Uitspraken

“1. Een verbinding met formule:

(…)

N-(3-pyridine)carbonyl-L-fenylalanine-L-leucine boronzuur,

N-(4-morfoline)carbonyl-L-fenylalanine-L-leucine boronzuur,

N-(4-morfoline)carbonyl-B-(l-naftyl)-L-alanine-L-leucine boronzuur,

N-(8-chinoline)sulfonyl- B -(l-naftyl)-L-alanine-L-leucine boronzuur,

N-(4-morfoline)carbonyl-L-tyrosine-L-leucine boronzuur,

N-(4-morfoline)carbonyl-[0-(2-pyridylmethyl)]-L-tyrosine-L-leucine boronzuur,

of farmaceutisch aanvaardbare zouten of boronaatesters ervan is.

“1. A compound having the formula:

(…)

N-(2-pyrazine)carbonyl-L-phenylalanine-L-leucine boronic acid,

N-(2-quinoline)sulfonyl-L-homophenylalanine-L-leucine boronic acid,

N-(3-pyridine)carbonyl-L-phenylalanine-L-leucine boronic acid,

N-(4-morpholine)carbonyl-L-phenylalanine-L-leucine boronic acid,

N-(4-morpholine)carbonyl-β-(1-naphthyl)-L-alanine-L-leucine boronic acid,

N-(8-quinoline)sulfonyl-β-(1-naphthy)-L-alanine-L-leucine boronic acid,

N-(4-morpholine)carbonyl-(O-benzyl)-L-tyrosine-L-leucine boronic acid,

N-(4-morpholine)carbonyl-L-tyrosine-L-leucine boronic acid,

N-(4-morpholine)carbonyl-[O-(2-pyridylmethyl)]-L-tyrosine-L-leucine boronic acid;

or pharmaceutically acceptable salts or boronate esters thereof.

(…)”

“ Summary of the Invention

[0011] The present invention provides previously unknown peptidyl boronic acid ester and acid compounds. The present invention also provides methods of using amino acid or peptidyl boronic ester and acid compounds, in general, as inhibitors of proteasome function.

(…)

Examples

(…)

Example 18: Kinetic experiments

[0217] Table II summarizes results from kinetic experiments that measured the inhibition of the 20S proteasome by compounds having the formula of compound (1) or (2). P, AA1, AA2, AA3, and Z1 and Z2 represent the structures present on formula (1) or (2). The protocol for the kinetic assay described in Tables II-V is as described in Rocket al., Cell 78: 761-771 (1994). In these tables, Ki values are reported, which are dissociation constants for the equilibrium that is established when enzyme and inhibitor interact to form the enzyme: inhibitor complex. The reactions were performed using SDS-activated 20S proteasome from rabbit muscle. The substrate used was Suc-LLVY-AMC.

(De voorzieningenrechter merkt op dat in tabel II een overzicht is opgenomen van ongeveer 90 geteste verbindingen waarbij de Ki-waardes variëren van 0.022 tot 998. Een lage Ki-waarde duidt op een sterker gebonden remmer. MG-341 heeft een Ki-waarde van 0.6 in tabel II).

(…)

[0219] Table III demonstrates that dipeptide boronic acids have lower Ki values than the corresponding dipeptide aldehydes.

(…)

[0221] Table IV demonstrates the markedly superior selectivity for the 20S proteasome over other proteases, e.g. Cathepsin B, exhibited by the boronic esters/acids as compared to the peptide aldehydes.

[0222] The selectivity of boronic acid inhibitors of the proteasome is further demonstrated in Table V.

Example 19: Inhibition of Protein Degradation in C2C12 Cells

[0223] C2C12 cells (a mouse myoblast line) were labelled for 48 hrs with 35S-methionine. The cells were then washed and preincubated for 2 hrs in the same media supplemented with 2mM unlabelled methionine. The media was removed and replaced with a fresh aliquot of the preincubation media containing 50% serum, and a concentration of the compound to be tested. The media was then removed and made up to 10% TCA and centrifuged. The TCA soluble radioactivity was counted. Inhibition of proteolysis was calculated as the percent decrease in TCA soluble radioactivity. From this data, an EC50 for each compound was calculated.

[0224] Data for compounds of formula (1) or (2) are presented in Table VI.

(…)

Example 23: Boronic Acid Compounds Block the DTH Response in Mice

[0228] Naive mice were sensitized by the application of 20 μL of a 0.5% (v/v) solution of 2,4-dinitrofluorobenzene in 4:1 acetone/olive oil to both of the rear limb footpads. This procedure is performed on two consecutive days, which are referred to as days 0 and 1.

[0229] The efferent phase of the contact sensitivity response was elicited on day 5 by the application of 10 μL of a 0.2% (v/v) solution of 2,4-dinitrofluorobenzene in 4:1 acetone/olive oil to both sides of the left ear. The contralateral control ear was treated on both sides with 10 μL of vehicle only. (…)

[0230] Test compounds were administered orally as a suspension in 0.5% methylcellulose (4000 centipoises Fisher Scientific) 30 minutes prior to the application of the challenge dose of 2,4-dinitrofluorobenzene to the ears. (…)

[0231] Approximately 18 hours after the challenge, ear swelling was determined by measuring both the control and the experimental ear using a Mitutoyo Digital micrometer. The absolute difference in thickness of the experimental (left) ears vs. the control (right) ears was determined for each treatment group. Efficacy was determined by comparing this difference in thickness to the difference calculated for the vehicle control group. Test results are provided in Table VII.

”

“ Summary of Invention

The present invention concerns the identification, characterization, and purification of two chymotrypsin-like serine proteases characteristic of AD (Alzheimer's disease, voorzieningenrechter), called "chymase" and "multicatalytic protease",that are derived from rat or human brain. Both chymase and the multicatalytic protease have an enzymatic activity capable of cleaving between Met and Asp, which activity is required to generate beta-amyloid. Methods are provided for quantifying the activities of chymase and the multicatalytic protease in brain (and other tissue samples and fluids) to allow diagnosis of AD, and for development of inhibitors as therapeutic agents to slow progress of the disease. Novel compositions have been prepared and identified as inhibitors of the two enzymes. Methods are also provided for cloning and sequencing of human brain chymase and the multicatalytic protease, and the preparation of nucleic acid and antibody probes to them.

Thus, in a first aspect the invention features a purified peptide (which term includes polypeptides and proteins) having an endopeptidase enzymatic activity which causes hydrolysis of a peptide bond between a methionine and an aspartic acid in a beta-amyloid precursor protein. By "purified" is meant that the peptide is separated from components of a cell or tissue in which it naturally occurs; preferably it is purified to represent at least 70% by weight, most preferably 90%, of the peptides present in a solution or preparation.

In a second aspect, the invention features a method for detection in a biological sample of an protease indicative of Alzheimer's disease. The method includes providing a substrate for the protease having the formula I:

I: R-A4-A3-A2-A1-R1

wherein R is an N-terminal blocking group or hydrogen; R1 is a reporter group; A4 is a covalent bond (i.e., R is directly bonded to A3), an amino acid or a peptide, e.g., including up to about five D- or L-amino acids; A3 a covalent bond (i.e., A4 is directly bonded to A2 or when A4 is a covalent bond R is directly bonded to A2), or is a D-amino acid, Phe Tyr, or Val or a conservative amino acid substituent of Val, e.g., Leu, Ile, Nle, Ala, and Nva; A2 is a hydrophobic amino acid, e.g., Leu, Ile, Nle, Phe, Val, Nva, Tyr, or Pro, or preferably lysine or a conservative amino acid substituent thereof, e.g., Arg, Orn, Cit, and Harg or when A4 includes at least two amino acids, A2 is any amino acid; and A1 is Met, Phe, Nle, Leu, Ile, Tyr, or 3-phenylproline; when A4 and/or A3 are a covalent bond, R may be an alkane dicarboxylic acid or alkane carboxylic acid of 2 to 20 carbon atoms, or a lower alkyl monoester thereof, optionally substituted by an alkyl, aralkyl, or aryl substituent; preferred substituents are isopropyl, isobutyl, benzyl, or phenyl, and preferred lengths are 3 to 10 carbon atoms. The method also includes contacting the biological sample with the substrate, and detecting cleavage of the substrate as an indication of the presence of the protease in the biological sample.

(…)

By "N-terminal blocking group" is meant a D-amino acid or an arylcarbony1, alkylcarbony1, alkoxycarbony1, aryloxycarbonyl, aralkyloxycarbonyl, aralkylsulfonyl, alkylsulfonyl, or arylsulfonyl peptide protecting group, or other equivalents known to those skilled in the art of peptide synthesis and which are known to protect molecules from degradation by aminopeptidases (Gross and Meienhofer, eds.. The Peptides, vol. 3, Academic Press, New York, 1981 pp. 3-81, describes numerous suitable amine protecting groups) . As used herein, either individually or as part of a larger group, "alkyl" means a linear, cyclic, or branched-chain aliphatic moiety of 1 to 20 carbon atoms; "aryl" means an aromatic moiety, e.g., phenyl, of 6 to 18 carbon atoms, unsubstituted or substituted with one or more alkyl, substituted alkyl, nitro, alkoxy, or halo groups; "substituted alkyl" means an alkyl group having a substituent containing a heteroatom or heteroatoms such as N, 0 or S; "halo" means Cl or Br, and "alkaryl" means an aryl moiety of 7 to 19 carbons having an aliphatic substituent, and, optionally, other substiuents such as one or more alkyl, substituted alkyl, alkoxy or amino groups. "Aralkyl" means a linear or branched chain aliphatic moiety of 7 to 18 carbon atoms including an aryl group or groups. Examples of suitable N-terminal blocking groups include formyl, acetyl, trifluoroacetyl, benzyloxycarbonyl (carbobenzyloxy) , substituted benzyloxycarbonyl, tertiary butyloxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl, phthaloyl, benzoyl, acetoacetyl, chloroacetyl, phenoxycarbonyl, methoxysuccinyl, succinyl, adipyl, suberyl, 2,4-dinitrophenyl, dansy1, p-methoxybenzenesulfonyl, p-toluenesulfonyl, methanesulfonyl, D-serine, and D-glutaitiic acid.

(…)

In a fourth aspect, the invention features an inhibitor of a protease, having the formula II:

II: R-A4-A3-A2-Y

where R, A4, A3, and A2 are as described above, and Y is a group reactive with the active site of the protease. These inhibitors have therapeutic utility derived from their ability to inhibit the enzyme "chymase" or the enzyme "multicatalytic protease" or related enzymes. These inhibitors include peptide sequences protected at their N-terminus by a protecting group, and attached at their C-terminus by a covalent bond to a moiety Y, which interacts strongly with functional groups located near the reactive site of the enzyme.

The N-terminal blocking group provides metabolic stability to the composition by reducing or eliminating its ability to be degraded by endogenous aminopeptidases. The carboxyl terminal moiety Y allows a strong chemical interaction, mediated by covalent or noncovalent bond formation, at the active site of the enzyme to effectively inactivate the enzyme.

Y is a group which strongly interacts with functional groups in or near the enzyme active site and is covalently attached to the carbonyl group of amino acid A2 by an amide bond. It is provided with a lipophilic side chain substituent R2, which interacts with the lipophilic substituent recognition site of the enzyme to be inhibited, e.g., chymase or the multicatalytic protease, and with a functional group R3, which can react with or interact with an adjoining serine hydroxyl group to form a transition-state analog (Rich, Protease Inhibitors, ed. Barrett and Salversen, Elsevier, 1986, p. 167.

Y may be derived from an amino acid analog Y-H in which Y has the formula III:

III: -HN-CH-R3”

|

R2

wherein R2 may be a saturated or unstaturated alkyl or substituted alkyl group of 2 to 10 carbon atoms, alicyclic or aromatic groups of 5 to 10 carbon atoms or arylalkyl groups of up to 11 carbon atoms. Examples of suitable R2 groups include ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl n-pentyl, n-hexyl, isohexyl, n-decyl, phenyl, benzyl, beta-phenylethyl, alpha-naphthyl, beta-naphthyl ethy1, cyclohexyl, cyclohexylmethy1, adamantyl, alpha-styryl, beta-styryl, and propargyl.

Examples of suitable R3 groups include the aldehyde and ketone functions -CHO, -CO-CH3, -C0-CH2C1, -CO-CH2Br, and boronic acid residues, e.g., -B-(OH)2,

where p and q are 2 or 3; as well as alphadiketones, e.g., -C-C-CH3, and alphaketoesters, e.g., -C-C-OCH3.

Other suitable R3 groups include -S02F, -C8H4-SO2F, and -PO(OR4)F where R4 is a lower alkyl or aralkyl substituent.

Additional suitable Y groups include the acylating heterocyclic ring systems described by Powers and Harper, in Proteinase Inhibitors, A.J.Barrett and G. Salversen, eds., Elsevier, New York, 1986, pp. 108-132. These substituents generally interact with the enzyme active-site serine and may subsequently react with other adjoining functionalities such as histidine side chains. Examples of such Y groups include N-substituted saccharins, as well as derivatives of benzoxazin-4-ones, 3-alkoxy-4-chloroisocoumarins, oxazine-2,6-diones, substituted isatoic acids, halomethylcoumarins, N-nitrosoisoquinolinones, haloenol lactones, isobenzofuranones, ynenol tetrahydro-2-furanones,

2-pyranones, chloropyrones, chloroisocoumarins and the like.

Other Y groups may be derived from alpha-aza amino acids and their amide, ester, and peptide derivatives of formula IV:

IV: H2N-NR2-CO-R5

where R2 is as defined in formula III and R5 may be derived respectively from an amine or ammonia, an alcohol, or a peptide sequence substituted at its amino terminus. Examples of the synthesis of such aza amino acids and azapeptides are reviewed by J. Gante, Synthesis, 405-413, 1989. Preferred values of R5 are amino acids or peptide sequences found at the amino terminus of beta-amyloid, e.g., asp, asp-ala, asp-ala-glu, asp-ala-glu-phe and their esters and amides.

(…)

b) Multicatalytic protease inhibitors

A strategy equivalent to that used to design chymase inhibitors was employed for the human brain multicatalytic protease. Figure 14 shows the relative hydrolysis rates of 48 peptide analog substrates, compared at a single substrate concentration. Note that substrate preferences differ from those of brain chymase. For example, while chymase prefers Phe at P1 and Lys at P2, the multicatalytic protease prefers Leu at P1 (although Phe, Nle, and Met are also excellent here) and Arg at P2. Among the residues tested thus far at the P3 position, He is the most-preferred. Thus, peptide analogs can be carefully tailored which target the multicatalytic protease but spare related chymotrypsin-like enzymes.

As illustrated above for chymase, preferred substrates of the multicatalytic protease can be modified at their carboxy termini to generate potent enzyme inhibitors. Figure 13, panel B depicts the inhibition of the multicatalytic protease by some peptide analogs and commercially available protease inhibitors. The novel peptide analog inhibitors are substantially more potent blockers of the multicatalytic protease chymotrypsin activity than any previously described inhibitor.

Bij WO 904 horen voorts - onder meer - de volgende figuren:

“Wij zijn verplicht u te informeren dat de voorzieningenrechter van de rechtbank te Den Haag bij vonnis van [datum] heeft beslist dat het door ons op de markt gebrachte generieke geneesmiddel “Bortezomib Teva” inbreuk maakt op Millennium Pharmaceuticals’s ABC (NL) 300151, en dat deze producten derhalve niet langer mogen worden aangeboden, verkocht of geleverd, dan wel gebruikt of in voorraad worden gehouden. Wij verzoeken u hierbij om alle voornoemde producten die zich onder u bevinden aan ons te retourneren. Wij zullen dan onmiddellijk de aankoopprijs en alle kosten in verband met de retournering van de producten aan u vergoeden.

De directie van Teva Nederland B.V.”

dan wel een brief van zodanige inhoud of vorm als de voorzieningenrechter in goede justitie zal bepalen, één en ander onder de verplichting om gelijktijdige kopieën van alle te verzenden brieven te verschaffen aan Millennium;

Obvious and consequently non-inventive selection among a number of known possibilities:

(i)

(ii)

(iii)

(iv) The invention consists merely in selecting particular chemical compounds or compositions (including alloys) from a broad field.

Example: The prior art includes disclosure of a chemical compound characterised by a specified structure including a substituent group designated "R". This substituent "R" is defined so as to embrace entire ranges of broadly-defined radical groups such as all alkyl or aryl radicals either unsubstituted or substituted by halogen and/or hydroxy, although for practical reasons only a very small number of specific examples are given. The invention consists in the selection of a particular radical or particular group of radicals from amongst those referred to as the substituent "R" (the selected radical or group of radicals not being specifically disclosed in the prior-art document since the question would then be one of lack of novelty rather than obviousness). The resulting compounds:

(a) are neither described as having nor shown to possess any advantageous properties not possessed by the prior art examples; or

(b) are described as possessing advantageous properties compared with the compounds specifically referred to in the prior art, but these properties are ones which the person skilled in the art would expect such compounds to possess, so that he is likely to be led to make this selection.

[40] A fundamental problem with this opinion is that the 904 Patent provided the person of

skill with a clear roadmap to bortezomib. Although choices were still required to be made for all of the elements identified by Dr. Wuest, those choices were rendered obvious by the teaching of the 904 Patent. The person of skill knew that, with the various substitutions recognized by the 904 Patent, a potent inhibitor of the proteasome would emerge. A person of skill is not doing anything inventive when he chooses options provided in a prior patent to build a molecule that he expects will work. Dr. Wuest’s assertions that the person of skill would still not know which of those choices would work “best” or would function in some “special” way is not germaine because the 936 Patent makes no such claim. Indeed, the 936 Patent does not assert that only a specific combination of elements will work or that the choices required to obtain bortezomib were the “best” of those that were available. The Patent claims millions of compounds made with a large array of structural options all of which are said to be potent inhibitors of the proteasome.

Anders dan de Canadese rechter is de voorzieningenrechter voorshands evenwel niet overtuigd van de voorspelbaarheid van de remmende eigenschappen van een verbinding (niet alleen qua activiteit maar ook qua selectiviteit) bij de speurtocht onder de miljoenen verbindingen van US 904.

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