Cross-References to Additional Materials This application incorporates by reference in their entirety for all purposes the following U. S. patents: No. 5,355, 215, issued October 11,1994 ; No. 6,486, 947, issued November 26,2002 ; No. 6,097, 025, issued August 1, 2000; No. 6,326, 605, issued December 4,2001 ; No. 6,488, 892, issued December 3,2002 ; No. 6,466, 316, issued October 15,2002 ; No. 6,483, 582, issued November 19,2002 ; No. 6,258, 326, issued July 10,2001 ; No. 6,503, 719, issued January 7,2003 ; No. 6,317, 207, issued November 13,2001 ; No. 6,297, 018, issued October 2,2001 ; No. 6,310, 687, issued October 30,2001 ;. and No. 6,159, 425, issued December 12,2000.

This application incorporates by reference in their entirety for all purposes the following U. S. patent applications: Serial No. 09/759,711, filed January 12,2001 ; Serial No. 09/349,733, filed July 8,1999 ; Serial No. 09/768,742 ; Serial No. 09/770,724, filed January 25,2001 ; Serial No. 09/777,/343, filed February 5,2001 ; Serial No. 09/710,061, filed November 10,2000 ; Serial No. 09/768,661, filed January 23,2001 ; Serial No. 09/596,444, filed June 19,2000 ; Serial No. 09/844,655, filed April 27,2001 ; Serial No. 10/000,172, filed November 30,2001 ; Serial No. 10/061,416, filed February 1, 2002; Serial No. 10/003,030 ; filed October 29,2001 ; Serial No. 10/108,656, filed March 27,2002 ; Serial No. 10/012,255, filed November 12,2001 and Serial No. 10/218,897, filed August 13,2002.

This application also incorporates by reference in their entirety for all purposes the following additional materials: K. E. van Holde, PHYSICAL BIOCHEMISTRY (2 ed. 1985); William Bains, BIOTECHNOLOGY FROM A TO Z (1993); Richard P. Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (6 ed. 1996); Paul Horowitz & Winfield Hill, THE ART OF ELECTRONICS

(1980); Joseph. Lakowicz, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY (2 ded. 1999); Bob Sinclair, EVERYTHING'S GREAT WHEN IT SITS ON A CHIP: A BRIGHT FUTURE FOR DNA ARRAYS, 13 THE SCIENTIST, May 24,1999, at 18; and Charles R. Cantor and Paul R. Schimmel, BIOPHYSICAL CHEMISTRY (1980).

Field of the Invention The invention relates to enzyme assays, and more particularly to protease assays using luminogenic protease substrates that include a rhodamine-based luminophore.

Background Protease enzymes include a number of families of proteolytic enzymes that are capable of catalytically hydrolyzing peptide bonds. Principal groups of proteases include metalloproteases, serine proteases, cysteine proteases, and aspartic proteases.

Proteases, in particular serine proteases, are involved in a number of physiological processes, such as blood coagulation, fertilization, inflammation, hormone production, the immune response, and fibrinolysis.

Numerous disease states are caused by and/or can be characterized by alterations in the activity of specific proteases and/or their inhibitors. Therefore, the measurement of these alterations is often clinically significant in the treatment and management of the underlying disease states. For example, emphysema, arthritis, thrombosis, cancer metastasis, and some forms of hemophilia result from the lack of regulation of serine protease activities (see, for example, Textbook of Biochemistry with Clinical Correlations, John Wiley and Sons, Inc. New York, 1993).

Viral proteases have been identified in cells suffering from viral infections.

Such viral proteases can include, for example, HIV protease, typically associated with AIDS, and/or NS3 protease, typically associated with Hepatitis C. These viral proteases may play a critical role in the life cycle of the infecting virus.

Certain proteases have also been implicated in cancer metastasis. Increased synthesis of the protease urokinase has been correlated with an increased ability to metastasize in many cancers. Urokinase acts on plasminogen to generate plasmin, which in turn can cause the degradation of the proteins in the extracellular matrix through which the metastasizing tumor cells invade. Plasmin can also activate

collagenases, thus promoting the degradation of the collagen in the basement membrane surrounding the capillaries and lymph system, also allowing tumor cells to invade into target tissues (see for example, Dano et al. Adv. Cancer. Res. 44,139, 1985).

The ability to measure changes in the activity of specific proteases may be clinically significant in the treatment and management of many underlying disease states. Proteases, however, have generally proven difficult to assay. Typical approaches may include ELISA assays (Enzyme-Linked ImmunoSorbent Assay), which use labeled antibodies that bind the protease enzyme, and RIA assays (Radioimmunoassays), which use various radioactive-labeled substrates. However, protease assays that employ naturally occurring substrates for enzyme detection may be both expensive and difficult to perform.

Some approaches to protease detection have employed chromogenic and/or luminogenic protease substrates (i. e. , substrates that exhibit a change in, or the occurrence of, color or luminescence when cleaved by protease activity). Such substrates typically rely upon a cleavage-induced change in the structure of a chromophore or luminophore that changes the spectroscopic properties of the chromophore or luminophore. In particular, amino-substituted fluorophores have been used to prepare enzyme substrates featuring a peptide side chain bound via an amide linkage. Protease-mediated cleavage of the amide regenerates the free fluorophore (see Scheme 1 below). Cleavage Site Cleavage Site Cleavage NH NH Peptide Cleavage Site Peptide NH \ O O I 3 Scheme 1. Fluorogenic protease substrates.

Luminogenic substrates generally are preferred over chromogenic substrates because luminescence-based assays generally are more sensitive than colorimetric assays. Coumarin-based and rhodamine 110-based fluorogenic substrates may be used to determine protease activities. However, coumarin-based substrates exhibit low extinction coefficients that limit their utility. Additionally, the autofluorescence exhibited by most biological samples generally leads to high background noise, particularly in microscopic assays, due to the short excitation and emission wavelengths of the coumarin cleavage products. These features seriously limit the assay sensitivity of coumarin-based protease substrates (See, A. Keppler-Hafkemeyer, U. Brinkmann, 1. Pastan, Biochemistry 1998,37, 16934; S. Kumar, Clin. Exp.

Pharmacol. Physiol. 1999,26, 295; A. G. Porter, R. U. Janicke, Cell Death Differ.

1999,6, 99; H. R. Stennicke, G. S. Salvesen, J. Biol. Chem. 1997,272, 25719).

Protease substrates that incorporate a rhodamine 110 fluorophore may be used (see U. S. Patent Nos. 4,557, 862 and 4,640, 893, both to Mangel et al. ) to help overcome the limitations associated with coumarin-based protease substrates.

However, the resulting bis-peptide-substituted rhodamine compounds also exhibit significant drawbacks. The rhodamine-based substrates feature two protease-cleavable peptide residues, requiring multiple independent hydrolytic steps to yield the free fluorophore. In addition, the intermediate product of the first hydrolysis is significantly less fluorescent than the free fluorophore (see Scheme 2). The multiple step hydrolysis combined with the weak fluorescence of the intermediate cleavage product creates complicated enzyme kinetics, making fluorescence measurements more difficult to perform, and limiting the linear dynamic range of the resulting enzyme assays. Further, rhodamine 110, the product that results from complete hydrolysis, is poorly retained in cells, greatly limiting the applicability of rhodamine 110-based substrates to flow cytometric analyses. Although lipophilic rhodamine 110- based protease substrates have been developed to address the above problems, the weaker fluorescence of their cleavage products significantly reduces the sensitivities of the resulting enzyme assays (see, for example, U. S. Patent No. 5,576, 424 to Mao et al. (1996); U. S. Patent No. 6,248, 904 to Zhang et al. (2001); U. S. Patent No.

6,342, 611 to Weber et al. (2002); and U. S. Patent No. 6,335, 429 to Cai et al. (2002)).

In addition, monopeptide rhodamine 110-based protease substrates may exhibit a poor enzyme turnover rate, and altered cellular localization profiles, in addition to their reduced sensitivities. peptide PeptideyNH NH Peptide HN 0,, NH Protease" 0 OH \OH Protease Non-fluorescent moderately fluorescent T HpN/O/NHZ 0 OH Strongly fluorescent

Scheme 2. Two-step enzymatic cleavage of (peptide) 2Rhl l0.

Summary The invention provides systems, including compositions, kits, and methods, suitable for performing enzyme assays, such as protease assays. The compositions may include derivatives of rhodamine 110 that are substituted at the xanthylium nitrogens by a peptide moiety and a morpholine-4-carbonyl moiety, respectively. These compositions may be luminogenic substrates for a large variety of protease enzymes, with utility in a variety of sensitive protease enzyme assays.

Brief Description of the Drawings Figure 1 is a graph showing the absorption spectrum of RhIlO-MPL in aqueous buffer (pH = 7. 0).

Figure 2 is a graph showing the fluorescence emission spectrum of RhllO- MPL in aqueous buffer (pH = 7.0).

Figure 3 is a graph showing the fluorescence emission spectrum of a sample containing Compound 9 before (bottom curve) and after (top curve) the addition of caspase-3, a protease enzyme involved in apoptosis.

Figure 4 is a graph showing the time-dependent increase in fluorescence intensity of Compound 9 upon addition of caspase-3, as described in Example 21.

Figure 5 is a graph demonstrating the significantly higher enzyme turnover rate exhibited by Compound 9 (Ac-DEVD-RhllO-MPL) (curve A) relative to (Ac- DEVD) 2Rhl 10 (curve B), as described in Example 22.

Figure 6 is a chart demonstrating the detection of caspase-3 activity related to apoptosis using Compound 9, as described in Example 23.

Figure 7 is a chart demonstrating the cell number response of detected caspase-3 activity in JurKat cells using Compound 9, as described in Example 24.

Figure 8 is a chart demonstrating the dose response of camptothecin on JurKat cell apoptosis measured using Compound 9, as described in Example 24.

Figure 9 is a chart showing the time course response of detected camptothecin- induced JurKat cell apoptosis measured using Compound 9, as described in Example 24.

Figure 10 is a chart showing the higher sensitivity of Compound 9 relative to to (Ac-DEVD) 2Rhll0 for detection of human recombinant caspase-3 activity in whole cells, as described in Example 25.

Figures 11-13 are reaction schemes showing a synthetic pathway for a stepwise synthesis of Ac-DEVD-RhllO-MPL (Compound 9), as described in Examples 1-9.

Figure 14 is a reaction scheme showing a synthetic pathway for a two-step synthesis of Ac-DEVD-Rhl 10-MPL (Compound 9).

Detailed Description The invention provides systems, including compositions, kits, and methods, suitable for performing protease assays.

The compounds of the invention may be useful as luminogenic protease enzyme substrates. The compounds may be used to detect protease activity in a sample. The compounds of the present invention generally comprise a peptide linked

to rhodamine moiety through a xanthylium amine group, and a morpholine moiety linked to the rhodamine through the remaining xanthylium amine group. The particular rhodamine may be selected so as to provide one or more desired spectral or other properties. The particular peptide alternatively or additionally may be selected so that it is recognized and cleaved by a particular protease.

The compounds of the invention may include protease substrates generally described by the formula:

where Rl is a covalently bound moiety that, upon cleavage by an appropriate enzyme, generates a luminescent rhodamine-based reaction product having the formula:

Substituent R'typically is a monovalent radical that may be formally derived by the removal of a hydroxyl from a carboxylic acid functional group on an amino acid, or from a carboxylic acid of an amino acid of a peptide. The carboxylic acid functional group may be the terminal carboxylic acid of the peptide chain (the C- terminus). The peptide may be a linear or branched peptide. Where R'includes more than one amino acid, Rl may be a peptide radical having less than about 50 amino acids, and more typically less than about 30 amino acids. Rl may be a peptide radical having 1-4 amino acids. Rl may include 2 to about 12 amino acids, 2 to about 10 amino acids, 2 to about 8 amino acids, 2 to about 6 amino acids, or 2 to about 4 amino acids, among others.

Substituents R2-R5 are independently hydrogen, halogen, Cl-C6 alkyl, Cl-C6 alkoxy, sulfonic acid, or carboxylic acid. As used herein,"sulfonic acid"and "carboxylic acid"may include the protonated functional group, the deprotonated and ionic functional group, and salts of the functional group. The carboxylic acid or sulfonic acid functional group may be"masked"or protected so as to render the resulting compound more cell-permeable. Typically, such protecting groups include esters of alcohols having 1-6 carbons or esters of acyloxyalkyl groups, preferably acetoxymethyl esters. Alternatively, one or more of R2-R5 may be-L-RX or-L-SC, where Rx is a reactive functional group, Sc is a conjugated substance, and L is a covalent linking moiety. At least two of substituents R2-R5 may be halogen substituents. For example, at least two of R2-R5 may be chloro substituents.

Alternatively, at least two of R2-R5maybe fluoro substituents.

If one or more of R2-R5 are nonhydrogen, the nonhydrogen typically is one or the other of R3 and R4. Derivatives of the dyes that are 5-or 6-carboxy derivatives or that are prepared from the 5-or 6-carboxy derivatives are generally more readily available, both commercially and due to their relative ease of synthesis. Although the compounds of the invention may be present as a mixture of 5-and 6-carboxy isomers or their derivatives, methods are available, including high-resolution chromatographic methods, for separating the 5-and 6-substituted isomers of the compounds of the invention, according to methods known in the art.

Where R'is, or is derived from, an amino acid, the amino acid may be a natural or synthetic amino acid, optionally modified on any free amine or carboxylic acid by a suitable protecting group. Suitable amine protecting groups include carbobenzyloxy (CBZ), 9-fluorenylmethyl carbamate (Fmoc), p-toluenesulfonyl (tosyl), succinoyl, glutaroyl, acetyl or other protecting groups that yield the corresponding amide. However, the other functional groups present on the amino acid may be modified by esterification or alkylation. Suitable protecting groups for peptide synthesis, among others, are provided in Table 1.

Table 1: Selected protecting groups useful for peptide synthesis. Protecting Group Protecting amino acid Removed by tert-butyl Asp/Glu (OtBu) TFA/HCOOH/HBr (tBu) Ser/Thr/Tyr (tBu) 1-adamantyl Asp (O-1-Ada TFA tert-butyloxycarbonyl Lys (Boc) TFA/HCOOH/HBr (tBOC) His (Boc) triphenylmethyl His (Trt) TFA Cys (Trt) HOAc/TFA/Hg (II)/I2 Asn/Glu (Trt) TFA tert-butoxymethyl His (Bum) TFA 4-methoxy-2,3, 6- Arg (Mtr) TFA trimethyl- benzenesulfonyl 2,2, 5,7, 8-pentamethyl- Arg (Pmc) TFA chroman-6-sulfonyl 2,4, 6-trimethoxybenzyl Asn/Gln (Tmob) TFA Cys (Tmob)

Where R'is, or is derived from, a peptide or polypeptide, the peptide or polypeptide may incorporate natural or synthetic amino acids, each of which may be modified on any free amine or carboxylic acid by a suitable protecting group, as described above. "Peptides"and"polypeptides", as used herein, include chains of amino acids whose a-carbons are linked through peptide bonds typically formed by a condensation reaction between the a-carboxyl group of one amino acid and the amino group of another amino acid. The terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other

end of the chain (carboxy terminal) has a free carboxyl group. As used herein, the term"amino terminus" (abbreviated N-terminus) refers to the free a-amino group on an amino acid at the amino terminal of a peptide or to the a-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide. Similarly, the term"carboxy terminus"refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide. Peptides also include peptide mimetics such as amino acids joined by an ether as opposed to an amide bond.

The polypeptides described herein may be written with the amino terminus at the left and the carboxyl terminus at the right. The amino acids comprising the peptide components of this invention are numbered with respect to the protease cleavage site, with numbers increasing consecutively with distance in both the carboxyl and amino direction from the cleavage site. The term"residue"or"amino acid"as used herein refers to an amino acid that is incorporated into a peptide. The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

The term"domain"or"region"may refer to a characteristic region of a polypeptide. The domain may be characterized by a particular structural feature, such as a (3-turin, an a-helix, or a (3-pleated sheet, by characteristic constituent amino acids (e. g. , predominantly hydrophobic or hydrophilic amino acids, or repeating amino acid sequences), or by its localization in a particular region of the folded three dimensional polypeptide. As used herein, a region or domain is composed of a series of contiguous amino acids.

Where the resulting compound is intended for use as an enzyme substrate, Rl may be derived from a peptide having a sequence suitable for cleavage by the desired enzyme. Generally, Rl is selected so that the resulting compound is a binding site and substrate for a protease enzyme (also known as a peptidase enzyme). Suitable enzymes include, without limitation, aminopeptidases and dipeptidyl peptidases, such as dipeptidyl peptidases I, II and IV, calpain, elastase, trypsin, chymotrypsin, granzyme A, thrombin, cathepsins, urokinase, kallikrein, human adenovirus

proteinase, plasminogen activator, interleukin converting enzyme, amyloid A4- generating enzyme, follipsin and leucine aminopeptidase. In particular, Rl may be selected so that the resulting compound is a caspase enzyme substrate.

The terms"protease activity"or"activity of a protease"typically refer to the cleavage of a substrate peptide by a protease enzyme. Thus, protease activity may comprise the"digestion"of one or more peptides into a large number of smaller peptide fragments. Protease activity of particular proteases may result in hydrolysis at particular peptide binding sites characteristically recognized by that particular protease. The particular protease may be characterized by the production of peptide fragments bearing particular terminal amino acid residues.

The amino acids or peptides referred to herein may be described in part using shorthand designations, as shown in Table 2. Other abbreviations that may be used herein include"Fmoc"for 9-fluorenylmethyoxycarbonyl, "Ac"for acetyl,"Boc"for tert-butoxycarbonyl,"Z"or"Cbz"for benzyloxycarbonyl,"MPL"for morpholinecarbonyl, "AA"for amino acids, "PP"for peptides, and"RhllO"for rhodamine 110. For example, a peptidyl-rhodamine 110-morpholinecarbonyl compound of the invention may be referred to using these shorthand designations as '(PP)-Rhl 10-MPL'.

Table 2: Amino acid nomenclature. Amino Acid 3 Letter Designator 1 Letter Designator Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu E Glutamin Gln Q Glycine Gly G Histidine His H Homoserine Hse Isoleucine Ile I Leucine Leu L Lysine Lys K Methionind Met M Methionine sulfoxide Met (O) Norleucine Nle Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

The compounds of the invention may include, among others, polypeptides that include the sequences listed in Table 3. The amino acid residues that comprise a protease binding site may be numbered relative to the peptide bond hydrolyzed by a particular protease. Therefore, as used below, the first amino acid residue on the amino side of the cleaved peptide bond is designated PI. The numbering of the residues increases with distance away from the hydrolyzed peptide bond.

Table 3: Selected particular peptide sequences. Enzyme Substrate Peptide Sequence Caspase 1 YVAD (SEQ ID NO. 1) DEHD (SEQ ID NO. 2) Caspase 2 VDVAD (SEQ ID NO. 3) DEVD (SEQ ID NO. 4) SHVD (SEQ ID NO. 5) DBLD (SEQ ID NO. 6) DGPD (SEQ ID NO. 7) DEPD (SEQ ID NO. 8) DGTD (SEQ ID NO. 9) DLND (SEQ ID NO. 10) DEED (SEQ ID NO. 11) DSLD (SEQ ID NO. 12) DVPD (SEQ ID NO. 13) Caspase 4 LEVD (SEQ ID NO. 14) Caspase 6 VEHD (SEQ ID NO. 15) VEID (SEQ ID NO. 16) Caspase 7 DEVD (SEQ ID NO. 4) Caspase 8 IETD (SEQ ID NO. 17) Caspase 9 LEHD (SEQ ID NO. 18) VEHD (SEQ ID NO. 15) AP AP FR Calpain LM AA Elastase AAAA (SEQ ID NO. 19) AR Plasminogen activator LGR Prostatin GPR Plasmin FR Kallirein PFR, PR Granzyme A GPR Granzyme B IEPD (SEQ ID NO. 20) R . AR Trypsin PR IPR AR Cathepsins B, L and O ARR FR FR Cathepsin C FR PFR Urokinase GR GGR Enzyme Substrate Peptide Sequence VLK Prohormone thiol protease FR VPR Thrombin GPR PR Human adenovirus LRGG (SEQ ID NO. 21) proteinase

As indicated above, one or more of the rhodamine substituents R-R'may be a reactive functional group (Rx) or a conjugated substance (Sc) that is bound to the compound of the invention by a covalent linkage, L. Covalent linkage L may be a single covalent bond, or L may incorporate a series of nonhydrogen atoms that form a stable linkage between the reactive functional group or conjugated substance and the compound. Typically, L incorporates 1-20 nonhydrogen atoms in a stable conformation. Stable atom conformations include, without limitation, carbon-carbon bonds, amide linkages, ester linkages, sulfonamide linkages, ether linkages, thioether linkages, and other covalent bonds well-known in the art. Preferred covalent linkages are single bonds, carboxamides, sulfonamides, ethers, and carbon-carbon bonds, or a combination thereof.

Any reactive functional group that exhibits appropriate reactivity to cross- react and conjugate with a desired substance is a suitable reactive functional group for the purposes of the invention. The choice of the reactive group used typically depends on the functional groups present on the substance to be conjugated. Typically, functional groups present on such substances include, but are not limited to, alcohols, aldehydes, amines, carboxylic acids, halogens, ketones, phenols, phosphates, and thiols, or a combination thereof. Suitable Rx groups include activated esters of carboxylic acids, aldehydes, alkyl halides, amines, anhydrides, aryl halides, carboxylic acids, haloacetamides, halotriazines, hydrazines (including hydrazides), isocyanates, isothiocyanates, maleimides, phosphoramidites, sulfonyl halides, and thiol groups, or a combination thereof. Typically, Rx is an activated ester of a carboxylic acid, an amine, a haloacetamide, a hydrazine, an isothiocyanate, or a maleimide group. In one aspect of the invention, Rx is a succinimidyl ester of a carboxylic acid.

The compounds of the invention that have a covalently bound reactive functional group may be used to prepare a variety of conjugates. In one aspect of the invention, the conjugated substance is a member of a specific binding pair. In another aspect of the invention, the conjugated substance is a molecular carrier. In yet another aspect of the invention, the conjugated substance is a biomolecule that is an amino acid, a peptide, a protein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, or a carbohydrate. Selected conjugated substances may aid the retention of the protease substrates within cell membranes, localize the protease substrates within a selected region of a cell, or bind the luminescent enzymatic cleavage product to a preselected component of a sample under investigation. Alternatively or in addition, the conjugated substance is a polar moiety, or a masked polar moiety. Alternatively or in addition, the conjugated substance is a solid or semi-solid matrix.

Where the conjugated substance Sc is an amino acid, a peptide, a protein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, or a carbohydrate, the conjugated substance may be a naturally occurring or a synthetically modified substance. The conjugated substance also may be a member of a specific binding pair or a molecular carrier. Specific binding pair members typically specifically bind to and are complementary with the complementary member of the specific binding pair. Conjugated members of a specific binding pair typically are used to localize the compound of the invention to the complementary member of that specific binding pair. Representative specific binding pairs are listed in Table 4.

Table 4: Representative specific binding pair members. antibody antigen avidin (streptavidin) biotin DNA aDNA enzyme enzyme substrate lectin carbohydrate receptor ligand RNA aRNA Where the conjugated substance Sc is a carrier, it typically is a biological or artificial polymer. Biological polymers include proteins, carbohydrates, and nucleic

acid polymers. Artificial polymers include polyethylene glycols and polymeric microparticles composed of polystyrene, latex, or other polymeric material.

Preferably, a conjugated carrier is a carbohydrate that is a dextran, or amino- substituted dextran, or a polymeric microparticle. Such carriers are useful for altering the solubility of the compound, enhancing its retention within cell membranes, or decreasing its compartmentalization within cells.

Where the conjugated substance is a polar moiety, the conjugated substance is typically substituted one or more times by a highly polar functional group, such as a carboxylic acid or sulfonic acid. To improve loading into cells, the polar moiety typically is masked or protected, temporarily rendering it more lipophilic and therefore more cell-permeant. One such masking group is an ester group that may be cleaved by esterases to release the free polar moiety within cell membranes, where they are well-retained. Typically, the polar moiety is a carboxylic acid, a dicarboxylic acid, or a tricarboxylic acid moiety that is protected as an ester, such as an acetoxymethyl ester.

Where the conjugated substance is a solid or semi-solid matrix, the conjugated substance may be a metal or glass surface, and may be, for example, the sides or bottom of a microwell, or a slide, or the surface of a chip. Conjugation of an appropriate luminogenic protease substrate to the interior surfaces of the wells on a multi-well microplate may generate a preassembled assay system for selected protease activity, requiring only the addition of sample solutions and/or appropriate control solutions to perform a selected assay. Selected buffers and/or other reagents may also be provided as dry components within the microplate wells, further facilitating the formulation of a microplate assay kit for protease activity. Such a kit would have particular utility for high-throughput assays.

Applications The compounds of the invention are converted by protease enzymes to a highly luminescent enzymatic product in a single step, as shown below in Scheme 3.

The instant substrates are more sensitive than the corresponding bis-peptide rhodamine derivatives, and, unlike existing lipophilic monopeptide-substituted rhodamine compounds, the enzymatic product'RhllO- (morpholine amide) 'is as fluorescent as free rhodamine 110 (the enzymatic product of the bis-peptide rhodamine substrates. Additionally, the instant substrates are better retained in cells than free rhodamine 110. o Peptide NH/ \ NH O \O Non-fluorescent Protease 09 N 1 o zig \oh Highly fluorescent

Scheme 3. One-step cleavage of (peptidyl-Rhl 10-MPL) substrates by protease enzymes.

The instant protease substrates exhibit a high luminescence signal upon cleavage, but a very low luminescence signal in their spirolactone form. Further, appropriate selection of peptide sequence may result in substrates that exhibit a high degree of protease specificity. In addition, the luminescence signal of the cleavage product typically occurs in the visible region, well-separated from cellular

autofluorescence, thereby rendering them highly suitable for use in biological samples.

The compounds of the invention may be used in combination with simple solutions of protease enzymes, or they may be used to stain samples that contain cells.

The sample typically is stained by passive means, i. e. , by incubation of the desired sample with a staining solution containing the selected compound of the invention.

Passive incubation is most useful for compounds of the invention that have been masked or protected and that therefore are nonpolar and cell-permeable. However, any other suitable method of introducing the compound of the invention into the sample, such as microinjection, can be used to aid or accelerate introduction of the dye into the sample.

Before use, a staining solution of the compound of the invention is prepared.

The compound of the invention typically may first be dissolved in an organic solvent, such as DMSO or DMF, to prepare a concentrated stock solution. The stock solution may then be diluted into the appropriate aqueous solution, typically a buffer solution, to prepare the staining solution for addition to the sample. The nonfluorescent spirolactone compounds of the invention are typically less soluble in aqueous solutions than the free fluorescent products of protease activity. Alternatively, the compound of the invention may be suspended in a semi-solid matrix, such as a polymer gel, for addition to tissue sections and other solid samples.

The sample is optionally combined with other solutions in the course of staining, such as wash solutions or solutions containing additional detection reagents.

An additional detection reagent is a reagent that produces a detectable response due to the presence of a specific cell component, intracellular substance, or cellular condition, according to methods generally known in the art. Where the additional detection reagent has spectral properties that differ from those of the subject substrates, multi-color applications are possible.

Typically, after the compound of the invention is added to the sample, the sample is incubated for a time sufficient for any protease enzymes in the sample to cleave the peptide region and convert the spirolactone form of the dye to the free form of the dye. At any time after sufficient cleavage product is present in the sample to

measure a detectable optical signal, the sample may be illuminated and the resulting optical signal detected.

Typically, the sample is illuminated at a wavelength of light that results in a detectable optical response and observed with a means for detecting the optical response. The optical response may be colorimetric or luminescent. That is, the free rhodamine compound strongly absorbs light in the visible region, and protease activity may be detected/quantified on that basis. Alternatively, a luminescence response may be used to detect/quantify protease activity. The illumination source typically is selected so that efficient excitation of the free compound is achieved.

Because of the high intensity luminescence of the cleavage products in the visible wavelengths, the protease substrates of the invention are particularly well suited for detection of protease activity in biological samples. The maximal excitation close to the 488 nm argon laser line makes the free dyes compatible with a variety of luminescence instruments, as the argon laser is used in most commercial luminescence instruments. In general, useful illumination sources include ultraviolet or visible wavelength emission lamps, arc lamps, or lasers. These illumination sources are optionally integrated into spectrophotometers, laser scanners, fluorescence microplate readers, standard or mini fluorometers, flow cytometers, fluorescence microscopes, or chromatographic detectors. The luminescence emission is optionally detected by visual inspection or by use of any of the following devices: CCD cameras, video cameras, photographic film, laser scanning devices, fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, any of which may optionally incorporate photomultiplier tubes to enhance the detectable luminescence signal. Hence, the luminogenic compositions of the invention facilitate detection of protease activity, whether intracellular or extracellular. Similarly, the compounds of the invention are particularly well suited for high-throughput screening techniques, particularly those involving automated methods and small sample volumes.

The observation of a detectable optical response in the sample is typically correlated with the appearance of free rhodamine luminophore in the sample, and therefore the presence of an appropriate protease enzyme. The correlation typically is

accomplished by comparison of the luminescence response to a standard, or calibration, curve. The standard curve is generated according to methods known in the art using various and known enzyme activity levels, or by comparison with a reference dye or dyed particle that has been standardized versus a known enzyme activity.

In one example, the invention provides methods for detecting the activity of a protease enzyme. The methods involve contacting the protease with one or more of the protease substrates described herein. In a particular embodiment, the"contacting" is in a histological section or in a cell suspension or culture derived from a biological sample selected from the group consisting of a tissue, blood, urine, saliva, or other biofluid, lymph, biopsy. The detection method can include a method selected from the group consisting of fluorescence microscopy, fluorescence microplate reader, flow cytometry, fluorometry, and absorption spectroscopy.

In another example, any of the compositions described above may be used in a method for detecting protease activity in a sample. The sample may be a sample of "stock"protease, such as is used in research or industry, or it may be a biological sample. Thus, this invention provides for a method of detecting protease activity in a sample by contacting the sample with any of the compositions described above and then detecting a change in luminescence of the luminogenic composition where an increase in luminescence indicates protease activity. The sample may be a biological sample which may include biological fluids such as sputum or blood, tissue samples such as biopsies or sections (including tumor biopsies), and cell samples either as biopsies or in cells or tissues in culture, or tissue sections where the section is unimbedded and unfixed. Particularly preferred samples are tissue sections, cultured cells, cultured tissues, and the like.

A. Detection of protease activity The present invention provides methods for utilizing the luminogenic protease substrates to detect protease activity in a variety of contexts. Thus, in one embodiment, the present invention provides for a method of using the luminogenic substrates to verify or quantify the protease activity of a stock solution of a protease used for experimental or industrial purposes. Verification of protease activity of stock

protease solutions before use is generally recommended as proteases often exhibit diminished activity over time (e. g. , through self-hydrolysis) or show varying degrees of activation when activated from zymogen precursors.

Assaying for protease activity of a stock solution simply requires adding a quantity of the stock solution to a luminogenic protease substrate of the present invention and measuring the subsequent increase in luminescence or decrease in excitation in the absorption spectrum. The stock solution and the luminogenic substrate may also be combined and assayed in a"digestion buffer"that optimizes activity of the protease. Buffers suitable for assaying protease activity are well known to those of skill in the art. In general, a buffer will be selected whose pH corresponds to the pH optimum of the particular protease. For example, a buffer particularly suitable for assaying elastase activity consists of 50 mM sodium phosphate, 1 mM EDTA at pH 8.9. The measurement is most easily made in a fluorometer, an instrument that provides an"excitation"light source for the luminophore and then measures the light subsequently emitted at a particular wavelength. Comparison with a control substrate solution lacking the protease provides a measure of the protease activity. The activity level may be precisely quantified by generating a standard curve for the protease/substrate combination in which the rate of change in luminescence produced by protease solutions of known activity is determined.

While detection of the luminogenic compounds is preferably accomplished using a fluorometer, detection may by a variety of other methods well known to those of skill in the art. Thus for example, since the luminophores of the present invention emit in the visible wavelengths, detection may be simply by visual inspection of luminescence in response to excitation by a light source, such as through a fluorescence microscope. Detection may also be by means of an image analysis system utilizing a video camera interfaced to an digitizer or other image acquisition system. Detection may also be by visualization through a filter as under a fluorescence microscope. The detected signal may also be recorded on photographic film or using a video analysis system. The signal may be quantified on a realtime basis using either an image analysis system or a photometer.

For example, a basic assay for protease activity of a sample may involve suspending or dissolving the sample of interest in a buffer (at the pH optima of the particular protease being assayed), adding to the buffer one of the luminogenic protease substrates of the present invention, and monitoring the resulting change in luminescence using a spectrofluorometer. The spectrofluorometer may be set to excite the free luminophore at the excitation wavelength of the luminophore and to detect the resulting luminescence at the emission wavelength of the luminophore The rhodamine-based substrates of the invention may display sensitivity, specificity and selectivity in their interactions with particular protease enzymes. The specificity exhibited by many proteases may depend to a large extent upon the interaction of subsite amino acids in the protease active site with extended amino acid residues in the peptide substrate. This interaction may be characterized through the use of synthetic substrates, by observing variations in the specificity constant upon substituting or altering a single residue in the substrate peptide sequence.

B. Assays of isolated biological samples The present invention provides for methods of detecting protease activity in isolated biological samples. This may be determined by simply contacting the sample with a luminogenic protease substrate of the present invention and monitoring the change in luminescence of the substrate over time. The sample may be suspended in a "digestion buffer"as described above. The sample may also be cleared of cellular debris, e. g. , by centrifugation before analysis.

C. In situ assays of histological sections The invention also provides for a method of detecting in situ protease activity in histological sections. This method of detecting protease activity in tissues offers significant advantages over previous methods (e. g. , specific stains, antibody labels, etc. ) because, unlike simple labeling approaches, in situ assays using the protease substrates indicate actual activity rather than simple presence or absence of the protease. Proteases are often present in tissues in their inactive precursor (zymogen) forms which are capable of binding protease labels. Thus traditional labeling approaches provide no information regarding the physiological state, via protease activity, of the tissue.

The in situ assay method generally includes providing a tissue section (preferably a frozen section), contacting the section with one of the luminogenic protease substrates of the present invention, illuminating the section, and detecting any resulting optical signal. The sections are preferably cut as frozen sections as fixation or embedding may destroy protease activity in the sample. Visualization may be accomplished utilizing a fluorescence microscope.

The luminogenic substrate may be introduced to the tissue sections in a number of ways. For example, the luminogenic protease substrate may be provided in a buffer solution, as described above, which is applied to the tissue section.

Alternatively, the luminogenic protease substrate may be provided incorporated in a semi-solid matrix, such as agar or other gel, that is then spread over the tissue sample and helps hold moisture in the sample while providing a signal in response to protease activity. The luminogenic protease substrate may also include a conjugated substance that is a polymer, such as a plastic film, which may be used in procedures similar to the development of Western Blots. The plastic film may be placed over the tissue sample on the slide and the luminescence resulting from cleaved substrate molecules may be viewed in the sample tissue under a microscope.

Typically a tissue sample should be incubated for a period of time to allow the endogenous proteases to cleave the luminogenic protease substrates of the invention.

Incubation times may range from a few minutes to several hours at temperatures up to and including 37 °C.

D. In situ assays of cells in culture and cell suspensions derived from tissues and biopsy samples This invention further provides for a method of detecting in situ protease activity of cells in culture or cell suspensions derived from tissues, biopsy samples, or biological fluids (e. g. , saliva, blood, urine, lymph, plasma, etc. ). The cultured cells are grown either on chamber slides or in suspension and then transferred to histology slides by cytocentrifugation. Similarly, the cell suspensions are prepared according to standard methods and transferred to histology slides. The slide is washed with phosphate buffered saline and coated with a semi-solid matrix or a solution containing the luminogenic protease substrate. The slide is incubated at 37 °C for the time

necessary for the endogenous proteases to cleave the protease substrate. The slide is then examined under a fluorescence microscope equipped with the appropriate filters as described above.

Alternatively, the cells are incubated with the protease substrates at 37 °C, then washed with buffer and transferred to a glass capillary tube and examined under a fluorescence microscope. When a flow cytometer is used to quantitate the intracellular enzyme activity, the cells with the luminogenic substrate are simply diluted with buffer after 37 °C incubation and analyzed.

E. Protease binding site and conformation determining regions The protease binding site and conformation determining regions form a contiguous amino acid sequence that is recognized and cleaved by a particular protease. It is well known that various proteases cleave peptide bonds adjacent to particular amino acids. Thus, for example, trypsin cleaves peptide bonds following basic amino acids such as arginine and lysine and chymotrypsin cleaves peptide bonds following large hydrophobic amino acid residues such as tryptophan, phenylalanine, tyrosine and leucine. The serine protease elastase cleaves peptide bonds following small hydrophobic residues such as alanine.

A particular protease, however, will not cleave every bond in a protein that has the correct adjacent amino acid. Rather, the proteases are specific to particular amino acid sequences which serve as recognition domains for each particular protease.

Without being bound by a particular theory, it is believed that a specific protease's preference for a particular cleavage site over many other potential sites in a folded globular protein may be largely determined by the potential cleavage sites'amino acid sequences and also their conformation and conformational flexibility.

Thus, for example, one obtains limited proteolysis products, e. g., ribonuclease-S (a noncovalent complex consisting of two polypeptide chains) from a single chain folded protein ribonuclease-A using a protease called subtilisin.

Similarly, one obtains a two chain noncovalent complex, Staphylococal nuclease-T, from single chain Staphylococcal nuclease by trypsin digestion. Another example of a specific protease's preference for one substrate over others is the human fibroblast- type collagenase. This protease prefers type I over type III soluble collagen even

though both substrates contain the same collagenase sensitive Gly-Ile or Gly-Leu bonds (see, Birkedal-Hansen et. al. Crit. Rev. in Oral Biology and Medicine 1993, 4,197-250).

Any amino acid sequence that comprises a recognition domain and can thus be recognized and cleaved by a protease is suitable for the"protease binding site"of the luminogenic protease substrate compositions of this invention. Known protease substrate sequences and peptide inhibitors of proteases possess amino acid sequences that are recognized by the specific protease they are cleaved by or that they inhibit.

Thus known substrate and inhibitor sequences provide the basic sequences suitable for use in the protease recognition region. A large number of protease binding region protease substrates and inhibitor sequences suitable for use as protease binding domains in the compositions of this invention are known (see for example, U. S.

Patent No. 6,335, 429 and U. S. Patent No. 6,342, 611), can be used for this invention.

One of skill will appreciate that other protease substrates or inhibitor sequences, including variations and modifications of previously disclosed sequences, may be used in the present invention.

F. Protease activity detection, cell apoptosis, cell cytotoxicity, and drug-screening kits The present invention also provides for kits for the detection of protease activity in samples. The kits comprise one or more containers containing the luminogenic protease substrates of the present invention. The substrates may be provided in solution or bound to a solid support. Thus the kits may contain substrate solutions or substrate"dipsticks", blotters, culture media, and the like. The kits may also contain substrate cartridges for use in automated protease activity detectors.

The kits additionally may include an instruction manual that teaches the method and describes use of the components of the kit. In addition, the kits may also include additional reagents, including but not limited to buffering agents, luminescence calibration standards, enzymes, enzyme substrates, protease inhibitors, nucleic acid stains, labeled antibodies, culture media, disposable cuvettes, and/or other additional luminescence detection reagents, and the like to aid the detection of

protease activity utilizing the luminogenic protease substrates of the present invention.

These kits optionally may include chemically reactive forms of the substrates to permit a user to label substances of interest and develop individual assays.

Alternatively, the kits may include conjugates of the desired substrate that are selected specifically for a particular assay, typically where the conjugated substance is a member of a specific binding pair. The compounds of the invention optionally are present in pure form as a lyophilized solid, or as a concentrated stock solution, or in a prediluted solution ready for use in the appropriate assay. Typically, the kit is designed for use in an automated and/or high-throughput assay, and so is designed to be fully compatible with microplate readers, microfluidic methods, and/or other automated high-throughput methods.

Kits may additionally or alternatively comprise any of the substrates described herein (e. g. , nucleic acid based substrates, oligosaccharide substrates, lipid substrates, etc). In this instance the kit will facilitate detection of the particular activities/compounds/interactions for which the particular substrate backbone is a substrate or binding agent.

G. Preparation of luminogenic protease substrates Preparation of the peptide sequences : Many of the peptides useful in the preparation of the instant compounds are commercially available. For special peptide sequences, solid-phase peptide synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the desired sequence is the preferred method for preparing the peptide backbone of the compounds of the present invention. Techniques for solid phase synthesis are well described in literature (see, J. D. Wade and G. W. Tregear, Solid phase peptide synthesis : recent advances and applications. Australas Biotechnol, 1993.3, 332-6; R. B. Merrifield, Solid-phase peptide synthesis. Adv Enzymol Relat Areas Mol Biol, 1969,32, 221-96; F. Albericio, P. Lloyd-Williams, and E. Giralt, Convergent solid-phase peptide synthesis. Methods Enzymol, 1997, 289,313-36 ; M. F. Songster and G. Barany, Handles for solid-phase peptide synthesis, Methods Enzymol, 1997,289, 126-74. )

For example, the peptide synthesis may be performed using Fmoc synthesis chemistry. The side chains of Asp, Ser, Thr and Tyr are preferably protected using t- Butyl protecting groups, while the side chain of the Cys residue is protected using S- trityl and S-t-butylthio protecting groups. Lys residues are preferably protected using t-Boc, Fmoc and/or 4-methyltrityl protecting groups. Many appropriately protected amino acid reagents are commercially available. The use of multiple protecting groups allows selective deblocking and coupling of a desired luminophore to any particular desired side chain. Thus, for example, t-Boc deprotection is accomplished using trifluoroacetic acid (TFA) in dichloromethane, Fmoc deprotection is accomplished using 20% (v/v) piperidine in DMF or N-methylpyrolidone, and 4- methyltrityl deprotection is accomplished using 1 to 5% (v/v) TFA in water or 1% TFA and 5% truisopropylsilane in DCM, S-t-butylthio deprotection is accomplished in aqueous mercaptoethanol (10%), t-butyl and t-boc and S-trityl deprotection is accomplished using TFA: phenol: water: thioanisol: ethanedithiol (85: 5: 5: 2.5 : 2.5), and t- butyl and t-Boc deprotection is accomplished using TFA: phenol: water (95: 5: 5).

Detailed synthesis, deprotection and luminophore coupling protocols are provided in Examples 1-16.

Alternatively, the peptide components of the luminogenic protease substrates of the present invention may be synthesized utilizing recombinant DNA technology.

Briefly, a DNA molecule encoding the desired amino acid sequence may be synthesized chemically using any of a variety of methods known to those of skill in the art, including the solid phase phosphoramidites method described by Beaucage and Carruthers, Tetrahedron Lett. 22,1859-1862 (1981), the triester method according to Matteucci, et al. , J. Am. Chem. Soc. , 103,3185 (1981), or by other known methods.

It is preferred that the DNA be synthesized using standard (3-cyanoethyl phosphoramidites on a commercially available DNA synthesizer using standard protocols.

The oligonucleotides may be purified, if necessary, by techniques well known to those of skill in the art. Typical purification methods include, but are not limited to gel electrophoresis, anion exchange chromatography (e. g. , Mono-Q column, Pharmacia-LKB, Piscataway, N. J. , USA), or reverse phase high performance liquid

chromatography (HPLC). Methods of protein and peptide purification are well- known. For a review of standard techniques see, Methods in Enzymology Volume 182: Guide to Protein Purification, M. Deutscher, ed. (1990), pp. 619-626.

The oligonucleotides may be converted into double stranded DNA either by annealing with a complementary oligonucleotide or by polymerization with a DNA polymerase. The DNA may then be inserted into a vector under the control of a promoter and used to transform a host cell so that the cell expresses the encoded peptide sequence. Standard methods of cloning and expression of peptides are well- known. See, for example, Sambrook, et al. , Molecular Cloning: A Laboratory Manual (2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory (1989) ), Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques (Berger and Kimmel (eds. ), San Diego: Academic Press, Inc. (1987) ), or Current Protocols in Molecular Biology, (Ausubel, et al. (eds. ), Greene Publishing and Wiley-lnterscience, New York (1987).

Linkage of the luminophores to the peptide backbone : The luminogenic protease substrates of the present invention are preferably prepared by first preparing a mono-amino acid-substituted rhodamine-MPL compound, and then coupling the remaining required amino acids using standard peptide synthesis methods in the desired sequence. Alternatively, the desired compound may be prepared by coupling the Rhl 10-MPL compound with a complete peptide sequence in a single step (see for example, Example 10). The latter procedure may exhibit a lower yield than the corresponding stepwise synthesis. These two synthetic procedures are illustrated by the two syntheses of Ac-DEVD-Rhl 10-MPL as set out in the Examples below and in Appendix A.

The assays of the invention optionally may be performed using apparatus, methods, and/or compositions described in the various patents and patent applications listed above under Cross-References and incorporated herein by reference. The apparatus include luminescence detectors and sample holders such as microplates, among others. The methods include photoluminescence methods, such as luminescence intensity, among others. The compositions include various energy transfer donors and acceptors, among others.

Examples The examples below are given so as to illustrate the practice of this invention.

They are not intended to limit or define the entire scope of this invention.

Example 1. Preparation of Compound 1.

Compound 1 Rhodamine 110 (2.2 g, 6.0 mmol) is dissolved in anhydrous DMF (40 mL). To the DMF solution is added N, N-diisopropylethylamine (3.2 ml) and the resulting mixture is stirred at room temperature for 30 min. To the resulting solution is slowly added 4-morpholinecarbonyl chloride (0.82 g, 5.5 mmol), and stirred at room temperature for 48 hr. The reaction mixture is evaporated under vacuum to give a crude product, which is further purified on a silica gel column using 10: 1 CHCl3/MeOH as eluent. The column chromatographic purification affords 1.65 g of the desired product (yield: 68%). MS, m/z = 443.67 (M+).

Example 2. Preparation of Compound 2.

Compound 2 Compound 1 (1.65g, 3.72 mmol) is dissolved in anhydrous DMF (40 mL). To the DMF solution is added anhydrous pyridine (5 mL), and the solution is stirred for 30 min. To the reaction mixture is added doubly-protected aspartic acid, Fmoc-Asp- (O-t-Bu)-OH (6.13 g, 14.9 mmol), and EDC (4.28g, 22 mmol). The reaction mixture is stirred at room temperature for 24 hr, and then treated with 5% HCl solution (150 mL). The resulted aqueous solution is extracted with 1: 1 chloroform/ethyl acetate (2 x 250 mL). The combined organic layers are evaporated under vacuum to give the crude product that is further purified on a silica gel column (using 1 : 1 CHC13/EtOAc as eluent) to give 1.18 g of the desired product (yield: 38% yield). MS, m/z = 836.9 (M+).

Example 3. Preparation of Compound 3.

Compound 3 Compound 2 (1.18g, 1.41 mmol) is dissolved in CH2Cl2 (24 mL). Piperidine (6 ml) is added, and the resulting solution is stirred at room temperature for 2 hr. The reaction mixture is then evaporated under vacuum to give the crude product, which is further purified on a silica gel column (using 1: 1 CHCl3/MeOH as eluent) to give 0.7 g of the desired product (yield: 89%). MS: m/z 559.27 (M+).

Example 4. Preparation of Compound 4.

Compound 4 Compound 3 (0.21 g, 0.376 mmol) is dissolved in anhydrous DMF (12 mL).

To the DMF solution, anhydrous pyridine (2 mL) is added, and the solution is stirred for 30 min. To this solution is added Fmoc-protected valine (Fmoc-Val-OH) (0.46 g, 1.36 mmol) and EDC (0.39 g, 2.0 mmol). The reaction mixture is stirred at room temperature for 24 hr, treated with 5% HCl solution (50 mL), and extracted with 1: 1

chloroform/ethyl acetate (2 x 200 mL). The combined organic layers are dried over anhydrous sodium sulfate and concentrated under vacuum. The resulting residue is purified using silica gel column chromatography using 1: 1 CHCl3/EtOAc as eluent to obtain 0.22 g of the desired product (yield: 63%). MS, m/z 936.33 (M+).

Example 5. Preparation of Compound 5.

Compound 5 Compound 4 (0.22g, 0.235 mmol) is dissolved in CH2Cl2 (4 mL). Piperidine (1 mL) is added, and the solution is stirred at room temperature for 2 hr. The reaction mixture is then evaporated under vacuum to give the crude product. Further purification on a silica gel column (using 10: 1 CHCl3/MeOH as eluent) yields 0. 15 g of the desired product (yield: 89%). MS, m/z = 714. 1 (M+).

Example 6. Preparation of Compound 6.

Compound 6

Compound 5 (60 mg, 0.084 mmol) is dissolved in anhydrous DMF (4 mL). To the DMF solution is added anhydrous pyridine (0.4 mL) and the reaction mixture is stirred for 30 min. To the resulted solution is added the doubly protected glutamic acid Fmoc-Glu (O-tBu)-OH (72 mg, 0.17 mmol) and EDC (65 mg, 0.34 mmol). The reaction mixture is stirred at room temperature for 24 hr, and then treated with 5% HCl solution (25 mL). The resulted aqueous solution is extracted with 1: 1 chloroform/ethyl acetate (2 x 100 mL). The combined organic layers are evaporated under vacuum to give the crude product, further purified on a silica gel column (using 2: 1 CHC13/EtOAc as eluent) to give 50 mg of the desired product (yield: 53%). MS, m/z = 1121. 4 (M+).

Example 7. Preparation of Compound 7.

Compound 7 Compound 6 (50 mg, 0.0446 mmol) is dissolved in CH2C12 (2 mL). To this solution is added piperidine (0.5 mL), and the resulting mixture is stirred at room temperature for 2 hr. The reaction mixture is then evaporated under vacuum to give a crude product which is further purified on a silica gel column (using 10: 1 CHCl3/MeOH as eluent) to give 30 mg of the desired product (yield: 75%). MS, m/z = 898.7 (M+).

Example 8. Preparation of Compound 8.

Compound 8 Compound 7 (30 mg, 0.0334 mmol) is dissolved in anhydrous DMF (2 mL).

Anhydrous pyridine (0.5 mL) is added, and the solution is stirred for 30 min. To the resulted solution is added acetyl-protected and t-butyl protected aspartic acid (Ac- Asp (O-tBu) -OH) (38 mg, 0.15 mmol) and EDC (38 mg, 0.2 mmol). The reaction mixture is then stirred at room temperature for 24 hr, and treated with 5% HCl solution (20 mL). The resulted aqueous solution is extracted with 1: 1 chloroform/ethyl acetate (2 x 50 mL). The combined organic layers are evaporated under vacuum to give a crude product that is further purified on a silica gel column (using 2: 1 CHC13/EtOAc as eluent) to give 25 mg of the desired product (yield: 68%).

MS, m/z = 1112. 4 (M+).

Example 9. Preparation of Compound 9.

Compound 9

Compound 8 (0.48 g, 0.43 mmol) is dissolved in a solution of CHC13 (10 mL) and anisole (0.5 ml). To this solution is slowly added trifluoroacetic acid (TFA) (10 mL), and the reaction mixture is stirred at room temperature for 6 hr. Following the reaction, the reaction mixture is evaporated under vacuum to dryness, and the resulting residue is washed with ethyl ether to give a crude product that is further purified by reversed-phase HPLC to give 0.28 g of the desired product (yield: 69%).

MS, m/z 944.27 (M+).

Example 10. Two-step preparation of Compound 8.

Compound 3 (0.9 g, 1.46 mmol) is dissolved in anhydrous DMF (40 mL).

Anhydrous pyridine (6 mL) is added, and the solution is stirred for 30 min. To this solution is added a protected Asp-Glu-Val tripeptide (Ac-Asp (OtBu)-Glu (OtBu)-Val- OH) (1.13 g, 2.2 mmol) and EDC (0.63 g, 3.3 mmol), and the mixture is stirred at room temperature for 24 hr. The reaction mixture is then treated with 5% HCl solution (100 mL), and extracted with 1 : 1 chloroform/ethyl acetate twice (2 x 200 ml). The combined organic layers are dried over anhydrous sodium sulfate, and concentrated under vacuum. The resulted residue is purified by silica gel column chromatography using 10: 10: 1 CHCl3/EtOAc/MeOH as eluent to obtain 0.9 g of the pure desired product (yield: 55%) as a white power. MS, m/z 1112.4 (M+).

Example 11. One-step preparation of Compound 8.

Compound 1 is treated with a protected Asp-Glu-Val-Asp tetrapeptide (Ac- Asp (OtBu)-Glu (OtBu)-V- (O-Boc-D)-OH) in a manner analogous to that described in Example 2 to give the desired product.

Example 12. Preparation of Compound 12.

Compound 12 Compound 1 is treated with the protected Asp-Glu-Val-Asp tetrapeptide Cbz- Asp (OtBu)-Glu (O'Bu)-V- (O-Boc-D)-OH in a manner analogous to that described in Example 2 to give the desired product.

Example 13. Preparation of Compound 13.

Compound 13 Compound 12 is deprotected in a manner analogous to that described in Example 9 to give the desired product.

Example 14. Preparation of Compound 14.

Compound 14 Compound 1 is treated with protected alanine (N-Fmoc-Ala-OH) in a manner analogous to that described in Example 2 to give the desired product.

Example 15. Preparation of Compound 15.

Compound 15 Compound 14 is deprotected using a method similar to that described in Example 3 to give the desired product.

Example 16. Preparation of Compound 16.

Compound 16 Compound 15 is coupled with a protected alanine (N-Cbz-Ala-OH) using a method similar to that described in Example 2 to give the desired product.

Example 17. Preparation of Compound 17.

Compound 17 Compound 3 is deprotected in a manner similar to that described in Example 9 to give the desired product.

Example 18. The luminogenic protease substrates of the invention provide a strong signal upon protease digestion.

To demonstrate that the luminogenic protease substrates of the invention are easily digested by protease enzymes, the degree of cleavage is determined by assaying for the appearance of substrate cleavage products in the presence of a protease.

Approximately 1 microgram of protease substrate substituted with an appropriate peptide (as partially listed in Table 1) is dissolved in a buffer consisting of the appropriate salts and other additives needed to preserve enzyme activity. To this buffer solution is added a protease such as elastase, trypsin and caspases etc, and the solution is incubated. The solution is then analyzed by a luminescence instrument such as microscope, microplate reader, or a spectrophotometer. Alternatively, the solution is analyzed by HPLC before and after the addition of the protease. The digestion is carried out at 37 °C. The HPLC-separated components are monitored either via absorption or luminescence. Figure 3 shows the fluorescence emission spectrum of Compound 9 (Ac-DEVD-RhllO-MPL) (a) before and (b) after addition of caspase-3, a representative protease.

Example 19. Uptake and retention of MLP-Rhl 10-PP derivatives in live cells.

Jurkat cells are placed in 5 mL of HBSS medium containing 1-25 uM of the desired rhodamine 110-based protease substrate. The cells are incubated for varying times at 37 °C in a C02 incubator, recovered by centrifugation, and washed in 50 mL of ice-cold medium. The cells are then re-centrifuged and the final pellet is resuspended in 50 pL of fresh medium. Aliquots of each cell suspension are placed on microslides and viewed on a Nikon inverted microscope with epifluorescent illumination. The cells exhibit good staining by the rhodamine-based enzymatic reaction products. Other cells are treated similarly as described for the Jurkat cells with appropriate modifications.

Example 20. Susceptibility of the peptide-rhodamine 110-MPL derivatives to protease activity.

Peptide-rhodamine 110-MPL activities as synthetic substrates for proteases are measured in a luminometric enzyme assay. Some enzymes are commercially available, and the commercially unavailable enzymes are prepared by expressing DNA clones encoding these enzymes in an insect host cell (See, Webb, N. R. et al., "Expression of proteins using recombinant Baculovirus, "Techniques 1990,2, 173- 188). Cleavage of the synthetic substrates of the invention by the enzymes results in a luminescence signal that is readily recorded using a spectrofluorometer, a microscope, a flow cytomer or a fluorometric microplate reader.

Example 21. Cleavage of Compound 9 by recombinant human caspase-3.

Compound 9 is incubated with various concentrations of purified active human recombinant caspase-3 (Sigma) at 37 °C. The caspase-3 is prepared in PBS buffer containing 0. 1% BSA. 100 gel of diluted caspase-3 are transferred into a 96- well black microplate (with clear bottoms) into each well of which an equal volume of 50 uM Compound 9 in 2X reaction buffer (20 mM HEPES buffer, pH 7.4, 0.2% CHAPS, 4 mM EDTA, and 10 mM DTT) is added. The enzymatic reaction kinetics are measured using excitation at 485 nm and detecting the luminescence emission at 525 nm for 1 hr at 20 second intervals using a FLEXstation fluorescence microplate reader with integrated microfludics system (Molecular Devices Corporation). Figure 4 shows the time-dependent luminescence increase after the addition of 1 unit of caspase-3. The enzyme concentrations (from the bottom curve to the top curve) are 0, 0.61, 1.25, 2.50, 5.00 and 10.00 ng/well, respectively.

Example 22. A comparison of Compound 9 with bis- (Acetvl-DEVD)- rhodamine 110 in the detection of recombinant human caspase-3.

The caspase-3 assay used is similar to that described in Example 21. 100 pL of purified active human recombinant caspase-3 (10 ng/well, Sigma) in PBS buffer containing 0.1% BSA is treated with an equal volume of 50 uM Compound 9 or (Ac- DEVD) 2Rhl 10 in 2X reaction buffer (20 mM HEPES buffer, pH 7.4, 0.2% CHAPS, 4 mM EDTA, and 10 mM DTT) at room temperature. The luminescence kinetics are

measured using a FLEXstationTM fluorescence microplate reader using excitation at 485 and emission detection at 525 nM for 1 hr at 20 seconds interval. Figure 5 shows that Compound 9 (curve A) exhibits a much higher sensitivity for detection of human recombinant caspase-3 activity than the bis-peptide derivative (curve B).

Example 23. Cleavage of Compound 9 by apoptotic cells.

A supply of JurKat cells at a concentration of about 100,000/well (100 pL) are plated into a 96-well black plate (with clear bottom) in RPMI medium containing 10% fetal bovine serum (FBS) and 1% L-glutamine. Sample cells are treated with camptothecin (2 uM) for 6 hours at 37 °C in a 5% C02 incubator to induce apoptosis, while untreated cells are used as a'control'. Both the control and apoptotic cells are incubated with an equal volume of 50 FM Compound 9 in 2X reaction buffer (20 mM HEPES buffer, pH 7.4, 0.2% CHAPS, 4 mM EDTA, and 10 mM DTT) at room temperature for 20,40, and 60 min. The luminescence kinetics are measured using a FLEXstationTM fluorescence microplate reader using excitation at 485 and emission detection at 525 nM. Figure 6 shows that caspase-3 activity in JurKat cells can be readily detected using the protease substrates of the invention.

Example 24. Detection of cell apoptosis using a microplate reader.

One of the earliest and most consistently observed features of apoptosis is the expression of a series of cytosolic proteases, the caspases, which cleave protein substrates and lead to apoptotic morphology. Caspase-3, also called appopain or CPP32, has been identified to be a key member of this caspase family of proteases.

(See, Abu-Qare, A. W. and M. B. Abou-Donia, "Biomarkers of apoptosis: release of cytochrome c, activation of caspase-3, induction of 8-hydroxy-2'-deoxyguanosine, increased 3-nitrotyrosine, and alteration of p53 gene. "J Toxicol Environ Health B Crit Rev 2001,4, 313-32; Eldadah, B. A. and A. I. Faden, "Caspase pathways, neuronal apoptosis, and CNS injury. "J Neurotrauma 2000,17, 811-29; Nicotera, P., "Caspase requirement for neuronal apoptosis and neurodegeneration."IUBMB Life 2000,49, 421-5. ). It has been suggested that activation of the ICE-family proteases and caspase-3 activity are required for several phenotypes associated with apoptosis in mammalian cells.

The luminogenic cleavage of Compound 9 by caspase is readily used to monitor apoptosis in whole cells. Specifically, different numbers of JurKat cells are seeded into 96 well black plate (with clear bottom). The cells are treated with camptothecin (2 uM) for 6 hours at 37 °C in a 5% C02 incubator to induce apoptosis, and untreated cells are used as a'control'. Both the control and induced cells are then incubated with an equal volume of 50 uM Compound 9 in 2X reaction buffer (20 mM HEPES buffer, pH 7.4, containing 0.2% CHAPS, 4 mM EDTA, and 10 mM DTT) at room temperature for 30 min. The luminescence is measured using a FLEXstation fluorescence microplate reader using excitation at 485 nm and emission detection at 525 nm.

Figure 7 shows the cell number response of caspase 3 activity in the JurKat cells. Figure 8 shows the dose response of camptothecin on the JurKat cell apoptosis.

The Z factors are 0.51, 0.88, 0.8, 0.86, 0.76, and 0.83 corresponding to camptothecin doses from 0.1 to 30 uM. Figure 9 shows time course response of camptothecin- induced JurKat cell apoptosis detected by Compound 9.

Example 25. The comparison of Compound 9 with bis- (Ac-DEVD)- rhodamine 110 for detection of cell apoptosis.

JurKat cells at the density of 100,000 cells/well (100 uL) are seeded into a 96-well black plate (with clear bottom). The cells are treated with camptothecin (2 uM) for 6 hours at 37 °C in a 5% C02 incubator to induce apoptosis, and the untreated cells are used as a'control'. Both the control and induced cells are then incubated with an equal volume of 50 pM Compound 9 or bis- (Ac-DEVD)- rhodamine 110 in 2X reaction buffer (20 mM HEPES buffer, pH 7.4, containing 0.2% CHAPS, 4 mM EDTA, and 10 mM DTT) at room temperature for 30 min. The incubations are performed either in the presence of Ac-DEVD-CHO, a caspase-3 inhibitor (20 uM) or in the absence of the inhibitor. The resulting luminescence is measured using a FLEXstation fluorescence microplate reader using excitation at 485 nm and emission detection at 525 nm. Figure 10 clearly demonstrates the much higher detection sensitivity made possible by the use of Compound 9.

Example 26. Detection of cell apoptosis by luminescence microscopy using the protease substrates of the invention.

The ability of the caspase substrates of the invention to detect caspase activation in intact cells is tested using apoptotic HL-60 and Jurkat cells. These whole-cell assays are carried out in two stages: 1) induction of apoptosis, and 2) incubation with the selected substrate. For HL-60 cells, apoptosis is induced by treatment with 10 Rg/mL vinblastine for 4 hours. Control samples are treated with DMSO. For Jurkat cells, apoptosis is induced by treatment with 500 ng/mL agonistic antiFas antibody for 2 hours. Control samples are treated with PBS. Following apoptosis induction, the cells are incubated with 50 uM Compound 9 in caspase assay buffer (40 mM PIPES, pH 7.4 ; 100 mM NaCI ; 10% sucrose; 1 mM EDTA; 10 mM DTT). The cells are than transferred to a glass microslide and viewed using epifluorescent illumination on a Nikon inverted microscope. It is observed that drug- treated cells exhibit higher luminescence than DMSO-treated cells (control). HL-60 cells treated with 50 uM the caspase-3 inhibitor Ac-DEVD-CHO show reduced luminescence signal, indicating that the staining observed in the treated cells is due to caspase-mediated cleavage. Jurkat cells induced to undergo apoptosis by antiFas also show intense staining when incubated with Compound 9 while control cells showed only minimal staining.

Example 27. Cleavage of Ac-IETD-RhllO-MPLbvcaspases 3, 6, 7, and 8.

A protease substrate of the invention is prepared having an acetylated tetrapeptide side chain having the sequence Ile-Glu-Thr-Asp (or IETD, SEQ ID NO.

17). The following assay evaluated whether the resulting compound is a substrate for human caspase-3, -6,-7, or-8.

The caspase assays are carried out at 37 °C in 96-well plates in 100 aL of buffer containing recombinant human caspase, 5 uM of Ac-IETD-RhllO-MPL and caspase assay buffer (50 mM PIPES, pH 7.2 ; 100 mM NaCI ; 10% sucrose; 0.1% CHAPS; 1 mM EDTA; 10 mM DTT). At the end of incubation period, the luminescence of the samples is determined on a Gemini fluorescence microplate reader (Molecular Devices Corporation) using excitation and emission wavelengths of 490 nm and 550 nm respectively. The control samples (where the corresponding

enzyme is omitted) are used to correct the background fluorescence of non-specific hydrolysis of the substrate. Known caspase inhibitors such as Ac-DEVD-CHO (Acetyl-L-Aspartyl-L-Glutamyl-L-Valyl-L-Aspart-1-al ; SEQ ID NO. 22) and Ac- IETD-CHO (Acetyl-L-Isoleucyl-L-Glutamyl-L-Threonyl-L-Aspart-l-al ; SEQ ID NO.

23) are used to further confirm the specificity of the substrate. It is observed that Ac- IETD-Rhl 10-MPL is cleaved by caspase 8.

Example 28. Cleavage of Cbz-AA-Rhl 10-MPL and Cbz-AAAA-Rhl 10- MPL by elastase.

Approximately 1 microgram of the substrates Cbz-AA-RhllO-MPL or Cbz- AAAA-RhllO-MPL is dissolved in a buffer (pH 8.9) consisting of 50 mM sodium phosphate and 1 mM EDTA. To this solution is added 1 unit of elastase, and the samples are incubated. The enzymatic reaction is followed using a fluorescence microplate reader substantially as described in Example 24 above.

Example 29. Flow cytometric analysis of cells incubated with apoptosis- related protease substrates.

A series of samples are prepared for subsequent flow cytometric analysis. The substrate of the invention is present in a concentration of 10 uM in RPM11640 medium containing from 4% to 10% fetal calf serum. Cell densities during incubation with the chosen substrates ranged from 50,000 cells/mL to 4,000, 000 cells/mL.

Incubation times are from 30 min to 3 hr at 37 °C, and incubation volumes are from 50 uL to 2 mL. After incubation with the selected substrate for 30 to 60 min, cell suspensions are diluted 10-fold with ice cold Hank's Buffered Saline Solution (HBSS) and then filtered through a nylon fabric sheet. This filtered cell suspension is then subjected to fluorescence-based flow cytometric analysis using a 488 nm excitation source. Control cells without substrate incubation and the sample with the greatest expected luminescence signals are used to set the instrument detector parameters. A small amount of background luminescence would be expected from non-specific hydrolysis or the intact substrate. Signals from the cells that had been activated with 1 ug/ml ofant-Fas antibody, CH11 clone for 1 to 6 hours indicated an increase in peak channel number. A large increase in luminescence intensity is observed.

Example 30. Screening for drugs that stimulate the caspase cascade.

Drugs that stimulate the caspase cascade in the absence of Fas ligand or camptothecin may be useful, for example, as anti-cancer chemotherapeutic agents.

The assay procedures substantially described in Examples 19-27 may be used to screen for drugs that stimulate the caspase cascade by carrying out the assay under similar conditions as in the above examples, except employing a known or unknown compound with known or unknown anti-cancer or anti-tumor activity in place of camptothecin or Fas ligand reagent.

Example 31. Screening for drugs that inhibit or potentiate the caspase cascade.

Drugs that inhibit the caspase cascade may be useful in treating degenerative and other diseases caused by or associated with an inadequate activation of the caspase cascade. Drugs that potentiate the action of another caspase stimulator, such as Fas ligand or an anti-cancer drug or agent, may be suitable to treat cancers or tumors caused by or associated with an inappropriate function of the caspase cascade.

The assays and reagents described in this invention may be used to screen for drugs that may inhibit or potentiate the caspase cascade in cells by performing the assay substantially as described in Examples 19-27 using camptothecin, Fas ligand, or any other agent that sufficiently stimulates the caspase cascade or other apoptosis pathway. Such an assay may include the presence of a test substance that may inhibit, potentiate, or acts synergistically with, the action of the first agent that induced apoptosis or caspase cascade.

Example 32. Evaluation of potential chemotherapeutic treatments.

It is well known that the same cancer in different patients shows a great variability to treatment with anti-cancer drugs. Therefore it is very difficult to predict whether a cancer in a patient is treatable with a particular anti-cancer drug before treatment is begun. The luminescence assays described in this invention permit chemosensitivity or drug resistance testing of cancer or tumor cells or tissue samples taken from individual cancer or tumor patients. To perform the chemosensitivity test, a luminescence assay using a cancer cell or tissue sample taken from a patient may be

conducted substantially as described in Examples 19-27. A drug having a known or unknown chemotherapeutic activity may therefore be tested for its capacity to stimulate the caspase cascade, potentially providing information useful in designing an optimal chemotherapeutic drug treatment regimen for the patient.

Example 33. Use of PP-Rhl 10-MPL in high throughput screening of drugs.

The protease substrates of the invention may be useful for high throughput screening of drugs. Such assays are typically performed similarly to those described in Examples 21,23 and 24, while typically utilizing a much smaller volume and employing automated methods. Such assays may be run in a high density multi-well microplate (such as 384-well or 1536-well plate), or a microarray device suitable for high-throughput screening.

Example 34. Reduction of extracellular and/or background fluorescence: The assays of the invention optionally may be performed using systems for reducing extracellular and/or background luminescence, including separating layers and/or masking compounds. These systems may be particularly useful in assays of intracellular protease activity, where they may be used to reduce detection of luminescence from free rhodamine dyes that have leaked from the cell interior into the extracellular medium.

A separating layer generally comprises any material that separates labeled cells from the majority of the extracellular medium (typically by being positioned between the labeled cells and the majority of the extracellular medium) and that reduces detection of light from the majority of the extracellular medium. The separating layer may reduce the detection of undesired light using any suitable mechanism, including (1) absorbing and/or reflecting excitation light before it excites extracellular luminophores, and/or (2) absorbing and/or reflecting luminescence emitted by extracellular luminophores. Exemplary separating layers may include (1) a thin film or coating of metal, beads, or other absorbent and/or reflective materials, which typically is permeable to the extracellular medium, and/or (2) a sample container insert that displaces medium away from the cells, which typically is

impermeable to the extracellular medium. Exemplary separating layers are disclosed in the following materials, which are incorporated herein by reference: U. S. Patent No. 5,601, 997, issued February 11,1997 ; and PCT Patent Application Serial No.

PCT/EP97/02662, filed May 23,1997.

A masking (or photon-reducing) compound generally comprises any composition such as a molecule or particle that reduces the amount of light detected from luminescent materials in the extracellular medium. The masking compound may reduce the amount of light using any suitable mechanism, including (1) absorbing excitation light before it excites extracellular luminophores, (2) reducing and/or quenching the luminescence of the extracellular luminophores (e. g. , by reducing their extinction coefficients and/or quantum yields), and/or (3) absorbing luminescence emitted by the extracellular luminophores, among others.

Exemplary masking compounds may include (1) binding partners, such as antibodies, (2) paramagnetic ions, such as Mn2+, Co2+, or Cu2+, (3) quenchers, such as static and dynamic quenchers, (4) energy transfer partners, such as complementary acceptors, and/or (5) dyes having overlapping spectra, among others. Dyes having overlapping spectra may include (1) acid dyes (e. g. , sulphan blue, acid violet, acid red, amido black, brilliant blue R, azocarmine G, xylidine P. 2R, orange G, and erythrosine, among others), (2) direct dyes (e. g. , trypan blue, Evans blue, vital red, thiazine red, and Congo red, among others), (3) basic dyes (e. g. , crystal violet, nightblue, malachite green, methylene blue, toluidine blue, azur A, Victoria blue, Nile blue, rhodanile blue, safranin, neutral red, and rosaniline, among others), and/or (4) other dye groups (e. g. , celestine blue, Alcian blue, carminic acid, haematoxylin, and phenol red, among others), among others.

In one aspect of the invention, the masking compound includes one or more of Acid Blue 45, Acid Blue 92, Acid Blue 93, Acid Green 25, Acid Red 106, Acid Red 112 (Ponceau S), Acid Red 40, Acid Violet 5, Acid Violet 7, Alcian Blue 8GX, Amaranth, Brilliant Black BN, Brompyrogallol Red, Direct Blue 71, Direct Red 75, Direct Violet 51, Erioglaucine (Food Dye Blue), Erythrosin B, Gallocyanine, Hydroxy Naphthol Blue, Indigo Carmine, Malachite Green, Phenol Red, Potassium Ludigotrisulfonate, QSY-7, Reactive Black 5, Reactive Blue 2, Sufonazo III, and

Violamine R. Additional exemplary masking compounds, as well as methods of use, are disclosed in the following materials, which are incorporated herein by reference: U. S. Patent No. 6,200, 762, issued March 13,2001 ; U. S. Patent No. 6,214, 563, issued April 10,2001 ; U. S. Patent No. 6,221, 612, issued April 24,2001 ; and PCT Patent Application Serial No. PCT/EP97/02662, filed May 23,1997.

The disclosure set forth above may encompass one or more distinct inventions, with independent utility. Each of these inventions has been disclosed in its preferred form (s). These preferred forms, including the specific embodiments thereof as disclosed and illustrated herein, are not intended to be considered in a limiting sense, because numerous variations are possible. The subject matter of the inventions includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and/or properties disclosed herein.