Patent Publication Number: US-2006003943-A1

Title: Protease enzyme inhibitors

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
      This application is a continuation of U.S. application Ser. No. 10/285,071, filed Oct. 31, 2002, which claims priority under Title 35, United States Code 119(e) from Provisional Application Ser. No. 60/338,038 filed Nov. 13, 2001. 
    
    
     FIELD OF THE INVENTION  
      The present invention relates to an enzyme inhibitor conjugate which is capable of reversibly inhibiting one or more enzymes. The inhibitors of the present invention are used in formulations comprising one or more enzymes wherein the activity of said enzyme is suppressed by said inhibitor until said formulation is utilized by the consumer.  
     BACKGROUND OF THE INVENTION  
      Enzymes make up the largest class of naturally occurring proteins and have found wide utility in consumer products. The specificity of enzymatic activity and their almost inexhaustible catalytic behavior, for example, their ability to hydrolyze proteins, has been exploited by incorporating both naturally occurring and genetically engineered enzymes into cleaning compositions. Of the various types of enzymes, proteases are ubiquitous and have a wide range of applications.  
      Until now, proteases have been utilized to a lesser extent in personal care compositions wherein each ingredient must bear the burden of efficacy and utility. Enzymes, especially protease enzymes, lead to inherent composition instability in that the enzyme can and will attack any ingredient which can serve as a substrate. Controlling the ability of an enzyme to attack other composition ingredients while not inhibiting the ability of the enzyme to function once applied, has been a problem confronting formulators of enzyme-containing, especially protease enzyme-containing, personal care compositions.  
      There is therefore, a long felt need in the art for a means for controlling the activity of protease enzymes while the enzymes are contained in a product matrix. The means for controlling the enzyme must be efficient, stable, and highly reversible once the product matrix is applied. The present invention satisfies a long felt need in that it provides a protease enzyme inhibitor which serves as a means for holding an enzyme&#39;s catalytic activity in abeyance until a time wherein the formulator or consumer desires the enzyme to begin functioning.  
     SUMMARY OF THE INVENTION  
      The present invention meets the aforementioned needs in that it has been surprisingly discovered that enzyme activity can be reversibly inhibited by certain polymer conjugates, said conjugates suitable for use in compositions which contact human skin, as well as inert surfaces. It has been surprisingly discovered that the high molecular weight of the novel conjugates described herein prevents the enzyme-active substrate portion from penetrating into the skin, but does not inhibit or abate the ability of the substrate component from interacting with the targeted enzyme. In addition, the selection of various substituent groups which comprise the substrate portion allows the formulator to modulate the level of inhibition and prepare compounds which inhibit a specific species of enzyme or, in another iteration of the present invention, prepare conjugates which inhibit a wide range of enzymes.  
      The present invention relates to new compositions of matter which are capable of inhibiting enzymatic activity and which are compatible with exposure to human tissue. The present invention relates to compounds having the general formula: 
 
[Poly]—[(L) z —[R—CHO]] y  
 
 wherein [Poly] is a water-soluble, non-peptidic polymer component, L is an optionally present linking group, and RCHO is a substrate capable of interacting with one or more enzymes to reversibly inhibit said enzyme, and wherein said substrate comprises a peptide or peptide-like aldehyde. The index z is 0 when linking groups are absent and z is 1 when linking groups are present. 
 
      These and other objects, features, and advantages will become apparent to those of ordinary skill in the art from a reading of the following detailed description and the appended claims. All percentages, ratios and proportions herein are by weight, unless otherwise specified. All temperatures are in degrees Celsius (° C.) unless otherwise specified. All documents cited are in relevant part, incorporated herein by reference. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
      As described herein, each of the general aspects of the present invention will themselves have a plurality of aspects relating to variations of the main compound structure and which will be set forth and further described herein by the various embodiments encompassed within these various aspects. The non-limiting embodiments described herein of each aspect will be further characterized and illustrated by various non-limiting iterations relating to each embodiment. Specific examples of the herein described iterations will provide suitable enablement to make and to use all aspects of the present invention.  
      The term “substituted” is used throughout the specification and for the purposes of the present invention the term “substituted” is defined as “replacement of a hydrogen atom, two hydrogen atoms, or three hydrogen atoms from a carbon atom to form a moiety, or the replacement of hydrogen atoms from adjacent carbon atoms to form a moiety.” For example, a substituted unit that requires a single hydrogen atom replacement includes halogen, hydroxyl, and the like. A two hydrogen atom replacement includes carbonyl, oximino, and the like. Three hydrogen replacement includes cyano, and the like. The term substituted is used throughout the present specification to indicate that a moiety, inter alia, aromatic ring, alkyl chain, can have one or more of the hydrogen atoms replaced by a substituent. For example, 4-hydroxyphenyl is a “substituted aromatic carbocyclic ring”, and 3-guanidinopropyl is a “substituted C 3  alkyl unit.” 
      The following are non-limiting examples of moieties, which can replace hydrogen atoms on carbon to form a “substituted hydrocarbyl” unit:  
      i) —NHCOR 30 ;  
      ii) —COR 30 ;  
      iii) —COOR 30 ;  
      iv) —COCH═CH 2 ;  
      v) —C(═NH)NH 2 ;  
      vi) —N(R 30 ) 2 ;  
      vii) —NHC 6 H 5 ;  
      viii) ═CHC 6 H 5 ;  
      ix) —CON(R 30 ) 2 ;  
      x) —CONHNH 2 ;  
      xi) —NHCN;  
      xii) —OCN;  
      xiii) —CN;  
      xiv) F, Cl, Br, I, and mixtures thereof;  
      xv) ═O;  
      xvi) —OR 30 ;  
      xvii) —NHCHO;  
      xviii) —OH;  
      xix) —NHN(R 30 ) 2 ;  
      xx) ═NR 30 ;  
      xxi) ═NOR 30 ;  
      xxii) —NHOR 30 ;  
      xxiii) —CNO;  
      xxiv) —NCS;  
      xxv) ═C(R 30 ) 2 ;  
      xxvi) —SO 3 M;  
      xxvii) —OSO 3 M;  
      xxviii) —SCN;  
      xxix) —P(O)H 2 ;  
      xxx) —PO 2 ;  
      xxxi) —P(O)(OH) 2 ;  
      xxxii) —SO 2 NH 2 ;  
      xxxiii) —SO 2 R 30 ;  
      xxxiv) —NO 2 ;  
      xxxv) trihalomethyl having the formula: —CF 3 , —CCl 3 , —CBr 3 ;  
      xxxvi) and mixtures thereof;  
      wherein R 30  is hydrogen, C 1 -C 20  linear or branched alkyl, C 6 -C 20  aryl, C 7 -C 20  alkylenearyl, and mixtures thereof; M is hydrogen, or a salt forming cation.  
      The present invention is directed to a polymer conjugate comprising a substrate which is a peptide aldehyde or peptide-like aldehyde residue. The compounds of the present invention have the general formula: 
 
[Poly]—[(L) z —[R—CHO]] y  
 
 wherein [Poly] is a water-soluble, non-peptidic polymer component, L is an optionally present linking group, and RCHO is a substrate capable of interacting with one or more enzymes to reversibly inhibit said enzyme, and wherein said substrate comprises a peptide or peptide-like aldehyde. As can be seen from the formula above, the [Poly] unit is bonded to one or more enzyme substrate units at the opposite terminus from the aldehyde functional group. When a linking unit is present, the [Poly] unit will be attached to the linking unit, L, which will be attached similarly to the enzyme substrate unit. The index y indicates the number of aldehyde comprising units which are bonded to the polymer component. For embodiments of the present invention wherein the index y&gt;1, not all —RCHO units must be attached by the same linking unit if a linking unit is present, or alternatively, some —RCHO units may be attached via an L unit while others are directly bonded to the [Poly] component. 
 
      The first aspect of the overall invention relates to conjugates comprising one substrate unit (y=1), said compounds having the formula: 
 
[Poly]—(L) z —[R—CHO]
 
 of which there are two embodiments, one comprising a linking unit and one having no linking unit. 
 
      The second aspect of the overall invention relates to conjugates comprising two substrate units (y=2), said compounds having the formula: 
 
[Poly]—[(L) z —[R—CHO]] 2  
 
 one embodiment of which includes conjugates wherein the aldehyde comprising unit is connected at the terminal ends of a [Poly] backbone, said conjugates having the formula: 
 
[OHC—R]—(L) z —[Poly]—(L) z —[R—CHO]
 
 said embodiment described further herein below. 
 
      The third aspect of the overall invention relates to conjugates comprising more than two substrate units (y&gt;2).  
      [Poly] units are any non-peptide polymer backbone. One aspect of the present invention relates to [Poly] units which comprise a poly(ethylene glycol) unit, herein referred to as PEG units. The PEG units of the present invention have the formula: 
 
R 1 O(CH 2 CH 2 O) n —
 
 wherein R 1  is hydrogen, C 1 -C 4  alkyl, and mixtures thereof. The index n is from about 3 to about 200 and represents the average number of ethyleneoxy units present. In one embodiment of this aspect R 1  is hydrogen, while in another embodiment each R 1  is methyl. 
 
      In the first embodiment, the PEG units derive from poly(ethylene glycols) having the formula: 
 
HO(CH 2 CH 2 O) n H 
 
 non-limiting examples of which include PEG 200 (n=4), PEG 300 (n=6), PEG 400 (n=9), PEG 600 (n=13), PEG 1000 (n=23), PEG 2000 (n=45), and PEG 3400 (n=77). 
 
      In another embodiment, R 1  is methyl and the poly(ethylene glycols) are therefore, methyl capped ethers, MPEG&#39;s, represented by the formula: 
 
CH 3 O(CH 2 CH 2 O) n H 
 
 non-limiting examples of which include MPEG 350 (n=7), MPEG 550 (n=12), MPEG 750 (n=16), MPEG 2000 (n=45), and MPEG 5000 (n=113). 
 
      Another aspect of the present invention relates to units having the formula: 
 
R 1 O(R 2 O) n —
 
 wherein R 2  comprises C 2 -C 4  alkylene, and mixtures thereof. The index n is from about 3 to about 200 and represents the average number of alkyleneoxy units present. In one embodiment of this aspect R 1  is hydrogen, iterations of which include R 2  units which are a mixture of 1,2-propylene and 1,3-propylene and in another iteration R 2  units which are a mixture of ethylene and 1,2-propylene units. These iterations which describe mixtures of R 2  units can be represented by various means, for example, the formula:  
                 
 
 wherein R′ is hydrogen when standing for ethylene units and R′ is methyl when standing for 1,2-propylene units or, for example, by the formula:  
                 
 
 when ethylene, 1,2-propylene, and 1,3-propylene units are present; n′+n″=n. This last iteration can be considered to include block copolymers having the formula:  
                 
 
 wherein x+y+z=n. A non-limiting example of this iteration includes a block copolymer having an average molecular weight of about 8400 available ex BASF as Pluronic® F-68. 
 
      This last iteration can also be considered to include block copolymers having the formula:  
                 
 
 wherein x+y+z=n. A non-limiting example of this iteration includes a block copolymer having an average molecular weight of about 4550 available ex BASF as Pluronic® 10-R8. 
 
      A third aspect of the present invention as it relates to [Poly] units encompasses R 2  units which entirely comprise, 1,2-propylene, 1,3-propylene, 1,2-butylene, or 1,4-butylene units and wherein R 1  is hydrogen. Non-limiting examples of the iteration of this aspect wherein R 1  is hydrogen and each R 2 unit comprises 1,2-propylene includes [Poly] units derived from the following poly(propylene glycols); PPG 425 (n=7), PPG 725 (n=12) and PPG 1000 (n=17) all of which are available ex Aldrich Chemical.  
      While in another embodiment of this third aspect, each R 1  is methyl, iterations of which include R 2  units which are a mixture of 1,2-propylene and 1,3-propylene and in another iteration R 2  units which are a mixture of ethylene and 1,2-propylene units.  
      A fourth aspect of the [Poly] units relates to branched polymer backbones and are useful for preparing conjugates of the third overall aspect of the present invention which are conjugates having y&gt;2 as described herein above. Said conjugates comprise units having the formula: 
 
—O(R 2 O) n′ (R 3 O) n″ —
 
 wherein R 3  units comprise a branched unit having the formula:  
                 
 
 wherein R 2  C 2 -C 4  alkylene, and each u is independently from 0 to 10 provided at least one u is not equal to 0. The branched backbone conjugates of the present invention can be conveniently derived from [Poly] backbone starting materials having the formula: 
 
HO(R 2 O) n′ (R 3 O) n″ H 
 
 wherein the indices n′+n″=n as defined herein above. 
 
      A non-limiting example of a conjugate utilizing this aspect of the [Poly] units has the general formula:  
                 
 
 wherein n′+n″+n′″=n. 
 
      L is a linking unit which is capable of providing a link between the [Poly] backbone and the aldehyde comprising unit [R—CHO]. Linking units can be any suitable combination of atoms except highly reactive or unstable combinations, non-limiting examples of which include, O—O bonds, N—O bonds, and the like. The index z is 1 when linking units are present.  
      Non-limiting examples of suitable linking units includes units selected from the group consisting of:  
      i) -[C(R 7 ) 2 ] p -; wherein p is from 1 to 22;  
      ii) -[C(R 7 )2] p (CH═CH) q -; wherein p is from 0 to 12; q is from 1 to 6;  
      iii) —C(X)-;  
      iv) —OC(X)-;  
      v) —C(X)O—;  
      vi) -[C(R 7 ) 2 ] q C(X)X(R 8 O) p -; wherein p is from 0 to 12; q is from 1 to 6;  
      vii) -(OR 8 ) p XC(X)[C(R 7 ) 2 ] q -; wherein p is from 0 to 12; q is from 1 to 6;  
      viii) —C(X)NR 7 —;  
      ix) —C(X)R 8 C(X)-;  
      x) —C(X)NR 7 C(X)-;  
      xi) —C(X)NR 7 R 8 NR 7 C(X)-;  
      xii) —NR 7 C(X)-;  
      xiii) —NR 7 C(X)NR 7 —;  
      xiv) —R 8 NR 7 C(X)NR 7 —;  
      xv) —R 8 NR 7 C(X)NR 7 R 8 —;  
      xvi) —R 8 NR 7 —;  
      xvii) —R 8 O—;  
      xviii) —(R 8 ) u C(X)(R 8 ) u -;  
      xix) -(R 8 ) u OC(O)(R 8 ) u -;  
      xx) -(R 8 ) u C(O)O(R 8 ) u -;  
      xxi) -(R 8 ) u OC(O)O(R 8 ) u -;  
      wherein R 7  is hydrogen, C 1 -C 22  linear or branched alkyl; C 1 -C 22  cycloalkyl; C 1 -C 22  linear or branched fluoroalkyl; C 2 -C 22  linear or branched alkenyl; C 6 -C 22  aryl; C 7 -C 22  alkylenearyl; and mixtures thereof; R 8  is C 2 -C 20  linear or branched, substituted or unsubstituted alkylene; C 7 -C 20  alkylenearylene; C 6 -C 20  substituted or unsubstituted arylene; X is oxygen, sulfur, ═NR 7 , and mixtures thereof; u is 0 or 1.  
      One aspect of the linking units of the present invention relates to L units which comprise a carbonyl unit. A non-limiting example of a linking group comprising conjugate of the first aspect of the invention wherein y=1 has the formula:  
                 
 
 wherein n is the same as defined herein above. 
 
      Another aspect of liking units relates to urea units, for example, formed by reacting an —RCHO unit with an activated MPEG as follows:  
                 
 
 which utilizes linking unit (xiv) described herein above. 
 
      Another aspect of liking units relates to secondary amine units, for example, formed by reacting an —RCHO unit with an activated MPEG as follows: 
 
CH 3 O (CH 2 CH 2 O) n CH 2 CH 2 —OMs+H 2 N—RCHO→CH 3 O(CH 2 CH 2 O) n CH 2 CH 2 —NH—RCHO 
 
 which utilizes linking unit (i) described herein above. 
 
      The aldehyde comprising units of the present invention are units having the capacity to reversibly inhibit enzyme activity, said units having the formula: 
 
—R—CHO 
 
 wherein R is a unit which facilitates the interaction of the aldehyde functionality with an enzyme. The aldehyde comprising unit is also referred to herein as “the substrate portion of the conjugate” which is capable of differential binding with enzymes. For example, one specific —RCHO unit may bind tightly with one particular protease enzyme while weakly interacting with another. Or alternatively, the substrate may bind well to all protease enzymes, as well as weakly to other enzymes, inter alia, lipase enzyme. 
 
      One aspect of R units, used herein to exemplify the relationship of R unit components, has the formula: 
 
—J—W—K—
 
 wherein the unit J is an amino acid residue having the formula:  
                 
 
 wherein for this aspect R 4  is selected from the group consisting of 1-methylethyl, 1-methyl-propyl, 2-methylpropyl, benzyl, substituted benzyl, phenyl, substituted phenyl, and mixtures thereof. W is a spacer unit as defined herein below. K is a unit which is part of the terminal aldehyde unit and when taken together with said aldehyde unit has the formula:  
                 
 
 wherein for this aspect R 5  units are selected from the group consisting of 1-methylethyl, 1-methyl-propyl, 2-methylpropyl, and mixtures thereof. It is, however, convenient to consider the R units to be taken together with the aldehyde functionality to form an —RCHO unit which has the formula:  
                 
 
 The elements J, W, and K which comprise the —RCHO units themselves function to position the substrate portion of the conjugate within the active site of the target enzyme and thereby provide a means for abating enzyme activity and a means for stabilizing enzyme comprising compositions. The formula herein above represents the broadest aspect of —RCHO substrate units which can be considered to comprise an amino acid linked to an amino aldehyde by a suitable W spacer unit. R 4  and R 5  are each independently a hydrophobic unit selected from the group consisting of substituted or unsubstituted: 
 
      i) phenyl;  
      ii) benzyl;  
      iii) naphthyl;  
      iv) linear or branched C 1 -C 7  alkyl;  
      v) and mixtures thereof.  
      The carbon atoms to which R 4  and R 5  are bonded may be chiral or a mixture of S and R amino acids can be used to form a racemic mixture. One aspect of the aldehyde substrate units relates to compounds derived from S-amino acids and S-amino aldehydes having the formula:  
                 
 
      Without wishing to be limited by theory, the enzymes, which are inhibited by the conjugates of the present invention, interact primarily with the terminal aldehyde moiety. However, it has also been discovered that the relative position and size of the R 4  and R 5  units are important to enzyme binding activity. W units are spacer units which modulate the distance between the R 4 , R 5  and —CHO units thereby affecting the manner in which the substrate portion of the conjugate aligns into an enzyme binding site. In general, the formulator may select any W unit which results in a substrate that is capable of providing reversible protease enzyme inhibition.  
      The first aspect of the present invention as it relates to the selection of W units encompasses units which are comprised of two amino acids. This selection results in a substrate portion of the conjugate which is a peptidic unit having the formula:  
                 
 
 wherein each R 6  is independently selected and can be any moiety which comprises amino acids, non-limiting examples of which include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, p-benzoyl-phenylalanine, β-(1-naphthyl)-alanine, β-(2-naphthyl)-alanine, β-cyclohexylalanine, 3,4-dichlorophenylalanine, 4-fluorophenyl-alanine, 4-nitrophenylalanine, 2-thienylalanine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, 3-benzothienylalanine, 4-cyanophenylalanine, 4-iodophenylalanine, 4-bromophenylalanine, 4,4′-biphenylalanine, ornithine, sarcosine, pentafluorophenylalanine, and β,β-diphenylalanine, and mixtures thereof. 
 
      Common amino acids result in R 6  units selected from the group consisting of hydrogen, methyl, hydroxymethyl, 1-hydroxylethyl, 1-methylethyl, 1-methyl-propyl, 2-methylpropyl, benzyl, 4-hydroxyphenyl, 2-pyrrolidinyl, thiomethyl, (methylthio)methyl, carboxymethyl, carboxyethyl, 3-indolylmethyl, 4-aminobutyl, 3-guanidinopropyl, 1H-3-imidazolyl-, and mixtures thereof.  
      One embodiment of this aspect, which selects W units comprising two amino acids, has the formula:  
                 
 
 wherein each R 6  is hydrogen, methyl, ethyl, and mixtures thereof. One iteration of this embodiment which utilizes three chiral amino acids and a chiral amino aldehyde results in a series of polymer conjugates having the formula:  
                 
 
 wherein W derives from the amino acids glycine and alanine (Gly-Ala) and L is a carbonyl linking unit. 
 
      The following are non-limiting examples of the reversible enzyme inhibitors of the present invention.  
                                   TABLE I                       n 1     R 4     R 5     n 1     R 4     R 5                    44   benzyl   1-Me-propyl   112   benzyl   1-Me-propyl       44   benzyl   2-Me-propyl   112   benzyl   2-Me-propyl       44   benzyl   1-Me-ethyl   112   benzyl   1-Me-ethyl       44   benzyl   methyl   112   benzyl   methyl       44   benzyl   ethyl   112   benzyl   ethyl       44   4-HO-phenyl   1-Me-propyl   112   4-HO-phenyl   1-Me-propyl       44   4-HO-phenyl   2-Me-propyl   112   4-HO-phenyl   2-Me-propyl       44   4-HO-phenyl   1-Me-ethyl   112   4-HO-phenyl   1-Me-ethyl       44   4-HO-phenyl   methyl   112   4-HO-phenyl   methyl       44   4-HO-phenyl   ethyl   112   4-HO-phenyl   ethyl                   1 The index n represents the average number of units present.             
 
      Table I relates to conjugates wherein R 4  comprises an aryl or substituted aryl unit, for example, an amino acid derived from phenylalanine or tyrosine.  
      However, the R 5  unit may also comprise a substituted or unsubstituted aryl unit. The following non-limiting examples relate to a second iteration of the embodiment of W units derived from (Gly-Ala).  
                                   TABLE II                       n 1     R 4     R 5     n 1     R 4     R 5                    44   benzyl   phenyl   112   benzyl   phenyl       44   benzyl   4-HO-phenyl   112   benzyl   4-HO-phenyl       44   benzyl   4-Me-phenyl   112   benzyl   4-Me-phenyl       44   benzyl   benzyl   112   benzyl   benzyl       44   benzyl   4-HO-benzyl   112   benzyl   4-HO-benzyl       44   benzyl   4-Me-benzyl   112   benzyl   4-Me-benzyl       44   4-HO-phenyl   phenyl   112   4-HO-phenyl   phenyl       44   4-HO-phenyl   4-HO-phenyl   112   4-HO-phenyl   4-HO-phenyl       44   4-HO-phenyl   4-Me-phenyl   112   4-HO-phenyl   4-Me-phenyl       44   4-HO-phenyl   benzyl   112   4-HO-phenyl   benzyl       44   4-HO-phenyl   4-HO-benzyl   112   4-HO-phenyl   4-HO-benzyl       44   4-HO-phenyl   4-Me-benzyl   112   4-HO-phenyl   4-Me-benzyl                   1 The index n represents the average number of units present.             
 
      Table II relates to conjugates wherein R 4  comprises an aryl or substituted aryl unit example, an amino acid derived from phenylalanine or tyrosine and R 5  comprises an amino acid having an aryl unit.  
      However, the R 5  unit may also comprise a substituted or unsubstituted alkyl unit. The following non-limiting examples relate to a third iteration of the embodiment of W units derived from (Gly-Ala).  
                                   TABLE III                       n 1     R 4     R 5     n 1     R 4     R 5                    44   1-Me-propyl   benzyl   112   1-Me-propyl   benzyl       44   2-Me-propyl   benzyl   112   2-Me-propyl   benzyl       44   1-Me-ethyl   benzyl   112   1-Me-ethyl   benzyl       44   methyl   benzyl   112   methyl   benzyl       44   ethyl   benzyl   112   ethyl   benzyl       44   1-Me-propyl   4-HO-phenyl   112   1-Me-propyl   4-HO-phenyl       44   2-Me-propyl   4-HO-phenyl   112   2-Me-propyl   4-HO-phenyl       44   1-Me-ethyl   4-HO-phenyl   112   1-Me-ethyl   4-HO-phenyl       44   methyl   4-HO-phenyl   112   methyl   4-HO-phenyl       44   ethyl   4-HO-phenyl   112   ethyl   4-HO-phenyl                   1 The index n represents the average number of units present.             
 
      Table III relates to conjugates wherein R 4  comprises an alkyl or substituted alkyl unit, example, an amino acid derived from leucine or isoleucine and R 5  comprises an amino acid having an aryl unit.  
      However, both the R 4  unit and R 5  unit may comprise a substituted or unsubstituted alkyl unit. The following non-limiting examples relate to a fourth iteration of the embodiment of W units derived from (Gly-Ala).  
                                   TABLE IV                       n 1     R 4     R 5     n 1     R 4     R 5                    44   1-Me-propyl   1-Me-propyl   112   1-Me-propyl   1-Me-propyl       44   2-Me-propyl   2-Me-propyl   112   2-Me-propyl   2-Me-propyl       44   1-Me-ethyl   1-Me-ethyl   112   1-Me-ethyl   1-Me-ethyl       44   methyl   methyl   112   methyl   methyl       44   ethyl   ethyl   112   ethyl   ethyl       44   1-Me-propyl   1-Me-propyl   112   1-Me-propyl   1-Me-propyl       44   2-Me-propyl   2-Me-propyl   112   2-Me-propyl   2-Me-propyl       44   1-Me-ethyl   1-Me-ethyl   112   1-Me-ethyl   1-Me-ethyl       44   methyl   methyl   112   methyl   methyl       44   ethyl   ethyl   112   ethyl   ethyl                   1 The index n represents the average number of units present.             
 
      A second aspect of the W unit according to the present invention relates to the selection of units which are isosteric or which provide sufficient special orientation between the R 4  unit and R 5  unit within an enzyme active site.  
      One embodiment of the aspect of the W unit relates to 4-aminobenzoic acid, 3-aminomethylbenzoic acid, and 2-aminoethylbenzoic acid, each of which can be further substituted.  
      Iterations of this aspect of W units include units selected from the group consisting of:  
      i) 4-aminobenzoic acids having the formula:  
                 
 
      ii) 3-aminomethylbenzoic acids having the formula:  
                 
 
      iii) 2-aminoethylbenzoic acids having the formula:  
                 
 
 wherein R 10  can be any moiety which can substitute for hydrogen as defined herein above. 
 
      However another embodiment of this aspect relates to W units wherein the carboxy terminus is extended from the aryl ring, for example a conjugate having the general formula:  
                 
 
      Another aspect of the W units relates to non-aryl carbocyclic and heterocyclic units suitable as a unit which provides the proper alignment of R 4  and R 5  within an enzyme receptor site. Non-limiting examples of rings, which can substitute for phenyl when properly substituted in a like manner of the second aspect of the W units, include cyclopentane, cyclohexane, furan, thiophene, pyrrole, imidazole, pyran, thiazole, pyridine, pyridazine, pyrimidine, pyrazine, piperidine, piperazine, morpholine, and the like.  
      As described herein above, the second overall aspect of the present invention relates to conjugates having two substrate units (y=2). Conjugates of this type can be readily formed from [Poly] starting materials having two reactive sites, for example PEG units described herein above. The first embodiment of [Poly] units relates to PEG units derive from poly(ethylene glycols) having the formula: 
 
HO(CH 2 CH 2 O) n H 
 
 which can be used to form second aspect conjugates having the formula: 
 
OHCR—(L) z —O(CH 2 CH 2 O) n —(L) z —RCHO 
 
 wherein the units R and L and the indices n and z are the same as defined hereinabove. 
 
      A non-limiting example of one embodiment of this aspect has the general formula:  
                 
 
      The following are non-limiting examples of procedures for preparing the conjugates of the present invention.  
     EXAMPLE 1  
     Preparation of N-Cbz-Phe-Gly-Ala-Leu-OCH 3    
      N-Cbz-Phe-Gly (7.06 g, 19.8 mmol), HCl Ala-Leu-OCH 3  (5 g, 19.8 mmol), HOBT (3.24 g, 21.8 mmol), and triethylamine (3.0 mL, 21.8 mmol) are dissolved in dichloromethane (133 mL). A solution of dicyclohexylcarbodiimide (DCC) (4.49 g, 21.8 mmol) in dichloromethane is added to the reaction mixture and the reaction mixture is stirred for at least 2 h. The solution is filtered to remove the solid dicyclohexylurea which forms and the dichloromethane solution is washed with water, NaHCO3, and 10% citric acid. The organic layer is dried over magnesium sulfate, the solution is filtered and the solvent is removed in vacuo. The off-white solid is re-crystallized from ethyl acetate to generate the desired product, N-Cbz-Phe-Gly-Ala-Leu-OCH 3  , in 36% yield.  
     Preparation of Phe-Gly-Ala-Leu-OCH 3    
      N-Cbz-Phe-Gly-Ala-Leu-OCH 3  (1.9 g, 3.4 mmol) is dissolved in ethanol (200 mL) and Pd/C is added. The heterogeneous mixture is purged of oxygen, then a balloon containing hydrogen gas is fitted to the flask and the reaction is stirred overnight at room temperature. The result solution is filtered through Celite and the solvent removed in vacuo to afford Phe-Gly-Ala-Leu-OCH 3  which is re-crystallized from ethyl acetate to give the desired product in 80% yield.  
     Preparation of MPEG-Phe-Gly-Ala-Leu-OCH 3    
      Poly(ethylene glycol) methyl ether having an average molecular weight of 5000 (MPEG 5000 ) (13.7 g, 2.75 mmol) is weighed into a reaction vessel and is dissolved into toluene (100 mL) at 40° C. Under inert atmosphere, a solution of phosgene in toluene (1.6 mL, 1.93 M) is added. After the addition is complete, the reaction mixture is stirred for 12-18 hours at 40° C. to form MPEG chloroformate. In a separate vessel, Phe-Gly-Ala-Leu-OCH 3  (1.27 g, 3.025 mmol) is dissolved in dichloromethane (70 mL) at 30° C. Once dissolved, poly(N-vinylpyridine) (3.75 g 33 mmol) is added. MPEG chloroformate is then added dropwise to the mixture. The reaction mixture is stirred for 12-24 hours. The solution is filtered to remove the solid phase base and the product is precipitated by adding the filtrate to 1000 mL of diethyl ether. The white precipitate is collected and 21.4 g (90% yield) of MPEG-Phe-Gly-Ala-Leu-OCH 3  is obtained.  
     Preparation of MPEG-Phe-Gly-Ala-Leucinol  
      MPEG-Phe-Gly-Ala-Leu-OCH 3  (10 g, 1.8 mmol) is dissolved in ethanol (20 mL) and CaCl 2  (0.4 g, 3.6 mmol) is added. The solution is purged with inert gas and cooled to −5° C. NaBH 4  (0.3 g, 7.2 mmol) is added in portions while maintaining the temperature below 0° C. After addition is complete, the reaction is stirred for two hours then 0.1 M HCl (3 mL) is added until gas evolution stops. The product is precipitated by adding the filtrate to 1000 mL of diethyl ether. The resulting solid is collected by filtration and washed 3×100 mL with diethyl ether. The product, MPEG-Phe-Gly-Ala-Leucinol, is thus afforded in 92% yield.  
     MPEG-Phe-Gly-Ala-Leu-H conjugate  
      M-PEG-Phe-Gly-Ala-Leucinol (5 g, 0.92 mmol) is dissolved in dichloromethane (11.2 mL), and a solution of sodium bromide (0.104 g, 1.0 mmol) in water (1.1 mL) is added. The heterogeneous solution is stirred and cooled with an ice bath at 0° C. 2,2,6,6-Tetramethyl-1-piperidinyloxy, free radical (TEMPO) (1.1 mg, 6.4 □mol) is added followed by potassium bicarbonate (0.534 g, 5.34 mmol). In a separate flask, a solution of sodium hypochlorite (0.58 g, 1.0 mmol, 13% solution) in water (1 mL) is pre-cooled to 0° C. and added drop wise to the heterogeneous reaction mixture. After stirring for 30 min, the reaction is quenched with a solution of sodium thiosulfate (0.33 g, 1.34 mmol) in water (3 mL). The organic layer is separated from the aqueous layer, and the aqueous layer is extracted 3×25 mL with dichloromethane. The combined dichloromethane fractions are washed with brine, and are dried over magnesium sulfate. The dichloromethane is concentrated in vacuo and precipitated onto 500 mL of diethyl ether. The M-PEG-Phe-Gly-Ala-Leu-H is isolated by filtration and washed 3×25 mL with fresh diethyl ether. The powder is dried under vacuum.  
     Formulations  
      The use of enzymes, especially in personal care compositions is known. The enzymes, particularly protease enzymes, are believed to provide a desquamatory benefit wherein the outer layers of skin are removed thereby providing a fresh health skin surface. The compositions of the present invention comprise one or more enzymes and one or more enzyme inhibitors according to the present invention depending upon the type of formulation or, importantly, the temperature of exposure (e.g. storage conditions).  
      The use of an enzyme inhibitor not only acts to stabilize the enzyme until it is needed, but the inhibitor stabilizes the enzyme towards auto-digestion of itself and other components. As a guideline for preparing compositions comprising protease enzymes and enzyme inhibitors, the relative inhibition constant, K i , of the inhibitor is taken into consideration when formulating said compositions. Inhibition constants need not be exact such that approximate values are readily adaptable to formulation parameters. One convenient method for measuring and obtaining K i  values is described in “Kinetics of Subtilisin and Thiosubtilisin”, M. Philipp et al.,  Molecular &amp; Cellular Biochemistry,  51, pp. 5-32 (1983 ).  
      One embodiment of the present invention utilizes inhibitors with a K i  of from 10 nM to 25,000 nM, while another aspect includes a narrower range of from 50 nM too 5,000 nM. A typical range which is convenient for most applications comprises a K i  within the range of from 100 nM to 1,000 nM.  
      Depending upon the K i  and respective molecular weights of the enzyme and inhibitor, the amount of inhibitor present will vary, however, it is convenient for the formulator to begin within the range of from 2 moles of inhibitor to 1 mole of enzyme to a ratio of 10 mole of inhibitor to 1 moles of enzyme.  
      For the purposes of the present invention the inhibition constant, K i , is defined herein as:  
         K   i     =         [   E   ]     ⁡     [   I   ]         [     E   ⁢           ⁢   I     ]           
 
 wherein [E], [I], and [EI] are the concentrations of the unbound enzyme, the free inhibitor, and the enzyme-inhibitor complex respectively. 
 
      The compositions of the present invention comprise:  
      a) from about 0.001% to about 0.5% by weight, one or more protease enzymes;  
      b) from about 0.001% to about 0.5% by weight, one or more protease enzyme inhibitors according to the present invention; and  
      c) the balance carriers and adjunct ingredients.  
      Non-limiting examples of adjunct ingredients include polyhydric alcohols, inter alia, glycerine; osmo-protectants; lyotropic stabilizers; pH modifiers and buffering agents; polymeric thickeners; skin active agents, inter alia, vitamins; emollients, silicone oils, emulsifiers, surfactants, anti-microbial; sun-screening agents; and the like  
      The following are non-limiting examples of compositions according to the present invention.  
     Moisturizing Body Wash  
     
       
         
           
               
               
             
               
                   
                 TABLE V 
               
             
            
               
                   
                   
               
               
                   
                   
               
               
                   
                 weight % 
               
            
           
           
               
               
               
               
               
            
               
                 Ingredients 
                 2 
                 3 
                 4 
                 5 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Glycerin 
                 4.0 
                 — 
                 — 
                 — 
               
               
                 PEG-6 Caprylic/Capric Glycerides 
                 4.0 
                 — 
                 — 
                 — 
               
               
                 Palm Kernal Fatty acids 
                 3.0 
                 — 
                 — 
                 — 
               
               
                 Sodium Laureth-3 Sulphate 
                 45.0 
                 12.0 
                 15.0 
                 8.0 
               
               
                 Cocamide MEA 
                 3.0 
                 — 
                 — 
                 — 
               
               
                 Cocamidopropyl betaine 
                 — 
                 8.0 
                 10.0 
                 15.0 
               
               
                 Sodium Lauroamphoacetate 
                 25.0 
               
               
                 APG Glucoside  1   
                 — 
                 — 
                 2.0 
                 1.0 
               
               
                 Soybean Oil 
                 10.0 
                 — 
                 — 
                 — 
               
               
                 Polyquaternium-10 (JR30M) 
                 0.7 
                 0.25 
                 — 
                 — 
               
               
                 Polyquaternium-7 (Mackam 55) 
                 — 
                 — 
                 — 
                 0.7 
               
               
                 Protease 
                 0.1 
                 0.025 
                 0.05 
                 0.1 
               
               
                 Conjugate  2   
                 0.1 
                 0.025 
                 0.05 
                 0.1 
               
               
                 Deionized water 
                 Balance 
                 Balance 
                 Balance 
                 Balance 
               
               
                 pH 
                 7 
                 6.5 
                 7 
                 8.5 
               
               
                   
               
               
                     1  Plantacare 2000 ex Cognis.    
               
               
                     2  Polymer conjugate according to Example 1.    
               
            
           
         
       
     
     Body Lotion  
     
       
         
           
               
               
             
               
                   
                 TABLE VI 
               
             
            
               
                   
                   
               
               
                   
                   
               
               
                   
                 weight % 
               
            
           
           
               
               
               
               
               
            
               
                 Ingredients 
                 6 
                 7 
                 8 
                 9 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Glycerine 
                 8.0 
                 8.0 
                 10.0 
                 12.0 
               
               
                 Isohexadecane 
                 3.0 
                 3.0 
                 3.0 
                 6.0 
               
               
                 Niacinamide 
                 — 
                 3.0 
                 5.0 
                 6.0 
               
               
                 Isopropyl isostearate 
                 3.0 
                 3.0 
                 3.0 
                 3.0 
               
               
                 Sepigel 305  1   
                 3.0 
                 3.0 
                 3.0 
                 3.0 
               
               
                 Petrolatum 
                 4.0 
                 4.0 
                 4.0 
                 2.0 
               
               
                 Nylon 12 
                 2.0 
                 2.0 
                 2.5 
                 2.5 
               
               
                 Dimethicone  2   
                 2.0 
                 2.0 
                 2.5 
                 2.5 
               
               
                 Sucrose polycottonseed oil 
                 1.5 
                 1.5 
                 1.5 
                 1.5 
               
               
                 Stearyl Alcohol 97% 
                 1.0 
                 1.0 
                 1.0 
                 1.0 
               
               
                 D-panthenol 
                 1.0 
                 1.0 
                 1.0 
                 1.0 
               
               
                 D,L-□-tocopherol acetate 
                 1.0 
                 1.0 
                 1.0 
                 1.0 
               
               
                 Cetyl Alcohol 95% 
                 0.5 
                 0.5 
                 0.5 
                 1.0 
               
               
                 Behynyl alcohol 
                 1.0 
                 1.0 
                 1.0 
                 0.5 
               
               
                 EMULGADE PL 68/50 
                 0.4 
                 0.4 
                 0.5 
                 0.5 
               
               
                 Stearic acid 
                 0.15 
                 0.15 
                 0.15 
                 0.15 
               
               
                 PEG-100 stearate  3   
                 0.15 
                 0.15 
                 0.15 
                 0.15 
               
               
                 Protease 
                 0.005 
                 0.005 
                 0.025 
                 0.1 
               
               
                 Conjugate  4   
                 0.005 
                 0.005 
                 0.025 
                 0.1 
               
               
                 Deionized water 
                 Balance 
                 Balance 
                 Balance 
                 Balance 
               
               
                 pH 
                 7 
                 7 
                 7.5 
                 7 
               
               
                   
               
               
                     1  Comprises .pPolyacrylamide, Isoparaffin, and Laureth-7 ex Seppic.    
               
               
                     2  DC1403 ex Dow Corning.    
               
               
                     3  MYRJ 59 ex Uniqema.    
               
               
                     4  Polymer conjugate according to Example 1.    
               
            
           
         
       
     
     Moisturizing Facial Cream or Lotion  
     
       
         
           
               
               
               
             
               
                   
                 TABLE VII 
               
             
            
               
                   
                   
               
               
                   
                   
               
               
                   
                 weight % 
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Ingredients 
                 10 
                 11 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Glycerine 
                 12.0 
                 5.0 
               
               
                   
                 Isohexadecane 
                 5.0 
                 5.0 
               
               
                   
                 Niacinamide 
                 5.0 
                 7.0 
               
               
                   
                 Isopropyl isostearate 
                 2.0 
                 2.0 
               
               
                   
                 Sepigel 305  1   
                 3.0 
                 3.0 
               
               
                   
                 PEG 4000 
                 — 
                 10.0 
               
               
                   
                 Polymethylsilsesquioxane 
                 2.0 
                 2.0 
               
               
                   
                 Dimethicone  2   
                 3.0 
                 2.0 
               
               
                   
                 Sucrose polycottonseed oil 
                 1.0 
                 1.0 
               
               
                   
                 Stearyl Alcohol 95% 
                 0.5 
                 0.5 
               
               
                   
                 D-panthenol 
                 1.0 
                 1.0 
               
               
                   
                 D,L-□-tocopherol acetate 
                 1.0 
                 1.0 
               
               
                   
                 Cetyl Alcohol 95% 
                 1.0 
                 1.0 
               
               
                   
                 Cetearyl glucoside 
                 0.5 
                 0.5 
               
               
                   
                 Titanium dioxide 
                 0.3 
                 0.3 
               
               
                   
                 Stearic acid 
                 0.15 
                 0.15 
               
               
                   
                 PEG-100 stearate  3   
                 0.15 
                 0.15 
               
               
                   
                 Protease 
                 0.05 
                 0.05 
               
               
                   
                 Conjugate  4   
                 0.05 
                 0.05 
               
               
                   
                 Deionized water 
                 balance 
                 balance 
               
               
                   
                 pH 
                 7 
                 7 
               
               
                   
                   
               
               
                   
                     1  Comprises .pPolyacrylamide, Isoparaffin, and Laureth-7 ex Seppic.    
               
               
                   
                     2  DC1403 ex Dow Corning.    
               
               
                   
                     3  MYRJ 59 ex Uniqema.    
               
               
                   
                     4  Polymer conjugate according to Example 1.    
               
            
           
         
       
     
     Facial Moisturizing Cream  
     
       
         
           
               
               
             
               
                   
                 TABLE VIII 
               
             
            
               
                   
                   
               
               
                   
                   
               
               
                   
                 weight % 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Ingredients 
                 12 
                 13 
                 14 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Glycerine 
                 3.0 
                 5.0 
                 10.0 
               
               
                   
                 Petrolatum 
                 3.0 
                 3.0 
                 — 
               
               
                   
                 Cetyl Alcohol 95% 
                 1.5 
                 1.5 
                 1.0 
               
               
                   
                 Dimethicone copolyol  1   
                 2.0 
                 2.0 
                 2.0 
               
               
                   
                 Isopropyl palmitate 
                 1.0 
                 1.0 
                 0.5 
               
               
                   
                 Carbomer 954 
                 0.7 
                 0.7 
                 0.7 
               
               
                   
                 Dimethicone  2   
                 1.0 
                 1.0 
                 1.0 
               
               
                   
                 Stearyl Alcohol 97% 
                 0.5 
                 0.5 
                 1.0 
               
               
                   
                 Stearic acid 
                 0.1 
                 0.1 
                 0.1 
               
               
                   
                 PEG-100stearate  3   
                 0.1 
                 0.1 
                 0.1 
               
               
                   
                 Titanium dioxide 
                 0.3 
                 0.3 
                 0.3 
               
               
                   
                 Protease 
                 0.005 
                 0.025 
                 0.1 
               
               
                   
                 Conjugate  4   
                 0.005 
                 0.025 
                 0.1 
               
               
                   
                 Deionized water 
                 Balance 
                 Balance 
                 Balance 
               
               
                   
                 pH 
                 7 
                 7 
                 7.5 
               
               
                   
                   
               
               
                   
                     1  DC 3225C ex Dow Corning.    
               
               
                   
                     2  DC 200/350 cs ex Dow Corning.    
               
               
                   
                     3  MYRJ 59 ex Uniqema.    
               
               
                   
                     4  Polymer conjugate according to Example 1.