive fibers as described in U.S. Patent 4,573,986, issued to Minetola et al. on March 4, 1986, which is incorporated herein by reference. An example of a bonding method is a network of fibers formed by multiple adhesive lines coiled into a spiral, as described in U.S. Patent 3,911,173, issued to Sprague, Jr. on October 7, 1975, and U.S. Patent 4,785,996, issued to Zwieker et al. 22.

November 1978 and U.S. Patent 4,482,666 issued to Werenicz on June 27, 1989. Each of these patents is incorporated herein by reference. Thermal bonds, pressure bonds, ultrasonic bonds, dynamic mechanical bonds, or other suitable bonding means or combinations thereof known in the art may also be used as the bonding means.

The proposed disposable absorbent article is a diaper, which contains at least in one part a protease inhibitor and/or a delivery system with a protease inhibitor and its surface contacting the user is treated with a dermatological composition containing an enzyme inhibitor. The term diaper as used herein means an absorbent article which is generally used by children and incontinent persons and which is worn around the lower torso. In other words, the term diaper includes baby diapers, training pants, adult incontinence articles, etc.

Figure 1 is a diagram of a diaper 50, which is shown in an expanded state (i.e., not pulled down by elastic portions) and without certain parts so that its structure is clearly visible and is turned with its outer surface towards the viewer. The diaper 50 of Figure 1 is typically composed of a permeable topsheet 520, an impermeable backsheet 530 connected to the topsheet 520, an absorbent core 540 having a garment facing surface 542, a body facing surface 544, a side edge 546, waist edges 548 and tabs 549. The diaper 50 typically further includes elastic thigh cuffs 550 and a variously shaped elastic waist portion 560 and, typically, variously shaped fastening means 570.

The diaper 50 shown in Figure 1 has an outer surface 52, an inner surface 54, which corresponds to the body-facing surface opposite the outer surface 52, a first waist region 56, a second waist region 58, and a circumference 51, which is defined by the outer edges of the diaper 50, where the longitudinal edges are designated 55 and the end edges 57. (It is common in the art to divide the diaper into two waist regions and a crotch region located therebetween, but in this application, for simplicity, only the division of the diaper 50 into waist regions containing the original crotch region is used.) The body-facing surface 54 of the diaper 50 is that portion of the diaper 50 that is adjacent to the body of the wearer during wear. The body-facing surface 54 is typically formed by at least a portion of the topsheet 520 and other portions that may be attached to the topsheet 520, such as the thigh cuffs 550, as well as areas that are not part of the topsheet but that contact the wearer, such as the waist portion 560, side portions, and the like. The outer surface 52 includes the portion of the diaper 50 that faces away from the wearer's body (i.e., the outer surface 52 is typically formed by at least a portion of the backsheet 530 and other portions that may be attached thereto). The first waist region 56 and the second waist region 58 extend between the end edges 57 of the circumference 51 and the transverse centerline 53 • 99 9 of the diaper 50. A longitudinal centerline 59 is also shown in FIG. 1 .

Figure 1 shows a proposed embodiment of a diaper 50 in which the topsheet 520 and the backsheet 530 have a length and width greater than the dimensions of the absorbent core 540. The elastic thigh cuffs 550 and the backsheet 530 extend beyond the edges of the absorbent core 540, thereby forming a perimeter 51 of the diaper 50.

The diapers of the present invention can be made in a number of known embodiments in which the absorbent cores are adapted to the present invention. Examples of embodiments are generally described in U.S. Patent 3,860,003 (Buell) filed January 14, 1975, U.S. Patent 5,151,092 (Buell et al.) filed September 29, 1992, and U.S. Patent 5,221,274 (Buell et al.) filed January 22, 1993. All of these patents are incorporated herein by reference. Another embodiment of a diaper to which the present invention can be readily adapted is described in U.S. Patent 5,554,145 (Roe et al.), the disclosure of which is incorporated herein by reference.

The topsheet 520, which is particularly suitable for use in the diaper 50, is carded and thermally bonded in a manner conventional in the art. A suitable topsheet is composed of long polypropylene fibers having a denier of about 2.2. The term long fibers as used herein means those fibers having a length of at least 15.9 mm. The weight of the topsheet should be from about 14 to about 25 grams per square meter. A suitable topsheet is manufactured by Veratec, Inc., Division of International Paper Company, Mass. under the designation P-8.

• 4 4 • 4 4

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The topsheet 520 of the diaper 50 is typically made of a hydrophilic material that will facilitate the rapid passage of fluids (e.g., urine) through the topsheet. If the topsheet is made of a hydrophobic material, at least portions of the topsheet must be modified to be hydrophilic and allow fluids to pass rapidly through the topsheet. This will reduce the likelihood that bodily exudates will drain from the topsheet rather than being absorbed from the topsheet by the absorbent core. The topsheet can be rendered hydrophilic by impregnation with a surfactant. Suitable methods of impregnation with a surfactant include spraying the surfactant onto the topsheet and dipping the fabric into the surfactant. A more detailed description of such modification and hydrophilicity is provided in U.S. Patents 4,988,344 and 4,988,345, both issued to Reising et al. on January 29, 1991, which are incorporated herein by reference.

The topsheet may also be in the form of an apertured film, which is preferred in feminine hygiene absorbent articles. Apertured films are suitable because they are permeable to body fluids but are non-absorbent and have less tendency to backflow with subsequent wetting of the skin. This means that the surface of the film that contacts the body remains dry, thereby reducing body soiling and creating a better user experience. Suitable films are described in U.S. Patent 3,929,135 (Thompson) filed December 30, 1975, U.S. Patent 4,324,246 (Mullane et al.) filed April 13, 1982, U.S. Patent 4,342,314 (Radel et al.) filed August 3, 1982, U.S. Patent 4,463,045 (Ahr et al.) filed July 31, 1982. 1984 and U.S. Patent 5,006,394 (Baird) filed on 9

4, 1991. Each of these patents is incorporated herein by reference.

Recommended microaperture films for topsheets are described in U.S. Patent 4,609,518 (Curro et al.) filed September 2, 1986 and U.S. Patent 4,629,643 (Curro et al.) filed December 16, 1986, which are incorporated herein by reference. A proposed topsheet for use in feminine hygiene products is the film described in one or more of the above-mentioned patents and marketed by The Procter & Gamble Company of Cincinnati, Ohio under the designation DRIWEAWE®.

The body-facing surface of the film may be hydrophilic, allowing body fluids to permeate through the topsheet more rapidly than if it were not hydrophilic, thereby reducing the likelihood that the fluid will drain from the topsheet instead of being absorbed. In a preferred embodiment, a surfactant is incorporated into the polymeric material of the topsheet film as described in U.S. Statutory Invention Registration No. H1670 (Aziz et al.) issued July 1, 1997, which is incorporated herein by reference. In other embodiments (not shown) of the preferred invention, the absorbent article may be provided with additives to improve contact between the topsheet and the skin of the wearer. In one embodiment, the absorbent article is provided with elastic portions, as disclosed in U.S. Patent 4,892,536 (DesMarais et al.) issued January 9, 1990, U.S. Patent 4,990,147 (Freeland) issued February 5, 1991, and U.S. Patent 07,993,198 (Freeland et al.) issued December 18, 1992, which lift the topsheet, thereby improving contact with the perianal region of the wearer. In another embodiment, as disclosed in U.S. Patent 5,171,236 (Drier et al.), filed December 15, 1992, the diaper is provided with a series of joints that lift the topsheet. In yet another embodiment, as disclosed in U.S. Patent 5,171,236 (Drier et al.), filed December 15, 1992, the diaper is provided with a series of joints that lift the topsheet. Statuory Invention Registration H1687, filed in the name of Roe et al on October 7, 1997, an absorbent article is provided with a buttock protection portion that raises the top layer into the gluteal crease of the wearer.

····

In the proposed diaper embodiment described herein, the backsheet 530 has an hourglass shape and extends beyond the absorbent core by at least 1.3 cm to 6.4 cm around the entire circumference of the diaper.

The absorbent core 540 may be of any size or shape that is compatible with the diaper 50. One proposed embodiment of the diaper 50 has an asymmetrical T-shaped absorbent core 540 with tabs in the first waist region, but a generally rectangular shape in the second waist region. Examples of absorbent materials that can be used to form absorbent cores of devices are described, for example, in U.S. Patent 4,610,678 (Weisman et al.) filed on September 9, 1986, U.S. Patent 4,673,402 (Weisman et al.) filed on June 16, 1987, U.S. Patent 4,888,231 (Angstadt) filed on December 19, 1989, and U.S. Patent 4,834,735 (Alemany et al.) filed on May 30, 1989. The absorbent core may further include a dual absorbent core system consisting of an absorbent/distribution core of chemically stiffened fibers that is positioned above an absorbent retention core, this system is described in more detail in U.S. Patent 5,234,423 (Alemany et al.) filed on October 10, 1989. August 8, 1993 and U.S. Patent 5,147,345 (Young, LaVon and Taylor) filed September 15, 1992. All of these patents are incorporated herein by reference.

In the proposed embodiment, the diaper 50 further includes elastic thigh cuffs 550 to provide improved containment of fluids and body exudates, an elastic waist portion 560 to provide improved fit and support, and a fastening means 570 that creates a lateral connection that maintains the first waist region 56 and the second waist region 58 so that they overlap, thereby creating a lateral pull around the circumference of the diaper that maintains the diaper on the wearer. The diaper 50 may also include, in the waist regions 56 and 58, elastic waist bands (not shown) and/or »444

494 4 9 99 9 i · 4 49444 <»<«7 · 44 4 44444·

4 4 4 4 4 * * * * «44 44 444 4444 49 44 elastic side sections (also not shown) which ensure greater comfort and fit as well as more efficient use of the diaper 50.

Elastic thigh cuffs 550 may have a variety of different embodiments, including those described in U.S. Patent 3,860,003, U.S. Patent 4,909,803 (Aziz et al.) filed March 20, 1990, U.S. Patent 4,695,278 (Lawson) filed September 22, 1987, and U.S. Patent 4,795,454 (Dragoo) filed January 3, 1989, the disclosures of which are incorporated herein by reference. Absorbent devices having elastic cuffs treated with a component that may be useful herein are described in U.S. Patent Applications 08/766,386 and 08/840,039, filed March 3, 1990.

12. 1996 and 24. 4. 1997, which are incorporated herein by reference.

The elastic waistband element is comprised of an elastic waistband strip (not shown) which can be formed in a variety of different embodiments, including those disclosed in U.S. Patent 4,515,595 (Kievit et al.) filed May 7, 1985, U.S. Patent 5,026,364 (Robertson) filed June 25, 1991, and the aforementioned U.S. Patent 5,151,092 (Buell et al.) filed September 29, 1992, the disclosures of which are incorporated herein by reference.

The elastic side panels can be formed in a variety of different embodiments. Examples of diapers with elastic side panels located in the diaper tabs are disclosed in U.S. Patent 4,857,067 (Wood et al.) filed August 15, 1989, U.S. Patent 4,383,781 (Sciaraffa et al.) filed March 3, 1983, U.S. Patent 4,938,753 (Van Gompel et al.) filed July 3, 1990, and U.S. Patent 5,151,092 (Buell et al.) filed September 29, 1992, which are incorporated herein by reference.

Examples of clamping means 570 are described in U.S. Patent 4,846,815 (Scripps) July 11, 1989, U.S. Patent 4,894,060 (Nestegard) ·* ·· ·· ·♦ · · * · · · • · · · · · • ····· • · 9 9 9 9

999 9999 99 99 filed on January 16, 1990, U.S. Patent 4,946,527 (Battrell) filed on July 7, 1990.

8, 1990, U.S. Patent 3,848,594 (Buell) filed November 19, 1974, U.S. Patent 4,662,875 (Hirotsu et al.) filed May 5, 1987, and U.S. Patent 5,151,092 (Buell et al.) filed September 29, 1992, the disclosures of which are incorporated herein by reference.

The diaper 50 is typically applied by placing one of the waistband regions of the diaper, preferably the second waistband region 58, across the wearer's back and pulling the remainder of the diaper between the wearer's legs with the second waistband region, preferably the waistband region 56, positioned in front. Fastening means are then used to secure the diaper at the sides.

It is understood that the protease inhibitor and/or the delivery system for delivering the inhibitor to the skin during wear of the device may be used in any type of absorbent device. The above description is therefore for illustrative purposes only.

The present invention may also utilize training pants as an absorbent means containing a protease inhibitor. As used herein, the term training pants refers to a disposable garment having fixed side panels and leg openings and adapted for use by children or adults. Training pants are donned by the wearer inserting the wearer's legs into the leg openings and the pants are pulled up to the wearer's lower torso. Suitable training pants are described in U.S. Patent 5,246,433 (Hasse et al.), filed September 21, 1999, filed by Hasse et al., U.S. Patent No. 5,246,433, 1993, U.S. Patent 5,569,234 (Buell et al.) filed October 29, 1996, U.S. Patent 4,940,464 (Van Gompel et al.) filed July 10, 1990, and U.S. Patent 5,092,861 (Nomura et al.) filed March 3, 1992, the disclosures of which are incorporated herein by reference.

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Other disposable absorbent articles which may be used in the present invention are incontinence articles. The term incontinence article is used to refer to pads, undergarments, absorbent pads, bed liners and the like, whether used by adults or other incontinent persons. Suitable incontinence articles are described in U.S. 4,253,461 (Strickland et al.) filed March 3, 1981, U.S. 4,597,760 and 4,597,761 (Buell), the aforementioned U.S. 4,704,115, U.S. 4,909,802 (Ahr et al.), U.S. 4,964,860 (Gipson et al.) filed October 23, 1990, and U.S. 5,304,161 (Noel et al.) filed April 19, 1994. The disclosures of these patents are incorporated herein by reference.

Other disposable absorbent articles that can be used in the present invention are feminine hygiene articles, such as a menstrual pad. Suitable feminine hygiene products are described in U.S. Patent 4,556,146 (Swanson et al.) filed December 3, 1985, U.S. Patent 4,589,876 (Van Tilberg) filed April 27, 1993, U.S. Patent 4,687,478 (Van Tilburg) filed August 18, 1997, U.S. Patent 4,950,264 (Osborn) filed August 21, 1990, U.S. Patent 5,009,653 (Osborn) filed April 23, 1991, U.S. Patent 5,267,992 (Van Tilburg) filed December 7, 1993, U.S. Patent 5,389,094 (Lavash et al.) filed February 14, 1995, in U.S. Patent 5,413,568 (Roach et al.) filed May 9, 1995, U.S. Patent 5,460,623 (Emenaker et al.) filed October 24, 1995, U.S. Patent 5,489,283 (Van Tilburg) filed February 6, 1996, U.S. Patent 5,569,231 (Emenaker et al.) filed October 29, 1996, and U.S. Patent 5,620,430 (Bamber) filed April 15, 1997. The disclosures of these patents are incorporated herein by reference.

• ·

V. Protease inhibition testing

Standard in vitro enzyme activity and enzyme inhibition assays are well known. The reagents for performing these assays are readily available commercially. They are simple systems consisting of a substrate specific for the enzyme which, upon hydrolysis by the enzyme, forms a colored product. Enzyme activity is measured spectrophotometrically as the degree of development of the colored product (i.e., the level of color change) over a specified period of time. Inhibition of enzyme activity is manifested as a measurable reduction in the level of color change over the same period of time in the presence of an inhibitor.

For the purified protease assay and the stool protease assays described below, the IC ₅ of each inhibitor tested can be determined according to the following equation:

IC ₅₀ = (I)/((v/ _Vi )-1)) where (I) is the concentration of the inhibitor being tested, v is the level of substrate cleavage in the absence of inhibitor, and V i is the level of substrate cleavage in the presence of inhibitor.

The following purified protease and stool protease assays are used to determine the inhibitory activity of putative protease inhibitors against a) purified proteases present in stool and b) the protease activity of stool itself. All substances that meet the criteria for IC ₅₀ in any of the following assays are considered protease inhibitors. In these assays, v and V are measured as the change in absorbance (optical density, OH) at a given wavelength/time (e.g. minutes).

• ·

• · • · ·

A. Testing of purified proteases

1. Purified Trypsin

To test the activity of protease inhibitors against purified trypsin, 0.05 ml of the putative inhibitor and 0.125 ml of 32 nM trypsin (e.g. Sigma, St. Louis, MO, cat. no. T6424) in trypsin buffer (50 mM TRIS, 20 mM CaCl ₂ , pH 8.2) are placed in a microcuvette. The cuvette is allowed to incubate at 25°C for about 10 minutes. To this mixture, 0.025 ml of substrate (4 mM Cbz-arginine-pnitroanilide, e.g. Sigma, St. Louis, MO, cat. no. C4893) in trypsin buffer is added to the cuvette, everything is mixed, and after 10 minutes the absorbance is measured at 25°C at 405 nm. The level of substrate cleavage in the presence of the inhibitor (vj) is the slope of the absorbance curve at 405 nm versus time. The same procedure is repeated without the addition of the putative inhibitor. The level of substrate cleavage, in the absence of inhibitor (v), is the slope of the absorbance curve at 405 nm versus time. The values of Vj and the inhibitor concentration (I) are used to calculate the IC ₅₀ according to the above equation.

2. Purified chymotrypsin

To test the effectiveness of protease inhibitors against purified chymotrypsin, 0.05 ml of the putative inhibitor and 0.125 ml of 16 nM chymotrypsin (e.g. Sigma, St. Louis, MO, cat. no. C8946) in chymotrypsin buffer (50 mM TRIS, 10 mM CaCl ₂ , pH 7.6) are placed in a microcuvette. The cuvette is allowed to incubate at 25°C for about 10 minutes. To this mixture, 0.025 ml of substrate (0.6 mM N-succ-ala-ala-pro-phe-p-nitroanilide, e.g. Sigma, cat. no. S7388) in chymotrypsin buffer is added to the cuvette, everything is mixed, and after 10 minutes • · • ·

measures the absorbance at 25°C at 405 nm. The level of substrate cleavage in the presence of inhibitor (vj) is the slope of the absorbance curve at 405 nm versus time. The same procedure is repeated without the addition of the putative inhibitor. The level of substrate cleavage, in the absence of inhibitor (v), is the slope of the absorbance curve at 405 nm versus time. The values of Vj and the inhibitor concentration (I) are used to calculate the IC ₅ o according to the above equation.

3. Purified leucine aminopeptidase

To test the activity of protease inhibitors against purified leucine aminopeptidase (LAP), 0.05 ml of the putative inhibitor and 0.125 ml of 0.06 U/ml LAP (e.g. Sigma, St. Louis, MO, cat. no. L5006) in LAP buffer (50 mM sodium phosphate, pH 7.2) are placed in a microcuvette. The cuvette is allowed to incubate at 25°C for about 10 minutes. To this mixture is added 0.025 ml of substrate (2.4 mM L-leucine p-nitroaniline, e.g. Sigma, St. Louis, MO, cat. no. L9125) in LAP buffer, mixed, and after 10 minutes the absorbance is measured at 25°C at 405 nm. The level of substrate cleavage in the presence of the inhibitor (v;) is the slope of the absorbance curve at 405 nm versus time. The same procedure is repeated without the addition of the putative inhibitor. The level of substrate cleavage, in the absence of inhibitor (v), is the slope of the absorbance curve at 405 nm versus time. The values of Vj and the inhibitor concentration (I) are used to calculate the IC ₅₀ according to the above equation.

B. Testing for specific proteases from stool

The following is a general description of how to obtain a stool sample that is suitable for use in stool protease testing.

• 9 · · ·

• ·

In order to establish positive controls to ensure that the set of stool samples exhibits the enzymatic activity required for the evaluation of protease inhibitor activity, the following procedure is performed for each of the assays. Pooled infant stool (at least five different samples), collected to be free of urine and contamination, is mixed with water in a weight/weight ratio (w/w) of e.g. 1:50. This mixture is then thoroughly mixed to obtain a homogeneous suspension by homogenization or sonication. The stool suspension is used as a source of protease activity as described below and will have a substrate conversion rate in the absence of inhibitor ranging from 0.005 _OD405 per minute to 0.020 OD405 per minute. (To ensure perfect linearity, the final absorbance should not exceed 1.5 _OD405 units). If the activity of the child's stool is outside this range, then it is not possible to accurately determine the IC50 values for the putative protease inhibitors. However, the range of enzyme activity can be adjusted by increasing or decreasing the dilution factor for each enzyme. If this is not possible, a different group should be selected for sample collection.

1. Trypsin activity in stool

To test the effectiveness of protease inhibitors against trypsin in stool, the inhibitor and trypsin buffer (50 mM TRIS, 20 mM CaCl ₂ , pH 8.2) are placed in a cuvette so that the final volume is 0.8 ml. To this mixture, 0.1 ml of substrate (3 mM Cbz-arginine-pnitroanilide) is added to the cuvette. The cuvette is mixed by rotation and incubated at 25°C for 5 minutes. About 0.1 ml of stool suspension is added to the cuvette, everything is mixed, and after 5 minutes the absorbance at 405 nm minus the absorbance at 490 nm is measured at 25°C. (The absorbance at 490 nm is the correction factor

for background absorbance due to faecal particles, i.e. interference). The level of substrate cleavage, in the presence of inhibitor (vj), is the slope of the curve of the difference in absorbance (i.e. absorbance at 405 nm minus absorbance at 490 nm) versus time. The same procedure is repeated without the addition of the putative inhibitor. The level of substrate cleavage, in the absence of inhibitor (v), is the slope of the curve of the difference in absorbance versus time. The values of Vj and v and the concentration of inhibitor (I) are used to calculate the IC50 according to the above equation.

2. Chymotrypsin activity in stool

To test the effectiveness of protease inhibitors against chymotrypsin in feces, the inhibitor and chymotrypsin buffer (50 mM TRIS, 10 mM CaCl ₂ , pH 7.6) are placed in a cuvette so that the final volume is 0.92 ml. To this mixture, 0.04 ml of substrate (1.25 mM Nsukc-ala-ala-pro-phe-p-nitroanilide) is added to the cuvette. The cuvette is mixed by rotation and incubated at 25°C for 5 minutes. About 0.04 ml of stool suspension is added to the cuvette, everything is mixed and after 5 minutes the r is measured at 25°C.

absorbance at 405 nm minus absorbance at 490 nm. The level of substrate cleavage, in the presence of inhibitor (v;), is the slope of the curve of the difference in absorbance (i.e. absorbance at 405 nm minus absorbance at 490 nm) versus time. The same procedure is repeated without the addition of the putative inhibitor. The level of substrate cleavage, in the absence of inhibitor (v), is the slope of the curve of the difference in absorbance versus time. The values of Vj and the inhibitor concentration (I) are used to calculate the IC ₅₀ according to the above equation.

3. LAP activity in stool • ·

To test the efficacy of protease inhibitors against LAP in stool, the inhibitor and LAP buffer (50 mM sodium phosphate, pH 7.2) are placed in a cuvette so that the final volume is 0.95 ml. To this mixture, 0.03 ml of substrate (6 mM L-leucine-p-nitroanilide) is added to the cuvette. The cuvette is mixed by rotation and incubated at 25°C for 5 minutes. About 0.02 ml of stool suspension is added to the cuvette, everything is mixed, and after 5 minutes the absorbance at 405 nm minus the absorbance at 490 nm is measured at 25°C. The level of substrate cleavage, in the presence of the inhibitor (v;), is the slope of the curve of the difference in absorbance (i.e. absorbance at 405 nm minus absorbance at 490 nm) with respect to time. The same procedure is repeated without adding the putative inhibitor. The level of substrate cleavage, in the absence of inhibitor (v), is the slope of the absorbance difference versus time curve. The Vj values and inhibitor concentration (I) are used to calculate the IC ₅₀ according to the above equation.

Using the purified protease and fecal protease assays described above, the protease inhibitory activity of selected protease inhibitors used in the absorbent compositions of the present invention was determined, and the test results are shown in Table 1.

Table 1

IC50 (μM) Purified proteases Specific Fecal Proteases Trypsin Chvmotrvpsin LAP* Trypsin Chymotrypsin LAP* Inhibitor soybean trypsin inhibitor 0.25 0.026 >10 <0.01 0.06 >20 aprotinin 0.168 1 >20 0.01 0.22 >20 hexamidine diisothionate 2.5 >1000 256 2.3 >1000 130 p-aminobenzamidine 13.8 >500 >500 20 >500 >500 leupeptin 0.14 >500 >500 0.11 >500 >500 pepstatin A 324 4.9 >500 >500 300 >500 chymostatin >500 <0.12 >500 >500 0.02 >500

*LAP = leucine aminopeptidase

Table 1 shows that each of the compounds inhibits at least one protease tested using purified proteases and/or fecal proteases.

C. General stool protease testing

The general method for obtaining a stool sample suitable for stool protease testing described above can be readily adapted, without undue experimentation, to obtain appropriate stool samples suitable for general stool protease testing.

To test the efficacy of protease inhibitors against protease activity from stool, 50 μΐ of inhibitor and 50 μΐ of stool suspension are added to a 1.5 ml tube. The tube is mixed by inverting and incubated at 25°C for approximately 45 minutes. Then 50 μΐ of protease buffer (200 mM TRIS buffer containing 20 mM CaCl ₂ , pH 7.8) is added to the tube. The tube is mixed again and incubated at 25°C for 45 minutes. Then 50 μΐ of protease substrate (0.4% casein-resorufin, e.g. Boehringer Mannheim, Indianapolis, IN, cat. no. 1 734 334) is added to the tube. The tube is mixed again and incubated at 37°C for 60 minutes to allow substrate cleavage. Then, 480 μΐ of trichloroacetic acid (5% w/v) is added to stop the reaction and precipitate the remaining casein resorufin. The tube is mixed by inversion at 37°C for 15 minutes. The tube is then centrifuged at a relative centrifugal force of 20,800 xg for 5 minutes. Then, 400 μΐ of the supernatant is added to 600 μΐ of assay buffer (0.5 M TRIS, pH 8.8) in a cuvette. The cuvette is mixed by inversion and the absorbance is measured at 574 nm. The same procedure is repeated ······«

JI ·····* • ·· · · ·····* 4 without putative inhibitor. A is the absorbance at 574 nm in the absence of inhibitor. Aj is the absorbance at 574 nm in the presence of inhibitor. Before the reaction begins, both A and Aj are almost zero. Therefore, the level of substrate cleavage in the presence of inhibitor (v i ) can be calculated by dividing the absorbance at 574 nm (Aj) by the reaction time. The level of substrate cleavage in the absence of inhibitor (v) can be calculated by dividing the absorbance at 574 nm (A) by the reaction time. The levels of Vj and v, and the concentration of inhibitor (I), can be used to calculate IC ₅ q according to the above equation.

Table 2

IC™ Values (uM) Inhibitor General Fecal Proteases soybean trypsin inhibitor 4.9 hexamidine 31 leupeptin >320 pepstatin A >32 chymostatin 64 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride 217

D. Absorbent testing

To determine the presence of an inhibitor in any part of an absorbent article (e.g., topsheet, absorbent core, backsheet and/or other layers, thigh cuffs, fastening sections, side sections, inserts or combinations thereof), a small sample of the article is taken from that part and the inhibitor is extracted. It is understood that a water-soluble inhibitor will be extracted with water or an aqueous solvent, while a fat-soluble inhibitor will be extracted with an organic solvent. The following description is merely an illustrative example of the determination of a protease inhibitor (trypsin) in an article and is not intended to be limiting, as this procedure can be used to determine inhibitors of other proteases and also because many other procedures are known in the art that can be used.

To detect trypsin inhibitor in the upper layer of the preparation, 1.5 cm diameter sections of the absorbent core are randomly punched out. The upper layer is removed from the punched out section and placed in a 1.5 ml centrifuge tube. The sample is left overnight in 0.75 ml of water or another solvent, such as the 50 mM TRIS, 20 mM CaCl ₂ , pH 8.2 buffer described above. All (0.125 ml) of the supernatant is removed and added to a cuvette containing 0.025 ml of freshly prepared 160 nM human pancreatic trypsin in TRIS-HCl containing 20 mM CaCl ₂ , pH 8.2 and allowed to incubate for 10 minutes at 25°C. A control sample containing only buffer is prepared in the same manner in a second cuvette. Cbz-arginine-p-nitroanilide substrate (0.025 ml of a 4 mM solution) is added to each cuvette and the test and control samples are incubated for 5 minutes. The change in absorbance of each sample at 405 nm is then monitored for 10 minutes. It is clear that this protocol can be used to detect protease inhibitory activity in other parts of the device, such as the absorbent core, etc.

An absorbent has protease inhibitory activity if the sample extract reduces the hydrolysis of the substrate by the protease by at least 10%, more preferably by 20%, more preferably by 50% and most preferably by 80%, relative to the control sample. The reduction in the hydrolysis of the substrate is generally • 4

4 ranges from 10% to 99%, more preferably from 20% to 99%, even more preferably from 50% to 99%, and most preferably from 80% to 99%.

The absorbent is also considered to have inhibitory activity if the sample contains an inhibitory substance, such as any of the previously described protease inhibitors, as well as substances that may not meet the IC ₅₀ criteria, e.g. substances such as L-chloro-3-(4-tosylamide)-amino-2-heptanone-HCl (TLCK), L-chloro-3-(4-tosylamide)-4-phenyl-2-butanone (TPCK), tranexamic acid, etc.

E. In vitro skin test for inhibition of IL-1α production

In this in vitro assay, which determines the efficacy of protease inhibitors against the development of a skin inflammatory response to stool and its enzymes, human keratinocytes, derived from epidermal tissue, are cultured in serum-free medium in plastic dishes, the surface of which is coated with a nylon mesh, until they are confluent. The meshed surface is then raised at the liquid-water interface to allow differentiation and the formation of multilayered organized structures analogous to those found in vivo, including a well-defined barrier layer of the stratum corneum. Any culture system that allows keratinocyte growth and differentiation can be used. A suitable commercial culture system is Epiderm (MatTek Corporation, Ashland, Massachusetts).

The stool of the children is collected in a way that is not contaminated with urine and diluted with phosphate buffered saline (PBS) (pH 7.2-7.4). The mixture is then thoroughly mixed by homogenization or sonication to form a homogeneous suspension. To determine the production of IL-Ια, due to the enzymatic activity of the stool, one part of the homogenate is diluted

4

PBS and added to the surface of the control culture in the culture vessel. To determine the inhibition of IL-1α production due to protease activity, a certain amount of the putative protease inhibitor is added to a second portion of the homogenate, which is diluted in the same way and then added to the surface of the test culture. The cultures are allowed to incubate in a controlled atmosphere. The control cultures, the test cultures with the inhibitor and the culture medium are collected after a certain period of time. The culture medium is tested for the presence of IL-100 in the usual way. A suitable test for IL-100 is, for example, an ELISA, which is commercially available as Quantikine from R&D Systems, Minneapolis, Minnesota.

The percentage reduction in IL-lot production, due to the presence of a protease inhibitor, is calculated as follows:

% reduction = IL-1α control culture minus IL-1α test culture X 100% IL-1α control culture

Using this standard assay, the ability of the serine protease inhibitors, soybean trypsin inhibitor and hexamidine diisethionate (hexamidine), to inhibit IL-1α production by cultured keratinocytes in the presence of feces was tested. The results showed that hexamidine, at concentrations of 1,000 μΜ and 100 μΜ, reduced IL-1α production by keratinocyte cultures exposed to feces by 51 to 88% and 5%, respectively. A concentration of soybean trypsin inhibitor, 10 μΜ, was sufficient to reduce IL-1α production by 56 to 75%. Heat treatment (90°C) of feces prior to testing resulted in almost complete suppression of IL-1α production, indicating that the main substance or substances inducing the cytokine response are · · * · ·«·· ··· ··· ··· ··♦» ·· ·· denaturable proteins. The results suggest that trypsin inhibitors (see Table 1) are also effective in reducing IL-1α production by skin cell cultures.

VI. Incorporation of inhibitors into the absorbent

1. Carrier component

The protease inhibitor for use in the absorbent article of the invention may be water- or fat-soluble and may be incorporated into the absorbent article in neat form, such as in powder or particulate form, or in solution, suspension, dispersion, emulsion or the like, in a pharmaceutically and dermatologically acceptable carrier that will not interfere with the inhibitory activity of the compound. The inhibitor may also be incorporated into another structure that is incorporated into the article during manufacture or assembly. The inhibitor may be coated or otherwise attached or attached to nanophase structures or other solid supports, such as glass, plastic or agarose beads, or may be contained in microcapsules that are pressure-ruptured or soluble, or contained in an absorbent material. It is understood that a variety of other materials and methods may be used to incorporate the inhibitor.

Inhibitor carriers include components that are in the form of solutions, creams, oils, ointments, powders, foams or gels, etc., and which may contain any ingredient commonly used in such components in the art. The ingredients of these ingredients will depend on the nature of the component, so that, for example, solutions will contain different ingredients than powders. Cosmetic components may further contain a wide range of possible ingredients, such as moisturizers, emollients, • ·· ·

9 ······ · · · 9 9 9 9 9 9 9 • 999 99 9999

999 99 999 9999 99 99 gelling agents, neutralizing agents, perfumes, coloring agents, etc.

Other ingredients, such as surfactants, etc., that may be present in the ingredient will be described in more detail in the Dermatological Ingredients section. Ingredients containing a protease inhibitor that are intended to pass through the skin should have a pH not lower than 4 and not higher than 7.5.

2. Inclusion

The protease inhibitor used in the absorbent article of the invention is incorporated into the article in such a way as not to interfere with the functions of other structures of the article (e.g., the absorbency of the core, the permeability of the topsheet, etc.). The inhibitor may be incorporated into any part or parts of the article, including, but not limited to, the topsheet, the backsheet, the absorbent core, any interlayer, the thigh cuff, the side panels, the waist area, the fastening panels, the insert panels, such as absorbent material inserted into the article during wear, specialized structures, such as a section whose function is to contain stool during bowel movements, etc. The inhibitor may be incorporated into the article separately or may optionally be included in a delivery system, described below, which is part of any of the preceding parts of the article, and which delivers the protease inhibitor directly or indirectly to the skin of the wearer during normal wear of the article.

Any amount of different protease inhibitors or mixtures thereof, whether or not incorporated into a distribution system, may be uniformly or non-uniformly distributed in the absorbent article. The absorbent article typically contains such an amount of protease inhibitor or mixture of inhibitors that it constitutes 0.0001 ·· ·· « · 9 9·· • 9 9 9 9 • 9 9 ·· ·

9 9 9 9

9999 99 ·· % to 30%, more preferably 0.0001% to 10%, even more preferably 0.001% to 5% and most preferably 0.001% to 1% by weight of the composition.

The inhibitor can be incorporated, directly onto the surface or into the structure of any type of topsheet, including woven, nonwoven and apertured, backsheet and/or absorbent core fabric, or other parts of the device, during manufacture or assembly, by various methods conventional in the art. For example, the inhibitor can be applied, after dispersion in a liquid or semi-solid carrier, to the topsheet, absorbent core or core side of the backsheet, by spraying, dipping, printing, soaking or other means. A number of other techniques that can be used include coupling polymerization, exposing the structural parts to steam to bond the inhibitor by hydrogen bonds that are readily broken upon exposure of the surface to body exudates, thereby releasing the inhibitor.

The inhibitor is applied to at least a portion of the surface that contacts the user and is available for automatic transfer to the skin of the user of the device, during contact, movement of the user and/or the action of body heat. Alternatively, the absorbent device may include a delivery system with an enzyme inhibitor that transfers at least a portion of the inhibitor to the skin of the user of the device during wear. In all of these embodiments, the inhibitor is transferred to the skin so that it can act at the skin/feces interface during bowel movements. In another preferred embodiment, the delivery system is a dermatological component containing a protease inhibitor and various emollients and immobilizing agents that is transferred directly from the user-contacting surface to the skin, where it performs its barrier function and inhibits fecal enzymes. The use or better yet, the repeated use of such means which transfers the protease inhibitor directly or indirectly to • ·«*· 4 4 4 44 99 ·· · 4 4 · 4 4 · 4 4 • 4 4 4 4 4 4 4

A A · ·♦·······

Η Τ’ ··· 9 · ···· «·· 44 444 44·· 44 4· the user's skin, should accumulate the protease inhibitor, thereby preventing the development of skin inflammation, which is caused by fecal proteases.

In another embodiment of the invention, the protease inhibitor is placed in the absorbent device separately or in the distribution system, in such a way and at such a location that it can inactivate proteases in stool, especially loose stools, trapped in the device before it is transferred to the skin.

In another embodiment of the invention, a protease inhibitor is placed within the absorbent article such that it can reduce or eliminate protease activity in fecal fluids or fecal-contaminated urine that have penetrated the interior of the absorbent article and which may, for some reason, later come into contact with a user-contact surface. The protease inhibitor may again be present alone or be part of a delivery system.

VII. Distribution systems

Protease inhibitors or components containing them may be part of any delivery system known in the art that directly or indirectly facilitates the transfer of the protease inhibitor to the skin of the user of the composition, thereby protecting it from irritation by fecal proteases. The delivery system may contain the protease inhibitor in pure form, such as a powder or particles, or in the form of a solution, suspension, dispersion, emulsion, etc., in a carrier or dermatological component. Upon release from the delivery system, the protease inhibitor is free to move from the location of the delivery system, to the surface that contacts the user, and then on to the skin. The delivery system may be a component of any part or parts • ·444 * ·4 ·· ** ·· 4 44 4 4 · · · ·

4 · 44444

4S · 44444444·

4·· 44 4444

444 4· 444 4444 44 44 absorbent article, including, but not limited to, a topsheet, a backsheet, an absorbent core, another interlayer, thigh cuffs, side panels, waist area, fastening panels, insert panels such as absorbent material inserted into the absorbent article during wear, specialized structures such as stool collection (e.g., bowel evacuation pockets), etc. The distribution system is preferably positioned close to the user's skin and should be part of the user-contacting surfaces of the article, such as the topsheet, side panels, thigh cuffs, waist area, fastening panels, etc. It is preferred that the distribution system incorporated into the topsheet is a dermatological component, as will be described below.

The delivery system may contain and/or deliver the protease inhibitor in any of the above-mentioned forms, including, powder, flakes or particles, or in the form of a solution, suspension, dispersion, emulsion, etc., in a pharmaceutically and dermatologically acceptable carrier.

Upon release from the distribution system, the inhibitor may be in an active functional form, such as in a solution, suspension, emulsion, etc., or it may be non-functional, such as in a powder or particulate form, but capable of being activated by exposure to moisture from urine or feces or by other means.

The types of delivery systems that can be used in the absorbent compositions of the present invention and facilitate the transfer of the protease inhibitor from any part of the composition to the skin are readily understood by those skilled in the art. Examples of delivery systems are, for example, pressure-bursting or dissolvable microcapsules that release the inhibitor or inhibitor component upon dissolution, due to moisture from urine or feces, or upon rupture due to φφφφ body pressure or manual disruption of the capsules by the user, prior to application of the composition to the user. For example, a water-soluble film that contains and upon exposure to moisture exposes the powder is described in U.S. Patent 4,790,836 and would be a suitable material for use in microcapsules containing the protease inhibitor in any form, such as powder, particulate, solid or semi-solid. Examples of pressure-disruptible microcapsules suitable for a protease inhibitor are described in U.S. Patent 3,585,998. Such microcapsules may be present in any part of the absorbent article, including the topsheet. U.S. Patent 4,623,339 describes an insertable exchange layer that can be activated by manual pressure to expel the agent through slots in its surface. The disclosures of all of the foregoing patents are incorporated herein by reference.

Other suitable delivery systems for inhibitors or inhibitor-containing components include, but are not limited to, chambers in the composition, which are closed or partially open spaces, of regular or irregular shape, that release the inhibitor upon exposure to moisture, heat or pressure, and water-soluble adhesives and other similar components that release the inhibitor upon exposure to moisture, etc.

Regardless of the delivery system used, the protease inhibitor or component containing it should be able to move from the site of its release, e.g., by urine flow, user movement, pressure, etc., or by viscosity reduction due to body heat, to other areas of the absorbent article. Protease inhibitors that are hydrophilic or are part of hydrophilic carriers can pass through hydrophilic structures of the absorbent article, such as hydrophilic pores or other openings that allow urine to flow from the topsheet into the core. Delivery systems containing inhibitors • 4 « · · · • · · · · · · · · · · · · · ···«

4·4· «4· protease or inhibitor components, however, are usually placed in proximity to the user's skin. In the proposed embodiment, the protease inhibitors are dissolved, suspended or emulsified components of the dermatological component, which can be placed anywhere in the composition, but preferably on a surface that contacts the skin, such as the surface of the top layer, side portion, waist area, thigh cuff, fastening portion, etc.

Dermatological components suitable for carrying a protease inhibitor are described below. In all of these embodiments, the dermatological component comprises from 0.01% to 50%, more preferably from 0.5% to 25%, and most preferably from 1% to 10% by weight of the protease inhibitor.

VIII. Dermatological ingredients

Dermatological ingredients suitable for use in the absorbent compositions of the present invention are described in U.S. Patent Nos. 08/926,532 and 08/926,533, both filed September 10, 1997, U.S. Patent No. 5,607,760 filed March 4, 1997, U.S. Patent No. 5,609,587 filed March 11, 1997, U.S. Patent No. 5,635,191 filed June 3, 1997, and U.S. Patent No. 5,643,588 filed July 1, 1997, the disclosures of which are incorporated herein by reference.

In addition to its function of providing an effective concentration of the protease inhibitor on the skin of the user, the dermatological composition containing the protease inhibitor may also contain ingredients that, for example, reduce stool adherence to the skin, form a skin/stool barrier (e.g., cover the skin to prevent stool adherence) while remaining relatively impermeable but breathable, or have other beneficial effects on the skin (e.g., soften the skin, improve or maintain skin quality), etc. The dermatological composition may take a variety of forms, such as an emulsion, solution, cream, ointment, balm, suspension, gel, etc.

The transfer of an effective concentration of enzyme inhibitor from the absorbent composition to the skin and the effective amount of dermatological component that is applied or transferred to the surfaces of the composition that are in contact with the skin of the user depends primarily on the dermatological component used. The amount of component should be, at least on the portion of the surface that contacts the skin, from 0.0078 mg/cm ² to 12 mg/cm 2 , more preferably from 0.16 mg/cm 2 to 6 mg/cm 2 and most preferably from 0.6 mg/cm ² to 4 mg/cm ² . However, these ranges are illustrative only and the skilled artisan will recognize that the nature of the component determines the amount that must be applied to transfer a sufficient concentration of enzyme inhibitor and that the required amount is determinable by routine experimentation.

The amount of dermatological component applied to the absorbent article is an important factor in the present invention, but more important is the amount of component transferred to the skin of the user during use of one or more of the compositions so prepared. Although the amount of dermatological component containing protease inhibitor transferred to the skin depends to some extent on the nature of the component used, relatively small amounts can be transferred that are still sufficient to produce a minimum inhibitory concentration of protease inhibitor on the skin. This is particularly true for the proposed components described in Example 1.

To determine the amount of protease inhibitor that is transferred to the skin of a user during the wearing of one or more of the modified absorbent articles, a method is provided below that determines the amount of dermatological ingredient transferred to the skin. Given the level of dermatological ingredient that is transferred to the skin during the wearing of one modified absorbent article for 3 hours (a typical wearing time), and particularly for the proposed dermatological ingredients described in Example 1, it is suggested that at least 0.0016 mg/cm , more preferably at least 0.0016 mg/cm , is transferred to the skin during the three hour wearing time.

2 but at least 0.0078 mg/cm and preferably at least 0.016 mg/cm of dermatological component. The usual amount of dermatological component transferred, by one modified composition, during three hours of wear, ranges from 0.0016 mg/cm ² to 0.78 mg/cm ² , more preferably from 0.0078 mg/cm ² to 0.47 mg/cm ² and most preferably from 0.016 mg/cm ² to 0.31 mg/cm ² .

For long-term use of the modified compositions (usually changed every 3 to 4 hours during the day and also before sleeping at night), such as for 24 hours, it is preferred to use components that are transferred to the user's skin in an amount of at least 0.0047 mg/cm 2 , more preferably at least 0.016 mg/cm 2 and most preferably at least 0.047 mg/cm 2 . The amount of component transferred in 24 hours is usually from 0.0047 mg/cm ² to 2.79 mg/cm ² , more often from 0.016 mg/cm ² to 10 mg/cm ² and most often from 0.047 mg/cm ² to 0.93 mg/cm ² .

It is apparent that, from the numerous substances suitable for the skin-transmitted enzyme inhibitor dermatological components of the invention, those substances that are considered safe and effective dermatological agents were logically selected. These substances are in Category I as defined by the U.S. Food and Drug Administration (FDA), (21 C.F.R. § 347), which now includes: allantoin, aluminum hydroxide gel, calamine, cocoa butter, dimeticone, fish oil (in combination), glycerin, kaolin, petrolatum, lanolin, mineral oil, shark oil, white petrolatum, talc, starch, zinc acetate, carbonate

zinc, zinc oxide, etc. Other suitable substances belong to category III, defined by the U.S. Food and Drug Administration (FDA), which now includes: live yeast derivatives, aldiox, aluminum acetate, microporous cellulose, cholecalciferol, colloidal oatmeal, cysteine hydrochloride, dexpanthenol, Peruvian balsam oil, protein hydrolysates, racemic methionine, sodium bicarbonate, vitamin A, etc.

A number of FDA-listed dermatological ingredients are now commonly used in commercially available dermatological products, such as A and D Ointment®, Vaseline®, Petroleum Jelly, Desitin® Diaper Rash Ointment and Daily Care® ointment, Gold Bond® Medicated Baby Powder, Aquaphor® Healing Ointment, Baby Magie® Baby Lotion, Johnson's Ultra Sensitive® Baby Cream. An effective concentration of a protease inhibitor can be incorporated into any of these commercial products and applied to an absorbent article to form a modified composition according to the present invention.

It will be further described that dermatological ingredients suitable for delivering enzyme inhibitors to the skin of a user are preferably, but not necessarily, ingredients with a melting profile that allows them to remain immobile at room temperature on a surface contacting the user and to readily transfer to the user at body temperature, while not liquefying during storage under extreme conditions. In this regard, the ingredients are, at least in part, transferable to the skin by contact, movement of the user and/or body heat. Since the ingredient is generally immobilized on the surface of the composition contacting the user, relatively low levels of the ingredient are sufficient to produce the desired effects on the skin. It may also be necessary to use special packaging materials for the modified compositions of the present invention.

In the proposed embodiment, the dermatological components are water-in-oil emulsions, where the protease inhibitor is in the aqueous phase. However, the dermatological component itself may be solid or more often semi-solid at 20°C, i.e. ambient temperature. The term semi-solid means that the component has rheological properties typical of pseudo-solid or ductile liquids. In the absence of friction, the components may have the appearance of a semi-solid, but may liquefy when friction is increased. This is because the component primarily contains both solid and liquid parts. The viscosity of the dermatological component containing the enzyme inhibitor should be from 1.0 x 10 ⁴ to 1.0 x 10 ⁶ g/cm.s at zero friction, but more preferably from 5.0 x 10 ⁴ to 5.0 x 10 ⁶ g/cm.s. The term zero friction viscosity refers to the viscosity measured at very low friction levels (e.g., 1.0 s- ¹ ) using a suitable viscometer (e.g., model CSL 100 from TA Instruments of New Castle, DE). One skilled in the art will recognize that components other than those with high melting points (see below) may be used which will have a comparable viscosity, which can be measured by extrapolating the viscosity curve, relative to the friction level for such component, to zero friction at a temperature of about 20°C.

The proposed components are at least semi-solid at room temperature to prevent the component from migrating into the absorbent prior to use. In addition, the components have a final melting point (100% liquefaction) above potential adverse storage conditions, which may be greater than 45°C (e.g., a warehouse in Arizona, a truck in Florida, etc.). Examples of components having these features are described in detail in U.S. Patent 5,643,588, U.S. Patent 5,607 • 4 4 4 • ·

4 · · 4 4 4 4 4 4 4 4 • · 4 44444

44 4 «44444

444 44 4444

444 44 444 4444 44 44

760, in U.S. Patent 5,609,587 and in U.S. Patent 5,635,191. The proposed components have the following melting profiles:

Property Suggested range The most suitable range % liquid at room temperature (20°C) 2-50 3-25 % of fluid at body temperature (37°C) 25-95 30-90 final melting point (°C) 38 45

The proposed enzyme inhibitor-containing components do not tend to flow at ambient temperatures and migrate to a greater extent to areas of the composition other than those to which they were applied. This means that a smaller amount of the dermatological component is required to achieve the expected therapeutic and protective effect.

To prevent the components from migrating to undesirable locations within the composition, the components must be immobilized by making their viscosity as high as possible. Unfortunately, in some cases, higher viscosity can reduce the transfer of the component to the user's skin. Therefore, a balance must be achieved that allows both the component to be retained on the surface of the composition and its transfer to the user's skin. Suitable viscosities of these components are in the range of 0.05 to 5 g/cm.s, more preferably 0.05 to 3 g/cm.s, and most preferably 0.05 to 1 g/cm.s, measured at 60°C using a rotational viscometer (a suitable viscometer is the Model 4357 from Lab Line Instruments, Inc. of Melrose Park, IL). The viscometer operates at 60 rpm using shaft No. 2.

• · · · · 4 4 4 4 « · 4 · 4 4 ·

4 · 4·····

44 4444 » 4444444 44 44

In addition to the dermatological ingredients adapted to have a therapeutic and/or protective effect on the skin and the positive effect of protease inhibitors, the other active ingredients of these ingredients are one or more preservatives or emollients. The term emollient, as used herein, is reserved for a substance that protects against moisture and irritation, softens, soothes, smoothes, coats, lubricates, moisturizes, protects and/or cleanses the skin. (It will be apparent that many of the substances mentioned above are emollients.) In the proposed embodiment, these emollients are malleable or liquid at ambient temperature, i.e. 20°C.

Examples of suitable emollients for use in the present invention include, but are not limited to, emollients derived from petroleum, sucrose esters of fatty acids, polyethylene glycol and its derivatives, humectants, fatty acid esters, alkyl ethoxylates, ethoxylated esters of fatty acids, fatty alcohol, polysiloxane, propylene glycol and its derivatives, glycerin and its derivatives, including glyceride, acetoglyceride and ethoxylated glycerides of C1- _C28 fatty acids, triethylene glycol and its derivatives, spongin and or other waxes, fatty acids, fatty alcohol esters, especially those having 12 to 28 carbon atoms in their chain, such as stearic acid, propoxylated fatty alcohols, other fatty esters of polyhydroxy alcohols, lanolin and its derivatives, kaolin and its derivatives, any dermatological agent mentioned above or mixtures of these emollients. Suitable emollients derived from petroleum include those hydrocarbons or mixtures thereof having a chain length of from 16 to 32 carbons. Petroleum hydrocarbons having this chain length include mineral oil and petrolatum. The term mineral oil generally refers to a less viscous mixture of hydrocarbons having from 16 to 20 carbons. The term petrolatum generally refers to a more viscous mixture of hydrocarbons having from 16 to 32 carbons. Petrolatum and mineral oil are preferred emollients for use in the dermatological composition of the present invention.

Suitable fatty acid ester plasticizers include those derived from C12-C28 fatty acids, more preferably saturated C16- _C22 fatty acids and short chain alcohols (C1- _C8 , more preferably C1-C3). Suitable examples of such esters are methyl palmitate, methyl stearate, isopropyl laurate, isopropyl myristate, isopropyl palmitate, ethylhexyl palmitate and mixtures thereof. Suitable fatty acid ester plasticizers can also be derived from esters of long chain fatty alcohols ( _C12 - _C28 , more preferably _C12 -C16) and shorter chain fatty acids, e.g. lactic acid, such as lauryl lactate and cetyl lactate.

Suitable alkyl ethoxylate-type plasticizers are C ₂ -C ₂₂ fatty alcohol ethoxylates having an average degree of ethoxylation of from 2 to 30. These plasticizers are selected from the group consisting of lauryl, cetyl and stearyl ethoxylates or mixtures thereof having an average degree of ethoxylation of from 2 to 23. Illustrative examples of such alkyl ethoxylates are laureth-3 (lauryl ethoxylate with an average degree of ethoxylation of 3), laureth-23 (with an average degree of ethoxylation of 23), ceteth-10 (cetyl alcohol ethoxylate with an average degree of ethoxylation of 10) and steareth-10 (stearyl alcohol ethoxylate with an average degree of ethoxylation of 10). These alkyl ethoxylate plasticizers are typically used in combination with petroleum-derived plasticizers, such as petroleum jelly, in a weight ratio of alkyl ethoxylate plasticizer to petroleum-derived plasticizer of from 1:1 to 1:5, more preferably from 1:2 to 1:4.

• · · · · · • · · · · • · · · · ·

Suitable fatty alcohol plasticizers are C12-C22 fatty alcohols, more preferably _C16 - _C18 fatty alcohols. Illustrative examples are cetyl alcohol, stearyl alcohol and mixtures thereof. These plasticizers are generally used in combination with petroleum-derived plasticizers, such as petrolatum, in a weight ratio of fatty alcohol plasticizer to petroleum-derived plasticizer of from 1:1 to 1:5, more preferably from 1:1 to 1:2.

Other suitable types of plasticizers are polysiloxane compounds. In general, those polysiloxane substances suitable for use in the present invention are those whose monomeric siloxane unit has the following structure:

R ¹

I —Si-0— where R ¹ and R ² may be, independently for each monomer unit, hydrogen or alkyl, aryl, alkenyl, alkaryl, arakyl, cycloalkyl, halohydrocarbon or other radical. All of these radicals may be mutually substituted. The radicals R ¹ and R ² of each individual monomer unit may be different from the respective groups of the adjacent unit. In addition, the polysiloxane may have a linear chain structure, a branched chain or a cyclic structure. The radicals R ¹ and R ² may also be independently other groups, such as siloxanes,

7 polysiloxanes, silanes and polysilanes. The radicals R and R can contain any of a number of organic groups including alcohol, carboxylic acid, phenyl and amine groups.

• ·

Examples of alkyl radicals are methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, decyl, octadecyl, etc. Examples of alkenyl radicals are vinyl, allyl, etc. Examples of aryl radicals are phenyl, diphenyl, naphthyl, etc. Examples of alkaryl radicals are toyl, xylyl, ethylphenyl, etc. Examples of aralkyl radicals are benzyl, alphaphenylethyl, beta-phenylethyl, alpha-phenylbutyl, etc. Examples of cycloalkyl radicals are cyclobutyl, cyclopentyl, cyclohexyl, etc. Examples of halogenated hydrocarbon radicals are chloromethyl, bromoethyl, tetrafluoroethyl, fluoroethyl, trifluoroethyl, trifluorooyl, hexafluorooxylyl, etc.

The viscosity of suitable polysiloxanes can vary widely, as can the viscosity of siloxanes in general, and therefore siloxanes can be flowable or can be modified to become flowable. That is, their viscosity ranges from 5.10' m/s (5 centistokes) (measured at 37°C using a glass viscometer) to 20 ^m2 /s (20,000,000 centistokes), but is not limited to these values. Polysiloxanes should have a viscosity at 37°C in the range of 5.10' ⁶ to 5.10' ³ m ² /s (5 to 5,000 centistokes), more preferably 5.10' ⁶ to 2.10' ³ m ² /s (5 to 2,000 centistokes), most preferably 10' ⁴ to 10' ³ m ² /s (100 to 1,000 centistokes). High viscosity polysiloxanes that are not flowable can be effectively applied to an absorbent article by, for example, forming an emulsion of the polysiloxanes in a surfactant or by forming a solution of the siloxanes using a solvent such as hexane, which is mentioned only by way of example. The individual methods of applying polysiloxane plasticizers to absorbent articles will be mentioned in more detail below.

Polysiloxane compounds proposed for use in this invention are described in U.S. Patent 5,059,282. Among

4· 4 »•44 ·

the most suitable polysiloxane compounds include polymethylsiloxane compounds with a phenyl group (e.g. Dow Corning 556 CosmeticGrade Fluid: polymethylphenylsiloxane) and or dimethicones with a cetyl or stearyl group, such as Dow 2502 and Dow 2503 polysiloxane fluids. In addition to these phenyl or alkyl group substitutions, effective substitution with amine, carboxyl, hydroxyl, ether, polyether, aldehyde, ketone, amide, ester and thiol groups can also be made. Of this range of effective substituents, phenolic, amino, alkyl, carboxyl and hydroxyl groups are preferred, but phenolic groups are most suitable.

Suitable fatty ester plasticizers are polyol polyesters, which are described in U.S. Patent No. 5,609,587. Examples of polyols include, but are not limited to, polyhydric compounds such as pentaerythritol, sugars such as raffinose, maltodextrose, galactose, sucrose, glucose, lactose, mannose, and erythrose, and sugar alcohols such as erythritol, xylitol, maltitol, mannitol, and sorbitol. Such polyols are formed from esters of fatty acids and/or other organic moieties having from 2 to 30 carbons. While it is not necessary that all of the hydroxyl groups of the polyols be esterified, the proposed polyol polyester plasticizers have substantially all (e.g., at least 85%) of the hydroxyl groups esterified. Sucrose polyol polyesters, such as sucrose polycotonate, sucrose polysoyate and sucrose polybehenate, are particularly suitable. Mixtures of these polyol polyesters are also suitable plasticizers for the present invention.

• J · *9 9 9 9 «· · • _ · * · · · · · · •99 99 999 9«99 ·· 99

Suitable moisturizing ingredients are glycerin, propylene glycol, sorbitol, trihydroxystearin, etc.

The amount of emollient that may be included in a dermatological composition depends on a number of factors, e.g. the type of emollient in question, the desired effect on the skin, other components of the composition, etc. The composition contains emollients from 0 to 100% by weight. However, a suggested range is from 10 to 95%, more preferably from 20 to 80%, and most preferably from 40 to 75% by weight.

Another useful component of the enzyme inhibitor-containing dermatological composition may be a substance that is capable of immobilizing the component (including the enzyme inhibitor, the proposed emollient and/or other protective agent) in a desired area within or on the treated article. Since some of the proposed components of the composition are malleable or liquid at 20°C, they will tend to flow or migrate even when subjected to only slight friction. If they are applied to a user-contacting surface or other area of the absorbent article in a dissolved or liquid state, they will generally not remain in that area because they tend to migrate and overflow into an undesired area of the article.

If any of the components of the dermatological component migrate into the interior of the composition, this may have an undesirable effect on the absorbency of the core of the composition due to the hydrophobic properties, e.g., plasticizers or other components used in the dermatological component of the composition of the present invention. This also means that a larger amount of the dermatological component must be applied to the composition to achieve the desired effect. Increasing the content of the dermatological component not only increases the cost, but also increases the undesirable effect on the absorbency of the core of the composition and the undesirable carryover of the component during processing of the modified composition.

The immobilizing agent counteracts the tendency of the components of the dermatological composition to migrate or flow away by retaining them on the surface or in the area of the composition to which they are applied. This is due in part to the fact that the immobilizing agent raises the melting point and/or viscosity of the component above that of the mobile components. Because the immobilizing agent is usually miscible with the emollient (or soluble in the emollient by means of a suitable emulsifier), it can retain the emollient on the surface that contacts the user or in the area to which it is applied.

It is advantageous to lock the immobilizing agent to the surface that touches the user or to the area of the device to which it is applied. This can be achieved by using immobilizing agents that solidify rapidly after application to the device. Furthermore, external cooling of the device using fans, blowers, etc. can be used to accelerate the crystallization of the agent.

The immobilizing agent should have a melting profile such that the component is solid or semi-solid at ambient temperature. Thus, proposed immobilizing agents should have a melting point of at least 35°C. At this temperature, the immobilizing agent will not tend to migrate or flow. Proposed immobilizing agents have melting points of at least 40°C. Typically, the immobilizing agent will have a melting point in the range of 50°C to 150°C.

The immobilizing agents may be selected from a wide variety of substances, provided that the inhibitory properties of the dermatological component have the beneficial effect on the skin described herein. It is found that certain emollients or classes of emollients have such melting properties that they are suitable as immobilizing agents. Such substances may serve both the functions of an emollient and an immobilizing agent. The proposed immobilizing agent will belong to the group consisting of C ₄ -C ₂₂ fatty alcohols, C ₂ -C ₂₂ fatty acids, C ₂ -C ₂₂ fatty alcohol ethoxylates and mixtures thereof, having an average degree of ethoxylation of from 2 to 30. The proposed immobilizing agents include C 1 -C 18 fatty alcohols, preferably crystalline substances with a high melting point selected from the group consisting of cetyl alcohol, stearyl alcohol, behenyl alcohol and mixtures thereof. (The linear structure of these substances may accelerate solidification on the treated absorbent.) Mixtures of cetyl alcohol and stearyl alcohol are preferred. Other proposed immobilizing agents are C ₆ -C ₁₈ fatty acids, preferably from the group consisting of palmitic acid, stearic acid and mixtures thereof. Mixtures of palmitic and stearic acid are preferred. Other proposed immobilizing agents include C ₆ -C ₁₈ fatty alcohol ethoxylates with an average degree of ethoxylation of from 5 to 20. The fatty alcohols and fatty acids should be linear. Importantly, these proposed immobilizing agents, such as C ₆ -C ₁₈ fatty alcohols, increase the level of crystallization of the component, thereby accelerating crystallization on the substrate surface.

Other types of immobilizing agents that can be used herein are polyhydroxy esters of fatty acids, polyhydroxy amides of fatty acids, and mixtures thereof. The proposed esters and amides have three or more free hydroxyl groups on the polyhydroxy group and are generally nonionic in nature. Given the possible skin sensitization of users of the compositions on

9

9999 ····

99

9 9 9

9 9 9

9 9 9 • 9 9 9

99 to which the ingredient was applied, these esters and amides should be relatively mild and non-irritating to the skin.

Polyhydroxy fatty acid esters suitable for use in the present invention have the following formula:

O

II

R-C-0—Y — —n where R is a C5-C31 hydrocarbon group, typically a linear C7-C19 alkyl or alkenyl chain, more preferably a linear C9-C17 alkyl or alkenyl chain, most preferably a linear C11-C17 alkyl or alkenyl chain or mixtures thereof. Y is a polyhydroxy hydrocarbon group with a hydrocarbon chain having at least 2 free hydroxyls directly attached to the chain and n is at least 1. Suitable Y groups can be derived from polyols such as glycerol, pentaerythritol, sugars such as raffinose, maltodextrose, galactose, sucrose, glucose, xylose, fructose, maltose, lactose, mannose and erythrose, sugar alcohols such as erythritol, xylitol, maltitol, mannitol and sorbitol and anhydrides of sugar alcohols such as sorbitan.

One class of suitable polyhydroxy fatty acid esters includes certain sorbitan esters, particularly sorbitan esters of saturated C ₆ -C ₂ 2 fatty acids. These sorbitan esters usually contain, depending on the method by which they are manufactured, mixtures of mono-, di-, tri-, etc. esters. Illustrative examples of suitable sorbitan esters are sorbitan palmitates (e.g., SPAN 40), sorbitan stearates (e.g., SPAN 60), and sorbitan behenates, which ...

·· *· ·· • · Although these sorbitan esters typically contain mixtures of mono-, di- and triesters, plus some tetraesters, the mono- and di-esters usually predominate in these mixtures.

Another class of suitable polyhydroxy fatty acid esters includes certain glyceryl monoesters, particularly glyceryl monoesters of saturated C16- _C22 fatty acids such as glyceryl monostearate, glyceryl monopalmitate and glyceryl monobehenate.

Mixtures of glyceryl monoesters, like sorbitan esters, usually contain some di- and triesters. However, to be useful in the present invention, these mixtures should contain primarily glyceryl monoesters.

Another class of suitable polyhydroxy fatty acid esters includes sucrose esters of fatty acids, especially sucrose esters of C12-C22 fatty acids. Particularly suitable are sucrose monoesters and diesters, as well as sucrose mono- and distearates and sucrose mono- and dilaurates.

44

4 4 4

4 4 4

4 4 4

4 4 4

44

Polyhydroxy fatty acid amides suitable for use in the present invention have the following formula:

o R ₂ II IR—cn—z where R ¹ is H, C1-C4 hydrocarbon, 2-hydroxyethyl, 2-hydroxypropyl, methoxyethyl, methoxypropyl or mixtures thereof, especially C1- _C4 alkyl, methoxyethyl or methoxypropyl, more preferably C1 or _C2 alkyl or methoxypropyl, most preferably C1 alkyl (i.e. methyl) or methoxypropyl and R is a C5-C31 hydrocarbon group, usually a linear chain of C7-C19 alkyl or alkenyl, more preferably a linear chain of C1-C19 alkyl or alkenyl, most preferably a linear chain of C1-C19 alkyl or alkenyl, or mixtures thereof and Z is a polyhydroxy hydrocarbon having a linear chain with at least 3 hydroxyl groups directly attached to the chain. See US Patent 5,174,927 which describes these polyhydroxy fatty acid amides and their preparation.

The Z group is derived from a reducing sugar in a reductive amination reaction, preferably glycityl. Suitable reducing sugars are glucose, fructose, maltose, lactose, galactose, mannose and xylose. In addition to these sugars, high dextrose corn syrup, high fructose corn syrup and high maltose corn syrup can also be used. A mixture of sugar components for the Z group can be formed from these corn syrups.

«*«· • BB Β·

BBB B * BB B

B B BBBB

Β BBBB·· • B · · · ·

BBB BBBB ·· ·♦

The group Z is selected from the group consisting of -CH ₂ -(CHOH)„CH ₂ OH, -CH ₂ (CH ₂ OH)-((CHOH) _n .,)-CH ₂ OH, -CH ₂ OH-CH ₂ (CHOH) ₂ (CHOR ³ )-(CHOH)-CH 2 OH, where n is an integer from 3 to 5 and R ³ is H or a cyclic or aliphatic monosaccharide. Glycityls where n is 4, especially -CH 2 -(CHOH) ₄ -CH ₂ OH, are particularly suitable.

In the above formula, R ¹ may be, for example, N-methyl, N-ethyl, N-propyl, N-isopropyl, N-butyl, N-2-hydroxyethyl, N-methoxypropyl or N-2-hydroxypropyl. R ² may be chosen, for example, to form cocamides, stearamides, oleamides, lauramides, myristamides, capricamides, palmitamides, tallowamides and the like. The group Z may be 1-deoxyglucityl, 2-deoxyfructityl, 1-deoxymaltityl, 1-deoxylactityl, 1-deoxygalactityl, 1-deoxymannityl, 1-deoxymaltotriotityl and the like.

The most suitable polyhydroxy fatty acid amides have this general formula:

R2 —

O

II

C-

Ri

AND

N—CH ₂ OH

AND

-CHch ₂ —oh where R ¹ is methyl or methoxypropyl, R ² is a Cn-Ci ₇ alkyl or alkenyl group with a linear chain. These include N-lauryl-N-methyl glucamide, N-lauryl-N-methoxypropyl glucamide, N-cocoylN-methyl glucamide, N-cocoyl-N-methoxypropyl glucamide, N-palmityl-N-methoxypropyl glucamide, N-tallowyl-N-methyl glucamide or N-tallowyl-N-methoxypropyl glucamide.

• 444 ······· vJ · · · · · ·

4 44 4444444 4

As previously mentioned, some immobilizing agents require an emulsifier to dissolve in the plasticizer. This applies in particular to some glucamides, such as N-alkyl-N-methoxypropyl glucamides with an HLB value of at least 7. Suitable emulsifiers are generally those with an HLB value of less than 7. In this regard, it has been found that sorbitan esters, such as sorbitan stearate, which have an HLB value of 4.9 and lower are suitable for dissolving glucamide immobilizing agents in petrolatum. Other suitable emulsifiers are steareth-2 (polyethylene glycol ethers of stearyl alcohol, which are formed according to the formula CH ₃ (CH 2 ) i 7 (OCH ₂ CH 2 ) _n OH, where the average value of n is 2, sorbitan tristearate, isosorbide laurate and glyceryl monostearate. The emulsifier may be present in an amount that allows the immobilizing agent to dissolve in the plasticizer and form a homogeneous mixture. For example, an approximately 1:1 mixture of N-cocoyl-N-methyl glucamide and petrolatum, which does not normally dissolve into a single-phase mixture, will dissolve into a single-phase mixture upon addition of 20% Steareth-2 and sorbitan tristearate as emulsifier.

Other types of additives that can be used as immobilizing agents, either alone or in combination with the immobilizing agents mentioned above, are waxes such as carnauba wax, beeswax, beeswax, candelilla, paraffin, ceresin, esparto, ouricuri, saw wax, isoparaffin and other known mined and mineral waxes. The high melting point of these substances allows the component to be immobilized on the desired surface or area of the composition. Microcrystalline waxes are also good immobilizing agents. Microcrystalline waxes help to lock the low molecular weight hydrocarbons within the dermatological component. Paraffin wax is suitable. An example of an alternative immobilizing agent is paraffin wax, such as

Parrafin S.P. 434 by Strahl and Pitsch lne.

The amount of immobilizing agent to be included in the composition depends on a number of factors, including the content of active ingredients (e.g., plasticizers), the type of immobilizing agent, if used, the other components of the composition, whether an emulsifier is needed to dissolve the immobilizing agent in the other components, and other similar factors. If an immobilizing agent is used, then its content in the composition is generally from 5 to 90%. More preferably, the content of the immobilizing agent in the composition is from 5 to 50%, most preferably from 10 to 40%.

It is desirable that at least a portion of the topsheet of the device be made of a hydrophilic material that facilitates the transfer of fluids (e.g., urine). It is also desirable that the dermatological component be sufficiently wettable to permit rapid transfer of fluids through the topsheet. Hydrophobic dermatological components may also be used, but only to the extent that the desired properties of the topsheet are not compromised. (One option is, for example, to apply the component to only a portion of the topsheet.) This will reduce the likelihood that body exudates will drain from the thus modified topsheet before they can be absorbed through the topsheet into the absorbent core.

If a hydrophilic component is required, it may be necessary to add a hydrophilic surfactant (or a mixture of hydrophilic surfactants) to improve wettability, depending on the respective components used in the component. However, some immobilizing agents, such as N-cocoyl-N-methoxypropyl glucamide, have HLB values above 7 and are sufficiently wettable even without the addition of a hydrophilic surfactant. Other ···· · ·· ·· » ·· 4 · • 4 4 4

4 4 4 4

4 4 4 4

444 44 449 9444 »· « 4 4

9 4

4 9

4 4 4

44 immobilizing agents, such as C ₆ -C ₈ fatty alcohols with HLB values below 7, may require the addition of a hydrophilic surfactant to improve wettability. Similarly, a hydrophilic surfactant must be added to a hydrophobic emollient, such as petrolatum, if the ingredient is required to be hydrophilic. Of course, there is no point in considering wettability if the user-contacting surface is not the top layer of the composition or if the desired properties of the top layer are provided in some other way (e.g., by partial coating).

Suitable hydrophilic surfactants should be miscible with the other components of the dermatological component to form mixtures. Due to the possible hypersensitivity of the skin of users of absorbent articles with a dermatological component applied, these surfactants should be relatively mild and non-irritating. These hydrophilic surfactants should be non-ionic in order not to irritate the skin, but also to avoid undesirable effects on other parts of the treated article. For example, reduction in tensile strength, quality of adhesive bonds, etc.

Suitable nonionic surfactants should be immobile after application of the component to the composition and their HLB values should be in the range of 4 to 20, more preferably 7 to 20. To be immobile, these nonionic surfactants typically have a melting point higher than the temperatures to which the absorbent compositions are exposed during storage, transportation, sale and use, e.g. at least 30° C. In this regard, nonionic surfactants typically have melting points similar to the immobilizing agents previously described.

Suitable nonionic surfactants for use in the components applied to the composition, at least in the outlet region, are: alkyl glycosides, alkyl glycoside ethers as described in U.S. Patent 4,011,389, alkyl polyethoxylate esters such as Pegosperse 1000MS from Lonza, Inc., Fair Lawn, New Jersey, ethoxylated sorbitan mono-, di- and/or tri-esters of _C12 - _C18 fatty acids with an average degree of ethoxylation of from 2 to 20, preferably from 2 to 10, such as TWEEN 60 (sorbitan esters of stearic acid with an average degree of ethoxylation of 4), and condensation products of aliphatic alcohols with 1 to 54 moles of ethylene oxide. The alkyl chain of the aliphatic alcohol is generally linear in configuration and contains from 8 to 22 carbon atoms. Particularly suitable are the condensation products of alcohols with an alkyl group containing from 11 to 22 carbon atoms with 2 to 30 moles of ethylene oxide per mole of alcohol. Examples of such ethoxylated alcohols are the condensation products of myristyl alcohol with 7 moles of ethylene oxide per mole of alcohol, the condensation products of coconut alcohol (a mixture of fatty alcohols with alkyl chains of 10 to 14 carbon atoms) with 6 moles of ethylene oxide per mole of alcohol,

ethylene oxide. A number of suitable ethoxylated alcohols are commercially available, including Tergitol 15-S-9 (a condensation product of linear C11- _C15 alcohols with 9 moles of ethylene oxide) sold by Union Carbide Corporation, KYRO EOB (a condensation product of linear C13-C15 alcohols with 9 moles of ethylene oxide) from The Procter & Gamble Co., NEODOL surfactants from Shell Chemical Co., particularly NEODOL 25-12 (a condensation product of linear C12-C15 alcohols with 12 moles of ethylene oxide) and NEODOL 23-6.5T (a condensation product of linear C12-C13 alcohols with 6.5 moles of ethylene oxide which has been distilled to remove impurities), and especially PLURAFAC surfactants from BASF Corp., particularly PLURAFAC A-38 (a condensation product of linear C18 alcohol with 27 moles of ethylene oxide). (Some hydrophilic surfactants, especially ethoxylated alcohols such as NEODOL 25-12, can act as alkyl ethoxylate plasticizers).

• ·· ·

Other suitable examples of ethoxy alcohol surfactants are Brij surfactants and mixtures thereof, particularly Brij 72 (i.e. Steareth-2) and Brij 76 (i.e. Steareth-10). Furthermore, mixtures of ethoxylated cetyl alcohol and stearyl alcohol with an average degree of ethoxylation of from 10 to 20 can also be used as hydrophilic surfactants.

Another surfactant suitable for use in the composition is Aerosol OT, dioctyl ester of sodium sulfosuccinic acid sold by American Cyanamid Company.

Other suitable surfactants are silicone copolymers such as General Electric SF 1188 (polydimethylsiloxane polyoxyalkylene ether copolymer) and General Electric SF 1228 (silicone polyether copolymer). These silicone surfactants can be used in combination with other types of hydrophilic surfactants mentioned above, such as ethoxylated alcohols. These silicone surfactants have been found to be effective at concentrations as low as 0.1%, but more preferably up to 0.25 to 1.0% by weight of the component.

If a hydrophilic component is to be used, the amount of hydrophilic surfactant required to increase the wettability of the component to the desired level depends in part on the HLB value and the amount of immobilizing agent, if used, the HLB value of the surfactant used, and other such factors. To increase wettability, the component may contain from 0.1 to 50% hydrophilic surfactant. However, to increase wettability, the component will typically contain from 1 to 25%, more preferably from 10 to 20% hydrophilic surfactant.

The composition may contain other components commonly present in emulsions, ointments, solutions, suspensions, etc., of this type. These components include water, viscosity modifiers, perfumes, disinfectant antibacterial agents, antiviral agents, vitamins, pharmaceutical agents, film-forming agents, deodorants, opacifiers, astringents, solvents, preservatives, etc. In addition, stabilizers such as cellulose derivatives, proteins and lecithin may be added to extend the expiration of the composition. All of these substances are well known in the art as additives and may be used in appropriate amounts in these compositions.

If a water-based dermatological component is used, a preservative must be used. Suitable preservatives are propylparaben, methylparaben, benzyl alcohol, benzylkonium, tribasic calcium phosphate, BHT or acids such as citric, tartaric, maleic, lactic, malic, benzoic, salicylic, etc. Suitable viscosity-increasing agents include some of the substances described as effective immobilizing agents. Other suitable viscosity-increasing agents are, for example, alkylgalactomannan, silicon dioxide, talc, magnesium silicate, sorbitol, colloidal silicon dioxide, magnesium aluminum silicate, zinc stearate, wool wax alcohol, sorbitan sesquioleate, cetylhydroxyethylcellulose and other modified celluloses. Suitable solvents are propylene glycol, glycerin, cyclomethicone, polyethylene glycols, hexylene glycol, diol and solvents with multiple hydroxyl groups. Suitable vitamins are A, D3, E, B ₅ and E acetate.

IX. Application of the component to the device

The absorbent device must be designed to transfer, during wear, at least a portion of the protease inhibitor-containing dermatological component from the device to the user's skin. Therefore,

the dermatological component is applied, either directly to one or more surfaces that are in contact with the wearer or is applied at other locations or in another way so that it is readily available for transfer from one or more surfaces that are in contact with the wearer during wear. (For example, fabrics placed under the surface that contacts the wearer, encapsulated components, etc.) It goes without saying that in order to make the transfer of the component to the areas most exposed to stress more effective, it is necessary to apply the component to the parts of the top layer and cuff that are in contact with the buttocks, genitals, groin area and anal area. Additionally, the component may be applied to other areas of the composition to provide transfer to the hips, abdomen, back, waist, thighs, etc. Suitable methods include spraying, printing (e.g., flexographic printing), coating (e.g., slot coating, screen coating), extrusion, or a combination of these application methods, e.g., spraying the dermatological component onto a rotating surface, such as a press roll, which then transfers the component to the desired portion of the composition. The enzyme inhibitor-containing dermatological component may be applied as a solid by any of a number of methods, e.g., extrusion.

If the component is hydrophobic in nature, it may happen that when applied to the top layer of the composition, the layer will not be completely saturated with the component in the area corresponding to the outlet area of the composition. If the top layer is saturated with the component in the outlet area, then there is a great risk that the component will block the openings of the top layer, thereby reducing the ability of the top layer to transfer fluids to the absorbent core below. Saturation of the top layer is also undesirable for achieving a therapeutic and/or protective effect. Similarly, saturation of other components of the composition may not be desirable. Suitable methods of application are those in which ♦······· /Z ·«···*<

··· ··· 4444 44 the component is applied primarily to the outer surface of the top layer of the composition.

The minimum amount of enzyme inhibitor-containing component applied to the user-contacting surface of the invention corresponds to an amount sufficient to provide a therapeutic, protective and/or skin-conditioning effect. The amount of component applied depends on various factors, including the substance of the composition being treated, the relative proportion of the user-contacting surface not covered by the component, the composition of the component, etc. Generally speaking, if relatively hydrophobic components are used and are applied to the entire top layer, then the amount of component applied is in the range of 0.016 mg/cm ² to 2.33 mg/cm ² , more preferably from 0.16 mg/cm 2 to 1.55 mg/cm 2 . It is clear that a larger amount of dermatological component can be applied to other parts of the composition where fluid distribution is not impaired (e.g. cuffs, waist portion, side panels, etc.). It is also clear that relatively hydrophilic components can be applied to the top layer in larger quantities without significantly affecting the distribution properties. Conversely, a larger amount of hydrophilic component may be undesirable if it is applied to parts (e.g. cuff, waist) other than the top layer, where exudates could be sucked to the edges and subsequent leakage of fluids.

Because the ingredient is completely immobilized on the surface, only a small amount of it is needed to transfer an effective amount of enzyme inhibitor. We believe that these small amounts are sufficient to achieve the desired effect on the skin, as the ingredient is continuously, spontaneously released during wear. This means that the ability to use relatively low levels of the dermatological ingredient makes it possible to

Bb Bb ··

Β Β · · Β Β • · · · ·

Β Β · Β Β · • Β Β Β Β

Β Β·99 99 99 the top layer of the product is able to maintain its transfer properties in the area of fluid outflow.

The component may be applied to the user-contacting surface non-uniformly. By non-uniformly is meant that the amount, location, distribution pattern, etc., of the component may be different on the user-contacting surface and may also be different in other areas of the device. For example, to maintain the distribution properties of the top layer, it may be necessary to apply the component non-uniformly, especially if it is hydrophobic in nature. For this reason, some parts of the treated surface of the device (and its areas) must have a greater or lesser amount of the component, including parts to which the component is not applied. In one such embodiment, where the component is relatively hydrophobic, the surface of the top layer has areas to which the component has not been applied, particularly in the area of the top layer that corresponds to the crotch area. The crotch area of the device, as used herein, is a rectangle, as defined below, which is centered longitudinally and transversely at the crotch point. The crotch point is determined by putting on the device with the user standing and pulling the thread around the legs in a figure eight shape. The point of the device that corresponds to the intersection of the fibers is considered to be the crotch point. (It is understood that the crotch point is determined by placing the absorbent device on the user in a specified manner and determining the location where the crossed fibers touch the device.) For incontinence devices (e.g. diapers, adult incontinence devices), the length of the crotch area corresponds to 40% of the total length of the absorbent device (i.e. in the y dimension). For menstrual pads, the length of the crotch area corresponds to 80% of the total length of the absorbent device. The width of the crotch area is the same as the width of the widest part in

of the absorbent core measured at the crotch point. (The parts of the absorbent core are the materials that participate in the absorption, transfer, distribution • · 4 4 * 4 4 4 and/or storage of body fluids. The term absorbent core does not include the topsheet or backsheet of the absorbent article.) An example would be an incontinence article with a length of 50 cm and a core width in the crotch point area of 10 cm, the crotch area being rectangular, centered on the crotch point and having a length of 20 cm and a width of 10 cm.

It is surprising that when the component is applied non-uniformly to the top layer or part thereof (e.g. microscopic or macroscopic areas where no component is applied), the component is transferred to the user even in areas of the skin that correspond to the untreated areas of the top layer or part thereof. The amount and uniformity of the component transferred to the skin depends on a number of influences, including, for example, the method of application of the dermatological component, the contact of the user's skin and the treated area, the friction that occurs during wear between the user's skin and the treated area, the heat generated by the user that enhances the transfer of the component, the properties of the component, the substances that make up the component, etc.

A variety of patterns may be used in non-uniform application of the component, including, for example, the application of small droplets (e.g., by spraying), tiny dots (e.g., by gravure printing), longitudinal or transverse stripes (formed by contact slot coating), spirals that move transversely or longitudinally, etc., pattern printing, etc. In these embodiments, where the topsheet contains separate, unprocessed regions, the extent of the open portion of the topsheet area that corresponds to the crotch area may vary considerably. (The extent of the open portion of the topsheet is determined by (i) measuring the surface area of the topsheet that falls within the crotch area, (ii) measuring the total surface area of the unmodified area in that portion of the topsheet, and (iii) dividing the result of the measurement from (ii) by the result of the measurement from (i). As used herein, the term unmodified means that the area of the topsheet contains less than 0.0016 mg/ ^cm2 of the ingredient. In this regard, the extent of the open portion may be from 1% to 99%, from 5% to 95%, from 10% to 90%, from 15% to 85%, from 20% to 80%, from 25% to 75%, from 30% to 70%, or from 35% to 65%. The extent of the open portion necessary to achieve the desired effects of the ingredient and the desired distribution properties of the top layer, is largely determined by the properties of the component (primarily its composition and the ratio of hydrophobic to hydrophilic properties). The skilled artisan will appreciate that the desired extent of the open portion can be readily determined by simple experimentation.

Components that are relatively hydrophobic and should not be applied to areas of the top layer are first applied to the top layer by

of the agent in an amount of from 0.0078 mg/cm to 5.43 mg/cm, but preferably

0 o from 0.16 mg/cm to 3.88 mg/cm and preferably from 0.62 mg/cm to 3.1 mg/cm . It is clear that components that are relatively hydrophilic can be applied in larger amounts without significantly affecting the distribution properties of the top layer. Of course, compositions with a relatively high percentage of open areas in the crotch can also have larger amounts of the component applied without significantly affecting the distribution properties of the top layer.

In one proposed embodiment, the topsheet is coated with longitudinal strips of the component. These longitudinal strips (or spirals) are separated from each other by longitudinal strips where little or no component is applied. In these embodiments, each strip of component typically has a width of from 3 mm to 20 mm, more preferably from 3 mm to 13 mm, and the width of the strips not containing the component is typically from 3 mm to 26 mm, more preferably from 4 mm to 13 mm. These ranges are applicable for • · · * tttt conventional baby diapers. For larger products, such as adult incontinence products, these ranges may be larger.

The dermatological component may also be applied in a non-uniform pattern to other parts of the device. In such cases, the open area may be calculated from a rectangle defined by the perimeter of the dermatological component.

The component may be applied to the device at any time during assembly. For example, the component may be applied to the finished absorbent article before it is packaged. The component may also be applied to a given part (e.g., topsheet, cuffs, side panels, waistband, etc.) at the point of exchange or by the fabric supplier before it is combined with other parts into the finished absorbent article. The component may also be applied to other areas of the article so that it may transfer to one or more user-contact surfaces during wear.

The component is usually applied to the composition in its heated state, since its melting point is significantly higher than the ambient temperature. The component is usually heated to a temperature in the range of 35°C to 150°C, but preferably from 40°C to 100°C, before being applied to the composition. The enzyme inhibitor can be added to the component before or after heating. If it is added before heating, the temperature must be chosen so as not to denature it. Alternatively, the enzyme inhibitor can be added to the preheated component, which has already been partially cooled to a temperature that does not damage the enzyme inhibitor, but is sufficient to apply the component to the composition. The liquid component, after being applied to the composition, is allowed to cool and solidify. The procedure for applying the component is usually adapted so that cooling is active.

• · • * ·· ·

When applying the component to the device, slot coating, spray coating, gravure coating and extrusion are preferred. One of these methods is slot coating the top layer of the composite finished device.

X. Testing methods

A. Transfer of dermatological ingredient and protease inhibitor to the user's skin

Overview

This method utilizes a removable skin analog that is placed on the skin of a wearer for a period of time. The skin analog is then removed and the amount of dermatological component or protease inhibitor in a solvent extract of the analog is determined using conventional analytical procedures. This method is described for use with baby diapers containing a dermatological component with an enzyme inhibitor. The skilled artisan will be able to determine appropriate changes for other dermatological components, protease inhibitors, absorbents, or wearer types.

Tested individuals

According to the following characteristics, two approximately equal groups of male and female children should be selected. A sufficient number of children should be selected to ensure that at least fifteen individuals complete all parts of each test.

Conditions of incorporation ··«·

a. Healthy child.

b. Caregiver willing to refrain from using solutions, creams, powders, or other dermatological products in the diaper area for the duration of the test.

c. Children who wear diapers all day.

d. Caregiver willing to bathe the child the day before the test, but not before it is completed.

e. A caregiver who is willing to forgo swimming for the child from the evening before the test until it is completed.

Conditions of separation

a. The child has been sick for the past four days.

b. Diarrhea (loose stools) within four days before the test.

c. Medications that may increase bowel movements (e.g. oral antibiotics, antifungals, corticosteroids).

d. Damaged skin at or around the testing site (e.g. burns, active skin lesions, etc.).

e. Known allergies or irritations from adhesive or dermatological ingredients.

Material used In vivo transfer

Skin analogue:

Sample container dermatological tape - TEGADERM Tape 1622W available from 3M Health Cares, St. Paul, MN glass with cap from VWR Scientific, West Chester, PA under the designation 15900242

44 4

4 4 44444

4 · 4 444444

444 44 4444

444 44 444 4444 44 44

Tape release powder: Baby powder (containing only talc and fragrance) to Johnson & Johnson, New Brunswick, NJ

Surgical gloves: from Best Manufacturing Co., Menlo GA, under the designation 6005PFM

Extraction and analysis of dermatological components

Solvent: dichloromethane from Sigma-Aldrich, St.

Louis, MO, 27056-3

Stearyl alcohol: Aldrich 25876-8

1-Hexadecanol: Aldrich 25874-1

Preparation bottle: 10 ml

Gas chromatograph: Hewlett ionization detector is suitable

Packard model 5890

Column: Chrompack CP Sil-5 capillary column

CB, 2 meters x 0.25 mm silica glass with 0.12 pm film thickness (without replacements)

Instrumentation data of the system: must allow repeated determination of areas with the highest values

Extraction and analysis of a typical inhibitor (e.g. hexamidine) of an enzyme (e.g. protease)

Solvent:

Column preparation bottle:

dichloromethane from Sigma-Aldrich, St. Louis, MO, 27056-3 10 ml

Hewlett Packard Zorbax SB-CN Narrow Bore 5 pm, 2.1 x 150 mm with Waters • · • ·· ·

Bondapack CN 10 pm, guard column

3.9 x 20 mm

Instrumentation data of the system: must allow repeated determination of the areas with the highest values

Way

In vivo transmission

A. Confirmation from the caregiver that the child has been bathed within the last 24 hours and that no solutions, powders, etc. have been applied to the diaper area since then.

B. Putting on surgical gloves, placing the child on the table, and removing their diaper.

C. Turning the baby onto his stomach.

D. Remove the TEGADERM tape release liner and lightly dust the adhesive surface with J&J Baby Powder (wearing surgical gloves to avoid contaminating the tape). Apply a sufficient amount of powder to the entire tape area, excluding the edges. (This step is done to prevent the tape from adhering too aggressively to the baby’s skin.)

E. Figures 2a and 2b illustrate the placement of the TEGADERM tape, referred to herein as tape 700. Tape 700 is applied to the infant’s right buttock. Tape 700 is applied to the highest point of the infant’s buttock near the gluteal crease. To measure transfer over two time periods or to measure the effect of an additional diaper, a • · ·» ·· ·· * · · · · 9 9 9 9 9

9 9 9 · 9 ·· 9 9 9 ··· ··· ··· ··· ··· ··· ··· ··· ··· second tape 700. If a second tape 700 is used, it is applied to the left buttock in the same way.

F. Diaper changes are performed according to the following protocol: 3 hour transfer time - 1 diaper, 6 hour transfer time - 2 diapers (change every 3 hours), 24 hour transfer time ad lib by caregiver. For 24 hour transfer time, the following additional instructions must be followed:

1. Use only water and a washcloth to clean the diaper area during the test. Do not use baby wipes. Do not touch the area around the straps with your hands or a cleaning tool.

2. Do not use dermatological products (solutions, ointments, creams, soaps, etc.) during the test.

3. Do not bathe the child during the test.

4. Use only tested diapers.

5. Record the time of defecation and wash the child with water and a washcloth.

G. Record the application time of each tested diaper.

H. Call the child before the designated transmission time expires.

I. Remove the test diaper. If the child has had a bowel movement, the 700 strip should be removed and discarded (the child has completed the test and the data will not be included in the analysis). If the child has urinated, the 700 strip is acceptable for the analysis below.

• ·· ·

• ·· ♦· ·

J. The test operator removes the 700 strip by grasping its edge with tweezers and gently peeling it away from the skin, all while wearing surgical gloves.

K. Prepare a jar and carefully label the sample for later identification.

L. After completion of the test, store all samples in the analysis jars described below.

1. Extraction and analysis of dermatological component test samples

This method is intended for testing the proposed dermatological component, the component from Table 4. The skilled artisan will know what adjustments are necessary for the extraction and analysis of other dermatological components. In general: 1) one of the major components of the dermatological component is extracted from the skin analog using an appropriate solvent, 2) a gas chromatograph or other suitable quantitative analytical technique is used to determine the amount of the major component in the extract, 3) the amount of the dermatological component is calculated from the unit area by the amount of the major component in the extract and the area of the tape.

Internal standard/solvent

Prepare the internal standard/solvent by accurately weighing 100±2 mg of 1-hexadecanol into a small beaker. Dissolve the 1-hexadecanol in dichloromethane and transfer to a graduated cylinder. Rinse the beaker 3 times with dichloromethane and transfer each time to a graduated cylinder. Make up to volume and mix well. This solution will be used to make the internal standard and extract the dermatological component from the strips. A container with

OO · 9 9 · 9 9 · ·

O~> « 9 · 9 9 9 9

999 99 999 9999 99 solvent should be tightly closed to prevent its evaporation.

Standard calibration

Prepare a calibration standard of known concentration by accurately weighing (±0.1 mg) 10 ± 1 mg of stearyl alcohol into a 100 ml graduated cylinder. Record the mass of stearyl alcohol used. Add the internal standard/solvent to the cylinder and mix until dissolved. Make up to volume and mix again. The solvent container should be tightly closed to prevent evaporation. This solution will be used to determine the response of stearyl alcohol and the 1-hexadecanol internal standard during instrument calibration.

Preparation and calibration of the gas chromatograph

Install the column and check the gas flow through the column at 100°C and the injection port and detector at operating temperatures. The flow chromatograph will operate under the following conditions:

Carrier gas: hydrogen (helium can also be used), flow rate 1.5 ml/min.

Injection port: 325°C, flow rate 30 ml/min, partition cleaning 2 ml/min, glass wool insert, Merlin microseal.

Injected volume: 2 μΐ divided.

FID detector: 350°C, set gas flow according to manufacturer's instructions. Typical gas flow is 400 ml/min for air, 30 ml/min for hydrogen and 30 ml/min for additive gas.

·· ·· ·· • 9 9 9 9 9 9 9 9 · · » · · · · · * #»♦··· ·· ♦ 9 9 9 9 9

99 999 9999 99 99

Column thermostat:

100°C with an increment of 15°C/min to 325°C, takes 10 minutes.

Make sure all connections are tight and free of leakage. Turn on the detector and allow it to stabilize. Leave the column at 325°C for 30 minutes. Clean the syringe with dichloromethane. The syringe should also be flushed several times after each injection. Perform several blind runs with dichloromethane until you get a good baseline without spurious peaks. If spurious peaks appear or the baseline is not good, try to correct the problem.

Set up the instrument according to the calibration standard previously prepared. Follow the manufacturer's recommendations to determine the correct sequence of operations. Calculations should be performed in a manner similar to that described below.

Sample analysis procedure

1) Remove the cap from the sample vial and add 10 ml of extraction solvent/internal standard solution using the dispenser. Close the cap and swirl the contents so that the 700 strip is not stuck to the sides of the vial and is completely immersed in the solvent. Repeat with all samples.

2) Leave the samples for 16 hours (usually overnight).

3) Mix the contents of the vial. Using a pipette, transfer all of the sample extract into the labeled vial. Cap the vial. Close the vial and leave it until the analysis is complete. Repeat with all samples.

• 4 444 • 4 ·

4 ·

• 4 4

44 4 4

M

4 4 4

4

4

4 •44 4444 «4

4 4

4 4

4 ·

4 4 4

44

4) Load the vials in random order into the test machine and start the assay under the conditions described above. The first vial should be a dichloromethane blank. Several control standards should be included (about every 20th sample) to verify the accuracy of the measurement.

5) After the measurement is complete, check each chromatogram to confirm the correctness of the analysis. If there is a problem, try to identify and eliminate it. If necessary, re-analyze the samples.

Calculations

The number of micrograms of stearyl alcohol in the extract of each sample is calculated based on the relative response of the stearyl alcohol peak to the peak of the internal standard l-hexadecanol. The ratio of the peak areas is multiplied by the relative response factor (determined during instrument calibration) and the micrograms of internal standard in the extract to obtain the total number of g of stearyl alcohol in the sample.

Instrument calibration

Determination of the relative instrument response factor for stearyl alcohol and the internal standard, which are derived from the peak areas of stearyl alcohol and 1-hexadecanol in the chromatogram of the calibration standard.

Reaction factor (R _č ) = — massyst mass _sa area _sa ^x where area _vst ⁼ peak area for internal standard. area _sa = peak area for stearyl alcohol.

• · « ···· tt <

• · • · *

9 · ·« « ·· • ·· ·· · · · • · • » • ·

999 9999

99

9 9

9 9

9 9

9 9 9

99 wt^ = micrograms of internal standard used to prepare the internal standard/extraction solvent.

mass ⁼ micrograms of stearyl alcohol used to prepare the calibration standard

Sample calculations

The number of micrograms of stearyl alcohol in each sample is calculated from the peak area on the sample chromatogram according to the following equation:

area

Number of pg SA ——- x area^ weight^ _

where area^ = peak area of the internal standard.

area = area of the stearyl alcohol peak. mass _vgt ⁼ micrograms of internal standard used to prepare the internal standard/extraction solvent.

Calculate the transferred amount of dermatological component in mg/ ^cm2 where:

Transferred component = _0.001 X mg stearyl alcohol_ (SA concentration in component) X (tape area)

In the method described above, the concentration of stearyl alcohol in the composition is 41% and the dimensions of the tape are 4.4 cm x 4.4 cm.

Transferred component = (0.001 x pg stearyl alcohol in component)/(0.41 x

4.4 cm x 4.4 cm) = 0.000126 x pg stearyl alcohol (mg/cm ² )

2. Extraction and analysis of protease inhibitor in the test sample

This method is adapted for use with a dermatological composition containing a protease inhibitor from Table 1. The skilled artisan will know what adjustments are necessary for the extraction and analysis of other protease inhibitors. In general: 1) the protease inhibitor is released from the skin analog by the action of an appropriate solvent, 2) HPLC or other quantitative analytical technique is then used to determine the level of inhibitor in the extract, 3) the amount of protease inhibitor is calculated as a unit area per the amount of inhibitor in the extract and the area of the tape.

Preparation of standards

To prepare a 10 pg/mL hexamidine standard solution, weigh out 0.10 grams +/- 0.02 grams of hexamidine diisethionate and dissolve it in the mobile phase (10% crystalline acetic acid and 17.5% methanol) HPLC solution. Prepare additional hexamidine standards from the 10 pg/mL standard solution, see Table 2, and make up to 100 mL with HPLC mobile phase solution.

Table 3

Preparation of standards* • ·

standard hexamidine standard solution (ml) final volume (ml) nominal concentration (pg/ml) 1 5.0 100 0.5 2 10.0 100 1.0 3 25.0 100 2.5 4 50.0 100 5.0

Sample preparation

1. Place the test transfer strip into a 40 ml glass vial.

2. Add 10 ml of dichloromethane and close the bottle tightly.

3. Let the bottle shake for 30 minutes.

4. Remove the vial from the shaker, remove the vial cap and add 10 ml of HPLC mobile phase solution. Close the vial and shake again.

5. Shake the sample for 30 minutes to dissolve the hexamidine in the aqueous phase.

6. Allow the sample to settle for at least 30 minutes and the layers to separate.

7. After sample separation, remove the aqueous (top) layer with a syringe and filter it through a 0.45 pm filter.

Sample analysis

Chromatograph the standards and samples according to the conditions described in Table 3.

Table 4

Chromatographic conditions • ·

Mobile phase flow: 0.25 ml/min 1 Mobile phase: 10% crystalline acetic acid, I 17.5% methanol Injected volume: 10ml UV detector wavelength: 254 nm UV detector sensitivity: 1,000 AUFS UV detector filter: Λ0 with Time: 10.0 minutes

Calculations

1. Standard concentration (mg/ml):

Si(mg/ml) = W(mg)/100*(V,/100) (1)

W = mass of hexamidine for the stock standard solution

Vj = volume of hexamidine stock solution used to prepare the standard (Table I)

2. Calibration curve

A. Create a table of mg/mL hexamidine in each standard (Si) and the responses (peak area or peak height), R _Í5 for each of the solutions.

B. Create a calibration curve according to equation (2).

Rj = mSj + b (2) • ·

3. Tested samples

A. Calculate the amount of hexamidine (Hj) in the sample extracts using the measured response R and the calibration equation:

ífí = (R-b)/m (3)

B. Calculate the amount of hexamidine (H) in the samples in mg according to Equation 4.

H = H, * 10 (4)

C. Divide the amount of hexamidine (H) by the area of the tape to determine the concentration of hexamidine per unit area of the skin analog.

VII. Examples of embodiments of the invention

The following are examples of (a) applying a dermatological component to a diaper topsheet and (b) methods of the present invention that utilize compositions containing such topsheets. A similar approach can be used to modify other parts of such a composition.

Example 1

Preparation and testing of an absorbent article with a top layer containing a dermatological ingredient and a protease inhibitor

A. Preparation of the dermatological component

The dermatological component (component A) is formed by mixing the following components: (i) 99 parts of a warmed (i.e., liquid) base composition containing 58 parts of petroleum jelly (from Witco Corp., Greenwich, • · ni ··· ·· ·· ·· ♦ yl ··· ·» ··· ··· ·· ·· ··

CT as White Protopet), 41 parts stearyl alcohol (from Procter & Gamble Co., Cincinnati, OH as CO1897) and 1 part aloe extract (from Madis Botanicals, Inc., S. Hackensack, NJ as Veragel Lipoid in Kaydol), with (ii) 1 part hexamidine diisothionate (from Laboratories Serobilogiques, Pulnoy, France as Elestab HP100).

B. Preparation of the composition by contact slot coating

The component from Table 1 is placed in a heating tank with an operating temperature of 77°C. The component is then applied with a contact applicator (e.g. Meltex EP45) to the top layer of the composition in strips that run lengthwise. Exactly 5 strips are applied, each of which has a width of 0.54 cm. (i.e. in the transverse dimension of the composition) and a length of 25.4 cm and the applied amount is 1.19 mg/cm . The distance between the strips is 0.67 cm.

C. Testing the inhibitory properties of the modified composition

This example describes a method for testing a diaper for protease inhibitory activity. This example is not limiting, as other portions of other absorbent articles may be tested and other methods may be used for testing, using other solvents and other substrate systems.

Ten holes of 1.7 cm diameter are punched in the core area of the absorbent material impregnated with component A and these holes are also punched in a control material containing no inhibitor. Each of the punched circles is then tested for trypsin inhibitory activity. The top layer is removed from the punched circle and placed in a 1.5 ml centrifuge tube. The sample is left overnight in 0.75 ml of water. 0.125 ml of the supernatant is removed and added to a cuvette containing 0.025 ml of 160 nM human pancreatic trypsin in TRIS-HCl with 20 mM CaCl ₂ , pH 8.2 and incubated for 10 minutes at 25°C. Cbzarginine-p-nitroanilide substrate (0.025 ml of a 4 mM solution) is added to each cuvette and the test and control samples are allowed to incubate for 5 minutes. The change in absorbance at 405 nm is then monitored for each sample after 10 minutes. The results of the assay, shown in Table 5, indicate that the absorption medium containing the inhibitor causes a decrease in the measured trypsin activity (relative to the control medium, which is identical except that it contains no inhibitor).

Table 5

Change OH/min x 10' ³ Control means 8.292±0.6 Second means 3.804±2

Example 2

Ways to improve skin condition

An incontinent adult weighing 82.5 kg, using absorbents and suffering from mild erythema, wears an adult incontinence device similar to the diaper of Example 1 for at least 5 days. The device is changed in the manner usual for the user (usually every 4 to 5 hours during the day and before bedtime). During this time, there is no intervention by the user (e.g.

• ··· application of dermatological preparations). Towards the end of these 5 days, a reduction or disappearance of the erythema is observed.

Example 3

Ways to improve skin condition

A child weighing 16 kg and suffering from diaper rash and erythema wears the diapers from Example 1 at night for at least 5 days (i.e. uses the untreated composition during the day). The diapers are changed by the caregiver in the usual way. During this time, no interventions by the caregiver (e.g. application of dermatological preparations) occur. Towards the end of these 5 days, a reduction or disappearance of the rash and erythema is observed.

Example 4

How to maintain skin condition

A child weighing 12.5 kg and without diaper rash or erythema is evaluated for otitis media and prescribed systemic antibiotics. Based on experience with conventional (untreated) diapers, the caregiver expects the child to develop erythema and/or diaper rash due to looser stools. The diapers described in Example 1 are worn continuously during the course of antibiotics. No user intervention (e.g., application of dermatological preparations) occurs during this time. No erythema or diaper rash is observed in the child during the administration of antibiotics.

Descriptions of all patents, patent applications (and all patents that relate to the subject matter, as well as relevant published ...

foreign patent applications) and publications mentioned in this specification are incorporated herein by reference. However, we do not admit that any of the documents mentioned herein describe the proposed invention.

While particular embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that various other changes and modifications may be made without departing from the scope of the invention. It is our intention, therefore, to cover in the appended claims all such changes and modifications that fall within the scope of the invention.

Claims (20)

PATENT CLAIMS 1. An absorbent composition characterized in that at least a portion thereof comprises a protease inhibitor having an IC ₅₀ for measuring protease purification method, 30 μΜ or less. Composition according to claim 1, characterized in that the IC ₅₀ is from 0.00001 μΜ to 30 μΜ, preferably from 0.0001 μΜ to 20 μΜ, and most preferably from 0.001 μΜ to 10 μΜ. 3. The absorbent composition characterized in that at least a portion thereof comprises a protease inhibitor having an IC ₅₀ at a measuring method specific fecal protease 90 μΜ or less. Composition according to claim 3, characterized in that the IC ₅₀ is from 0.00001 μΜ to 90 μΜ, preferably from 0.0001 μΜ to 30 μΜ, and most preferably from 0.001 μΜ to 10 μΜ. 5. The absorbent composition, a portion of which comprises a protease inhibitor characterized in that it has an IC ₅₀ at a measuring method generally fecal protease 500 μΜ or less. The device according to claim 5, characterized in that the IC ₅ není is not more than 500 μΜ, preferably not more than 300 μΜ and most preferably not more than 100 μΜ. The composition of claims 1-6, wherein the protease inhibitor is selected from the group consisting of a serine protease inhibitor, a metalloprotease inhibitor, a cysteine protease inhibitor, an aspartyl protease inhibitor, and mixtures thereof. • 99 · 99 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9,999,9999 · · · · The composition of claims 1-7, wherein the protease inhibitor is selected from the group consisting of a soybean trypsin inhibitor, a limestone protease inhibitor (Phaseolus limensis), a corn protease inhibitor, a Bowman-Birk inhibitor, a human trypsin inhibitor from pancreas, bovine pancreatic trypsin inhibitor, egg white trypsin inhibitor, egg white ovomucoids containing ovoinhibitors, chymostatin, aprotinin, leupeptin and analogs thereof, amastatin and analogs thereof, antipain, antithrombin III, hirudin, cystatin, E-64 , _2- macroglobulin, α-antitrypsin, pepstatin and its analogs, apstatin, (2R) -2-mercaptomethyl-4-methylpentanoyl-b- (2-naphthyl) -Ala-Ala amide, (2R) -2-mercaptomethyl-4 -methyl-pentanoyl-Phe-Ala amide, Nacetyl-Leu-Leu-methioninal, N-acetyl-Leu-Leu-norleucinal, paminobenzoyl-Gly-Pro- _D -Leu- _D- Ala hydroxamic acid, 2 (R) (N) - (4-Methoxyphenylsulfonyl) -N- (3-pyridylmethyl) amino) -3-methylbutanhydride oxamic acid, hexamidine and its salts, pentamidine and its salts, benzamidine and its salts and derivatives, p-aminobenzamidine and its salts and derivatives, guanidinobenzoic acid and its salts and derivatives, and mixtures thereof. The composition of claim 5 or 6, wherein the protease inhibitor is 4- (2-aminoethyl) -benzenesulfonyl fluoride hydrochloride. Composition according to claim 1-9, characterized in that it comprises a protease inhibitor of at least from 0.0001% to 30% by weight, preferably from 0.0001% to 10% by weight. 4 4 4 4 4 44 4 4 · 4 4 4 4 7/444 44 4444 444 44 444 444 Composition according to claims 1-10, characterized in that the extract in part causes a reduction in the hydrolysis of the substrate by the protease in the absorbent composition test method. The composition of claims 1-11, further comprising a distribution system comprising a protease inhibitor and transferring the inhibitor to at least a portion of the skin of the user of the composition. The composition of claim 12, wherein the distribution system is a dermatological component that comprises a protease inhibitor of from 0.01% to 50% by weight and which is transmitted, at least partially, to the skin of the user during wear of the composition. Composition according to claim 12 or 13, characterized in that the dermatological component comprises a protease inhibitor of from 0.05% to 25% by weight, preferably from 0.1% to 10% by weight. The composition of claims 1-14, further comprising a user-contacting surface, a portion of which comprises a dermatological component with a protease inhibitor, and preferably the user-contacting surface is a topsheet. Absorbent composition, characterized in that the extract of a portion thereof reduces the hydrolysis of the substrate by the protease in the absorbent composition test method by at least 10%, more preferably by 20% and most preferably by 50 to 90%. 17. An absorbent composition comprising a substance selected from the group consisting of a soybean trypsin inhibitor, a limase protease inhibitor (Phaseolus limensis), a protease inhibitor Řice φ φ from maize, Bowman-Birk inhibitor, human pancreatic trypsin inhibitor, bovine pancreatic trypsin inhibitor, egg white trypsin inhibitor, egg white ovomucoids containing ovoinhibitors, chymostatin, aprotinin, leupeptin and analogs, amastatin and its analogues antipain, antithrombin III, hirudin, cystatin, E-64 and its analogs, and _2- macroglobulin, α-antitrypsin, pepstatin and its analogs, apstatin, (2R) -2-mercaptomethyl-4-methylpentanoyl-b- (2-naphthyl) - Ala-Ala amide, (2R) -2-mercaptomethyl-4-methylpentanoyl-Phe-Ala amide, N-acetyl-LeuLeu-methioninal, N-acetyl-Leu-Leu-norleucinal, p-aminobenzoylGly-Pro- _D -Leu- _D- Ala hydroxamic acid, 2 (R) - (N- (4-Methoxyphenyl sulfonyl) -N- (3-pyridylmethyl) amino) -3-methylbutanohydroxamic acid, hexamidine and its salts, pentamidine and its salts, benzamidine and its salts and derivatives, p-aminobenzamidine and its salts and derivatives, guanidinobenzoic acid and its salts and derivatives, TLCK, TPCK, acid tranexamic acid and mixtures thereof. 18. A composition according to claim 17 wherein the agent is selected from the group consisting of trypsin inhibitor soya, aprotinin, hexamidine, p-aminobenzamidine, leupeptin, pepstatin A, chymostatin, guanidinobenzoic acid derivatives, and mixtures thereof. 19. A method of reducing the proteolytic activity of a stool protease in an absorbent composition comprising (i) inserting a protease inhibitor into the stool-containing portion of the absorbent composition and (ii) applying the composition to a user so that the protease inhibitor contacts the stool protease . A method of reducing the proteolytic activity of a stool protease on the skin portion contacting an absorbent composition comprising (i) • 4,444 • 4,444 4 4 4 4 4 4 4 4 4 4 5 4 4 4 4 4 Inserting a protease inhibitor into a distribution system of an absorbent article, wherein the distribution system is capable of transferring at least a portion of the enzyme inhibitor to the skin of the user of the device and (ii) applying the absorbent article to the skin of the user.