WO2011039171A1

WO2011039171A1 - Vasoactive peptide and derivatives thereof

Info

Publication number: WO2011039171A1
Application number: PCT/EP2010/064342
Authority: WO
Inventors: Axel Kretschmer; Jochim Jankowski; Vera Jankowski; Walter Zidek; Markus Van Der Giet
Original assignee: Bayer Schering Pharma Ag; Charité - Universitätsmedizin Berlin
Priority date: 2009-09-30
Filing date: 2010-09-28
Publication date: 2011-04-07

Abstract

The present invention is in the field of pharmacological active peptides and peptide analogs which can be used as therapeutics to treat diseases characterized by dysfunctional vascular physiology. The peptides modulate gene expression in blood vessel cells and they regulate vessel tonus thus influencing blood pressure and the function of main cardiovascular organs like kidney, heart and lung as well. The therapeutic mode of action of the vasoactive peptides results in amelioration of organ blood perfusion and antagonism of cellular degenerative processes within these organs. These modes of action of the peptides result in organ-protective effects and ameliorate the health conditions of patients suffering from cardiovascular diseases.

Description

Vasoactive peptide and derivatives thereof

Technical field of the invention The present invention is in the field of pharmacological active peptides and peptide analogs which can be used as therapeutics to treat diseases characterized by dysfunctional vascular physiology. The peptides modulate gene expression in blood vessel cells and they regulate vessel tonus thus influencing blood pressure and the function of main cardiovascular organs like kidney, heart and lung as well. The therapeutic mode of action of the vasoactive peptides results in amelioration of organ blood perfusion and antagonism of cellular degenerative processes within these organs. These modes of action of the peptides result in organ-protective effects and ameliorate the health conditions of patients suffering from cardiovascular diseases.

Background of the invention

Fatal cardiovascular diseases often develop from prolonged hypertension and gradually progressive loss of function of vascular cells, and of renal degeneration processes or affected heart functions. Key hormone mediators for chronic-progressive heart or kidney disease emerge from the renin- angiotensinogen-aldosterone system (RAAS), which is frequently activated in cardiovascular disease patients. Increased levels of angiotensin-ll and/or aldosterone in the circulation result in raised blood pressure and/or volume overload of the circulation thus affecting heart and renal functions which eventually decline by tissue remodeling and organ degeneration processes. These tissue degeneration processes are being elicited often by abnormal high angiotensin-ll (Ang-ll) and/or aldosterone levels. Noteworthy, increased angiotensin-ll level in the circulation stimulate the excessive release of aldosterone from adrenal glands into the circulation. This in turn enhances the decline of heart and kidney functions by fibrotic tissue remodeling and degeneration. Tissue remodeling affects pump functions of the heart and diminishes the renal blood filtration capacity. The outcome of these organ dysfunctions may result in chronic renal failure or heart failure or both. These cardiovascular diseases contribute to a major proportion of fatal illnesses in the industrialized countries.

The invention describes the novel endogenous octapeptide Pro-Glu-Val-Tyr- lle-His-Pro-Phe (Angioprotectin) and derivatives thereof, which modulate gene expression in vascular cells and in tissues of the kidneys and the heart. Angioprotectin and its derivatives modulate angiotensin-ll receptor apart from other biological activities which regulate the expression of certain genes in cells of the vasculature and the end-organs of the circulation i.e. heart and kidneys. Angioprotectin provides physiological antagonism of vasoconstrictor actions of Ang-ll via the angiotensin AT1 receptor. Therefore, angioprotectin and its derivatives will provide new options for the treatment of cardiovascular diseases. Furthermore, angioprotectin is of use and contributes as an essential tool to the identification of targets for the development of new therapy regimens in the field of cardiovascular diseases including renal diseases. In this context, angioprotectin is also a key component to be used for any kind of screening methods or methods for optimization of medicinal chemistry products in drug development processes, because angioprotectin is necessarily being used as reference control compound or as a model compound for drug optimization procedures.

Angioprotectin plasma concentrations in healthy human volunteers were about 15% and in renal failure patients up to 50% of plasma Ang-ll concentrations. A commercially available Ang-ll antibody did not differentiate Angioprotectin and Ang-ll and thus Angioprotectin can contribute to Ang-ll concentrations measured by antibody-based assays. As a consequence, the analyses of circulating Ang-ll levels in patients are likely to be perturbed by the overlaying effect of angioprotectin and this may lead to misinterpretations regarding the status of circulating Ang-ll levels in a given patient. In this regard, Angioprotectin is also crucial tool to develop diagnostic means for measuring angioprotectin in blood or urine samples. This will allow to determine circulating Ang-ll levels more precisely for its differentiation versus angioprotectin apart from the need to determine angioprotectin itself for monitoring the disease status of a patient. Description of the invention

Angioprotectin, a new vasoactive octapeptide of the structure Pro-Glu- Val-Tyr-lle-His-Pro-Phe

The octapeptide angioprotectin was first isolated by chromatography from blood of patients suffering from chronic renal failure. The isolation of peptides from clinical serum or plasma samples and the elucidation of the molecular structures of such peptides by means of mass spectroscopy have been described earlier (V. Jankowski et al. Mass-spectrometric identification of a novel angiotensin peptide in human plasma; Arterioscler Thromb Vase Biol 2007; 27:297-302). One of the isolated peptides was in the range of the molecular weight of known angiotensin peptides like angiotensin-ll or angiotensin (1 -7), however its molecular mass of 1001 .5 Dalton differed distinctly from known angiotensins. The molecule of the mass signal m/z 1001 .5 Dalton was analyzed in detail and the peptide sequence of angioprotectin was deduced from the findings of high resolution mass spectroscopy. In depth analysis of the signal of the molecular mass of m/z 1001 .5 Dalton and the analyses of its degradation products which are being formed under the conditions of mass spectrometry resulted in the structure elucidation of an octapeptide which was later named angioprotectin because of its vasoactive properties. The peptide sequence of this octapeptide is defined by the following amino acids Pro-Glu-Val-Tyr-lle-His-Pro-Phe. Subsequently to the sequence analysis by mass spetrometry, angioprotectin was synthesized by conventional peptide synthesis in order to proof the predicted molecular structure. It could be shown that the analytical data which determine the molecular structure of the peptide were identical for both, the natural angioprotectin isolated from human blood samples and the chemically synthesized octapeptide.

For subsequent tests of the biological activity of angioprotectin, both compounds i.e. the naturally occurring angioprotectin isolated from blood samples and the chemically synthesized angioprotectin have been used for these tests whenever possible, in order to demonstrate biological equivalence of the natural angioprotectin and the chemically synthezised angioprotectin. Since only a limited amount of angioprotectin could be isolated from blood samples, this naturally occurring angioprotectin was used predominantly in the cellular tests where low amounts were sufficient to demonstrate the biological effects of this peptide. Likewise, the synthetic peptide analogs where the first two amino-terminal amino acids Pro-Glu were substituted by N-methyl-Ala-Asp or alpha-methyl-Pro-Asp were also used in assays to determine their biological activity on vascular cells and renal cells.

Angioprotectin antagonized the contractile actions of Ang-ll on rat aortic rings. Equimolar angioprotectin concentrations reduced Ang-ll-induced constrictions by about 30 %. It could be also shown that angioprotectin antagonizes the angiotensin-ll receptor (AGTR1 ) expressed on human renal cells. Furthermore, angioprotectin modulates the Ca2+-influx of vascular smooth muscle cells this highlights the role of angioprotectin as a regulator of vascular function. In this line of evidence, angioprotectin induces the expression of genes in vascular cells like primary human endothelial cells of the microvasculature of the skin (human dermal endothelial cells hDEC) that are involved in the regulation of endothelial cell function and the physiological responsiveness of blood vessels. The following genes among others are being regulated in hDEC when the cells are being exposed to angioprotectin: Dual specificity phosphatase and pro isomerase domain containing 1 (DUPD1 ), extracellular leucine-rich repeat and fibronectin type III domain containing 2 (ELNF2), and the neuronal L-type calcium channel gamma-4 subunit (CACNG4). An extended list of genes regulated by angioprotectin in hDEC is being presented in Table 2 and in the following chapter "Detailed description of the invention". Since angioprotectin has not been identified as a naturally occurring component of circulating blood, and, since angioprotectin plays a pivotal role in the regulation of blood vessel tonus and further endothelial functions, quantitative measurement of angioprotectin in body fluids from patients becomes a prime target for the development of a diagnostic kit or device to monitor angioprotectin concentration levels as a biomarker in the context of diseases were the function of vascular cells play a role. Furthermore, the newly identified and pharmacological active octapeptide angioprotectin can be used as a critically important component to develop and conduct screening assays or test methods to identify and further optimize chemical compound structures for therapeutic use.

Summary of the invention

The invention relates to the use of ANGIOPROTECTIN or peptide derivatives thereof as therapeutically active compounds to treat cardiovascular diseases resulting from an activated renin-angiotensin- aldosterone system (RAAS) or resulting from dysfunctionally regulated blood vessel functions. Angioprotectin can also being used as a biomarker in cardiovascular diseases developing from activated RAAS or as a biomarker indicating a disease status or organ function that depends on the blood perfusion of an organ like heart, kidneys, lung or brain. The invention also relates to novel disease associations resulting from gene regulation processes influenced in cells by abnormal exposure to ANGIOPROTECTIN concentrations. In addition, the invention relates to novel methods of screening for therapeutic agents for the treatment of cardiovascular diseases, hematological diseases, neurological diseases, respiratory diseases, gastroenterological diseases and urological diseases in a mammal by use of ANGIOPROTECTIN or derivatives thereof. The invention also relates to pharmaceutical compositions for the treatment of cardiovascular diseases, or other diseases where blood supply of organs or tissues is being affected as seen in hematological diseases, neurological diseases, respiratory diseases, gastroenterological diseases or urological diseases. ANGIOPROTECTIN was shown to be used as a therapeutically active compound in humans and mammals. The invention further comprises methods of diagnosing cardiovascular diseases, hematological diseases, neurological diseases, respiratory diseases, gastroenterological diseases and urological diseases in a mammal by using angioprotectin as a biomarker. Furthermore, the detailed description of the biological activity of angioprotectin allows to use this octapeptide for the development and conduction of screening assays and test methods in the process of drug development. Detailed description of the invention

Isolation of ANGIOPROTECTIN from human serum or plasma samples

The blood samples (9,0 - 10,0 ml_ venous blood drawn from median cubital vein within the fold of the elbow was collected in Sarstedt serum-tubes (Sarstedt S-Monovette white cap serum tube with clot activator Catalog No. 02.1063) or by use of equivalent serum blood tubes like BD Vacutainer® Blood Collection Tube Catalog No. 367820 or 366430 or 366441 . Directly after blood collection the tubes were incubated for clotting at room temperature or at 37°C during a period of 30-60 minutes, and then placed at 4^QC for further 30 min. Subsequently the supernatant was collected after centrifugation at room temperature or at 4°C for 10 minutes at about 2600g. Serum samples were kept frozen until the isolation of ANGIOPROTECTIN. Likewise, ANGIOPROTECTIN can be isolated from donor blood plasma samples by use of Sarstedt Kalium- / Potassium EDTA-tubes (Sarstedt S- Monovette red cap K-EDTA tube Catalog No. 02.1066) or Sarstedt citrate- tubes (Sarstedt S-Monovette green cap citrate tube Catalog No. 02.1067) according to the use manual of the supplier or by means of other blood plasma separation methods.

The plasma samples were directly fractionated by size-exclusion- chromatography.

Size-exclusion chromatography gel (Sephacryl S-100 High Resolution; 1000 x 16 mm, S100 HR, Pharmacia BioTech, Uppsala, Sweden) was calibrated with 0.9 % NaCI in water. The plasma was loaded onto the column and the eluent (0.9 % NaCI in water) was pumped the with a flow rate of 2.5 ml_ min- 1 . The eluate was monitored with a UV-detector at 280 nm. Subsequently analytical reversed-phase chromatography was applied. The eluate of the size-exclusion chromatography was loaded onto a monolithic reversed phase chromatography column (ChromolithTM, Performance RP-18e; 100 x 4.6 mm I.D., Merck, Darmstadt, Germany), and a 0.1 % trifluoroacetic acid (TFA) in water solution was used as an equilibration buffer at a flow rate of 2 ml_ min-1 . The retained substances were eluted with a mixture of 0.1 % TFA in water and acetonitrile (20:80, v/v; eluent B), when using the following gradient: 0-2 min: 0 % eluent B, 2-32 min: 0-75 % B, 32-32.5 min: 75- 100 % eluent B; 32.5-33.5 min: 100 % B; flow rate: 2.0 ml_ min-1 . The eluate was monitored with a UV-detector at 280 nm. Eluate fractions were lyophilized for mass spectrometry analyses.

Structure elucidation of ANGIOPROTECTIN by mass spectrometry

The lyophilized fractions from the reversed-phase chromatography were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and MALDI-ime-of flight (MALDI-TOF/TOF) fragment ion analysis. The lyophilized fractions were re-suspended in 10 L H20. 1 μΙ_ of each fraction was prepared on a pre-structured MALDI sample support (MTP AnchorChipTM 400/384, Bruker Daltonics, Germany) using the a-4- hydroxycinnamic acid affinity sample preparation method 14. All mass spectrometric measurements were performed on a Bruker Ultraflex-lll TOF/TOF instrument (Bruker-Daltonics, Bremen, Germany). The instrument was equipped with a Smart beamTM laser operating with a repetition-rate of 100-200 Hz. On average, the presented spectra are the sums of 200 single- shot spectra for MS mode, and 600 for MS/MS mode. Mass spectra of positively charged ions were analysed in the reflector mode using delayed ion extraction. Fragment ion spectra were recorded using the LIFT option of the instrument. The calibration constants were determined using standard peptides prepared on positions adjacent to the sample, resulting in an error of <100 ppm for the recorded mass spectra. Peptide identification using the obtained fragment ion mass data was performed using the software package Mascot (Matrix Science, London, UK) as well as by RapideNovo 3.0.1 sequencing Tool (Bruker-Daltronic, Bremen, Germany). The adrenocorticotropic hormone (ACTH) fragment 18-39 (Arg-Pro-Val-Lys-Val- Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe) or saralasin (Sar-Arg-Val-Tyr-Val-His-Pro-Ala) (10 g / sample) was added to the sample as internal standard in the case of kinetic measurements by using MALDI mass spectrometry. Hereby, local differences in the peptide concentration on the MALDI spot were neutralized. The analytical procedure has been described in detail also by Vera Jankowski et al. published in Arteriosclerosis Thrombosis Vascular Biology Vol. 27, 297-302 in 2007, under the title "Mass-spectrometric identification of a novel angiotensin peptide in human plasma". A so far unknown peptide co-eluted in the same size-exclusion chromatographic fractions as the known angiotensin A (Ang A) (Figure 1 .A), which was separated from Ang A using reversed phase chromatography (Figurel .B). The reversed-phase chromatography allows the desalting of the eluate from size-exclusion chromatography and, at the same time, provides fractionation of the eluate. The peptide was obtained in a homogenous fraction (labelled by an arrow in Figure 1 .B), and this fraction was applied to MALDITOF/TOF mass spectrometry analyses. From these assays, the peptide was determined to have a molecular mass of 1001 .5 Da (Figure 1 .C). This peptide was fragmented by MS-MS-TOF-TOF-mass spectrometry to obtain sequence information (Figure 1 .D). Since no match for this peptide mass was found by a search in the MS/MS database Mascot (www.matrixscience.com) a de novo sequencing approach led to the octapeptide sequence structure of

Pro-Glu-Val-Tyr-lle-His-Pro-Phe. The amino-acid sequence was confirmed by MALDIFT mass-spectrometry. Moreover, this result was confirmed by comparison with a fragment ion spectrum of the identical synthetic octapeptide. This new octapeptide structure of Pro-Glu-Val-Tyr-lle-His-Pro- Phe was hereafter refered to "angioprotectin". Synthesis of the octapeptide angioprotectin

The identified peptide was synthesized automatically by the solid-phase method using standard Fmoc chemistry in continuous flow mode (TentaGel S Random-Access Memory (RAM) resin 0.21 mmol g^"1 for peptide amides, TentaGel S p-hydroxybenzoic acid (PHB) resin (Rapp Polymere, Tuebingen, Germany) for the free acid of angioprotectin, o-benzotriazole-N,N,N',N'- tetramethyluronium-hexafluoro-phosphate (HBTU), 2 equiv of n,n- diisopropylethylamine (DIEA), coupling 20 min, deblocking with 20% piperidine in Ν,Ν-dimethyl formamide (DMF) for 15 min, final cleavage with 95% TFA/5% water for 3 h). Purification of crude peptide was carried out by preparative HPLC on PolyEncap A300 (10 μιτι particle size, 250 mm x 20 mm, Bischoff Analysentechnik GmbH, Leonberg, Germany) in water with increasing concentrations of acetonitrile as mobile phase. An eluent gradient of 5-70 (v/v-%) acetonitrile/water (0.1 % TFA) over 70 min with a flow rate of 10 ml_ min^"1 was used. The purified peptide was lyophilized. The peptide was characterized by MALDI mass spectroscopy on a Voyager-DE STR BioSpectrometry Workstation MALDI-TOF mass spectrometer (Perseptive Biosystems, Framingham, US) using R-cyano-4-hydroxycinnamic acid and sinapinic acid as matrix and gave the expected [M + H]+ mass peaks. This method has been described previously (Beyermann, M., Fechner, K., Furkert, J., Krause, E. & Bienert, M. A single-point slight alteration set as a tool for structure-activity relationship studies of ovine corticotropin releasing factor. J Med Chem 39, 3324-3330 (1996). The product of the synthesis is presented by the peptide sequence given in SEQ ID NO:1 . Synthesis of the angioprotectin derivates N-methyl-Ala-Asp-Val-Tyr-lle- His-Pro-Phe and alpha-methyl-Pro-Asp-Val-Tyr-lle-His-Pro-Phe

The sequences of angioprotectin derivatives are presented by SEQ ID NO:2 (N-methyl-Ala-Asp-Val-Tyr-lle-His-Pro-Phe) and SEQ ID NO:3 (alpha-methyl- Pro- Val-Tyr-lle-His-Pro-Phe). These angioprotectin derivatives have been synthesized as described for the chemical synthesis of angioprotectin with the exemption that the non-proteinogenic amino acid alpha-methyl-proline was used as tert-butyloxycarbonyl (BOC) protected precursor for the synthesis. N-methyl-alanine and alpha-methyl-proline were purchased from Sigma Aldrich Chemie GmbH, Taufkirchen, Germany. The fmoc protection group was introduced to N-methyl-alanine using Fluorenylmethyloxycarbonyl chloride (FMOC-CI) under standard conditions known in the art. The BOC amino protection groups was introduced to alpha-methyl-proline by the use of 1 ,1 ,1 ,3,3,3-hexafluoroisopropanol (HFIP) as solvent and catalyst which allows the chemoselective mono-N-BOC protection with di-tert-butyl dicarbonate. Standard cleavage conditions were used for deprotection of the BOC group from the peptide. The molecular mass of the synthetic N-methyl- alanine- and alpha-methyl-proline- peptides derivatives was confirmed by electrospray ionisation (ESI) mass spectrometry identifying their mass signals 975,5 [M+H]+ and 1001 ,5 [M+H]+, respectively. Pharmacological characterization and properties of angioprotectin Example 1 : Angioprotectin antagonizes angiotensin-ll aortic contraction

Angioprotectin had no direct contractile effects on rat isolated aortic rings in physiologically relevant concentrations when applied to aortic rings in a tissue bath under spring tension. However, Angioprotectin antagonized the contractile actions

of Ang-ll on rat aortic rings. Equimolar Angioprotectin concentrations reduced Ang-ll-induced constrictions by about 30 % (Figure 1 .E). Contractile effects of vasoconstriction by Angioprotectin or Ang-ll were assayed using aortic rings freshly isolated from rat aorta. Male Wistar rats (14-16 weeks old) were killed by exposure to C02. An aortic segment immediately distal to the left subclavian arterial branch was isolated, and rings (3 mm in 16 length) were prepared according to the literature (Douglas, S.A., et al. Differential vasoconstrictor activity of human urotensin-ll in vascular tissue isolated from the rat, mouse, dog, pig, marmoset and cynomolgus monkey. Br J Pharmacol 131 , 1262-1274, 2000). Briefly, the endothelium was removed by gentle rubbing of the intimal surface, and aortic rings were suspended in tissue baths (10 ml) containing Krebs-Henseleit solution of the following composition (each mmol L^"1): 1 15 NaCI, 4.7 KCI, 1 .2 MgS04, 1 .5 KH2P04, 2.5 CaCI2, 25 NaHC03, and 10 glucose. The bathing solution was maintained at 37 °C and aerated with 95% 02, 5% CO2 (pH 7.4). Changes in tension force generation were recorded using an automated system (Emka Technologies, Paris, France). The viability of each aortic ring was determined by measuring contraction to potassium chloride (60 mmol L-1 KCI). After equilibration for 30 min, tissues were exposed

to Angioprotectin for 20 min. A cumulative concentration-response curve on the attenuation of the tension force generation by Angioprotectin (1 nmol L^"1 to 1 μιτιοΙ L^"1) was then obtained.

Example 2: Modulation of calcium-ion influx into vascular smooth muscle cells

Angioprotectin decreases partly the Ca2+-influx into vascular smooth muscle cells (VSMC) (Figure 1 .F). This reduction of Ca2+ ion influx into VSCM may explain at least in part the decrease in contraction force measured with the aortic ring tension assay by the presence of angioprotectin. In constrast, Ang-ll caused a marked increase in the intracellular Ca2+ concentration which coincidences with increased ring tension force. The cellular calcium ion influx was measured using a fluorometric image plate reader (FLIPR). Vascular smooth muscle cells (VSMC, obtained from Cell Systems Corp. Cell Systems, Kirkland, USA) were loaded with 1 μιηοΙ L^"1 Fluo4-AM and analyzed by a fluorescent image plate reader (FLIPR) in the absence or presence of increasing amounts of Angioprotectin or Ang-ll. The VSMC were seeded into 96-well plates at a density of 2.5 x 10⁴ cells/well. The cells were incubated overnight, and then loaded with Fluo4-AM (Molecular Probes, Eugene, US). Serial dilutions of palosuran in Hanks' balanced salt solution, including 0.35 g L^"1 NaHC03, 20 mmol L^"1 HEPES, 0.1 % BSA, and 0.5% DMSO final concentration, were added to the cells, and the cells were then incubated for 20 min. Angioprotectin or Ang-ll were subsequently added at a final concentration of 10-100 nmol L^"1 , and the fluorescence output was measured at 500 to 560 nm. An extinction wavelength of 485 nm and an emission wavelength of 535 nm were used to detect the fluorescence signal.

Example 3: identification of target receptors of angioprotectin

In a first attempt, angiotensin-ll (Ang-ll) displacement experiments were made in order to identify the target receptor that may mediate the physiological effects of angioprotectin. Using fluorescently labeled (FAM) peptides of Ang-ll and angioprotectin, it was found by fluorescence light microscopy on endothelial mouse vessel cells, that angioprotectin displaced Ang-ll from these cells, and. Angioprotectin displaced also labeled Angioprotectin from these endothelial cells. However, Ang-ll had no significant effect on the binding of labeled Angioprotectin to endothelial cells. This finding demonstrates that despite the fact that a considerable homology between the amino-acid sequences of Ang-ll and Angioprotectin exists, the physiological effects of Angioprotectin and Ang-ll obviously are mediated by different receptors. Example 4: Pharmacological effects of angioprotectin are mediated by regulation of gene expression

In order to identify the capability of angioprotectin and the related peptide analogs of the SEQ ID No. 1 - 3, to effect a pharmacological action by influencing the regulation of mRNA expression in cells of the vasculature and cells from tissues of the cardiovascular target organs heart and kidneys, angioprotectin was incubated with such cells and differences in relative mRNA levels were recorded in comparison to untreated control cells either by using Affymetrix microarray detection or by real-time polymerase chain reaction (RTPCR) . It could be shown, that angioprotectin has an effect on the regulation of gene expression in endothelial cells and kidney cells. Noteworthy, genes that are being involved in processes of gene expression and cellular growth have been identified to be regulated dependent on angioprotectin concentration applied to these cells (Table 2). The influence of angioprotectin on the relative amount of mRNA expression was determined as follows.

For Affymetrix microarray experiments total RNA was extracted from cells and purified using an affinity resin column (RNeasy; Qiagen, Hilden, Germany). The RNA was quantified by spectrophotometry (absorbance 260 nm), and the quality of RNA was assessed by microfluidics electrophoretical separation with a Bioanalyzer (Agilent Technologies, Palo Alto, USA). Purified total RNA (1 μg) was converted to cDNA using the Superscript Choice cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA), incorporating a T7-(dT)24 primer. Double-stranded cDNA was then purified by affinity resin column (Clean up Kit, Qiagen, Hilden, Germany) with ethanol extraction. Purified cDNA was used as a template for in vitro transcription reaction for the synthesis of biotinylated cRNA using an Enzo BioArray HighYield RNA transcription labeling kit (Affymetrix, Santa Clara, CA), and further purified using an affinity resin column (Clean up Kit, Qiagen, Hilden, Germany). After purification, in vitro cRNA was fragmented in buffer containing magnesium at 95°C for 35 min. Fragmented cRNA was hybridized onto the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. Briefly, 15 μg fragmented cRNA was added along with control cRNA (BioB, BioC, and BioD), herring sperm DNA (10 mg/ml), 10% DMSO, and acetylated BSA (50 mg/ml) to the hybridization buffer. The hybridization mixture was heated at 99 °C for 5 min, incubated at 45 °C for 5 min, centrifuged for 5 min at 13.000 rpm, and injected into the microarray. After hybridization at 45 °C for 16 h rotating at 60 rpm, the array was washed and stained with the Affymetrix Fluidics Protocols-antibody amplification for Eukaryotic Targets, and scanned using an Affymetrix microarray scanner (GeneChip Scanner 3000 7G system) at 570 nm. The data analyses from microarray experiments and data scaling were performed using Microarray Suite 5.0 software (Affymetrix), and normalization and further analysies were conducted by expressionist Pro 3.0 (Genedata) software. Results for HG-U133 Plus 2.0 arrays were subjected to global scaling with a target intensity of 100. Base-2 logarithms were calculated for all expression values and taken for subsequent statistical analysis.

For determination of relative mRNA expression by RTPCR, total cellular RNA was isolated from cells by a standard method using the Tri-Reagent protocol according to the manufacturer's specifications (Molecular Research Center, Inc., Cincinatti, Ohio). Total RNA prepared by the Tri-reagent protocol was then treated with DNAse I to remove genomic DNA contamination. For relative quantification of mRNA expression regulated by ANGIOPROTECTIN, total RNA from each cell or tissue source was first reverse transcribed. Total RNA was reverse transcribed using 1 mole random hexamer primers, 0.5 mM each of dATP, dCTP, dGTP and dTTP (Qiagen, Hilden, Germany), 3000 U RnaseQut (Invitrogen, Groningen, Netherlands) in a final volume of 680 I according to the manual of the supplier. The first strand synthesis buffer and Omniscript reverse transcriptase (2 u/ I) were from Qiagen, Hilden, Germany. The reaction was incubated at 37°C for 90 minutes and cooled on ice. The volume was adjusted to with water, yielding a final concentration of 12.5 ng/ I of starting RNA. The Applied Bioscience 7900HT Sequence Detection system was used according to the manufacturer's specifications and protocols. RTPCR reactions were set up for relative mRNA quantification of genes influenced by ANGIOPROTECTIN in relation to mRNA of housekeeping genes HPRT (hypoxanthine phosphoribosyltransferase), GAPDH (glyceraldehyde-3- phosphate dehydrogenase), -actin, and others for normalization. Forward and reverse primers and probes of ANGIOPROTECTIN regulated genes were designed using the Applied Bioscience ABI Primer Express™ software and using the gene sequences published for the genes of interest in the data bank of National Center for Biotechnology Information (NCBI), Gene Bank (http://www.ncbi.nlm.nih.gov/genbank/). The primers and probes were synthesized by Eurogentec (Belgium), and the probes were labelled with FAM (carboxyfluorescein succinimidyl ester) as the reporter dye and TAMRA (carboxytetramethylrhodamine) as the quencher. The following reagents were prepared in a total of 20 I : 1 x qPCR-MasterMix (Eurogentec; Belgium) and probe, forward and reverse primers each at 200 nM, 200 nM FAM/TAMRA-labelled probe, and 5 I of template cDNA. Thermal cycling parameters were 2 min at 50 °C, followed by 10 min at 95 °C, followed by 40 cycles of melting at 95 °C for 15 sec and annealing/extending at 60 °C for 1 min. The calculation of the relative mRNA expression is described in detail in the User Bulletin #2 of the ABI Prism 7700 Sequence Detection System of Applied Biosystems (Life Technologies Corp. Carlsbad, California, USA).

Some examples of genes which are being regulated by the addition of ANGIOPROTECTIN are listed in Table 2. This list is not intended to represent a complete list of genes regulated by angioprotectin application to cells or tissues.

Example 5: Correlation of circulating angioprotectin with disease status of patients suffering from renal failure

To assess the relevance of Angioprotectin to human pathophysiology, the concentration of this peptide and of Ang-ll was measured in the plasma of healthy subjects and in the plasma of end-stage renal failure (ESRD) patients (Figure 2). The concentration of the peptides was determined using a mass-spectrometry-based technique as described previously (Vera Jankowski et al. Detection of angiotensin II in supernatants of stimulated mononuclear leukocytes by MALDI-TOF-TOF-mass analysis. Hypertension 46, 591 -597, 2005). The clinical data of the patients and the healthy control subjects included in the study are given in Table 1 . In ESRD patients, Angioprotectin plasma concentrations significantly increased up to five-fold compared to healthy control subjects (Figure 2). Therefore, angioprotectin may serve as a biomarker in the diagnosis of a disease status of a patient. In contrast, plasma Ang-ll concentrations did not differ in healthy subjects and end-stage renal failure patients within the accuracy of the method (Figure 2).

Example 6: Diagnostic deficits to detect and quantify angioprotectin and angiotensin-ll separately

A commercially available Ang-ll antibody did not differentiate between Ang-ll and Angioprotectin (Figure 3). Thus, it is possible that other immunoassay kits for Ang-ll could similarly provide falsely high Ang-ll concentrations in patients with vascular disease. This may be clinically relevant, since Angioprotectin modulates vascular tone in an opposite manner compared with Ang-ll, however, the diagnostic test detects the sum of Ang-ll and angioprotectin in a given sample and this test result may be critically misleading in terms of a medication regimen for the patient based on these imprecise determination of Ang-ll levels. The quantification of Ang-ll in the presence or absence of angioprotectin was measured by a commercially available enzyme immuno assay (EIA). The angiotensin II enzyme immunoassay kit of SpiBio (Societe de Pharmacologie et d'lmmunologie, Massy, France) was used to test the cross-reactivity between Angioprotectin and Ang-ll. Briefly, a specific monoclonal anti-Ang-ll antibody was immobilised on a 96 well plate. After immunological reaction with Ang-ll and washing, the trapped molecule was covalently linked to the plate by glutaraldehyde crosslinking via amino groups. After washing and denaturing treatment, Ang-ll binds again to the second acetylcholinesterase- labelled monoclonal antibody used as detection antibody. The plate was washed and Ellman's reagent (enzymatic substrate for AchE and chromogen) was added to the wells. The intensity of the color from the chromogen was determined by spectrophotometry and was proportional to the amount of angiotensin II. For photometric measurement, we used a photometric microplate absorbance reader at 414 nm wavelength. To test the cross-reactivity between Angioprotectin and Ang-ll we used samples with different concentrations (as pmol L^"1) of Ang-ll and Angioprotectin: 100 pmol L^"1 Ang-ll or 100 pmol L^"1 Angioprotectin, or 50 pmol L^"1 Ang-ll plus 50 pmol L^"1 Angioprotectin, or 90 pmol L^"1 Ang-ll plus 10 pmol L^"1 Angioprotectin, or 10 pmol L^"1 Ang-ll plus 90 pmol L^"1 Angioprotectin, respectively.

Brief Description of the Drawings

FIGURE 1A

Size-exclusion chromatogram of human plasma (conditions: Sephacryl S- 100 highresolution, Pharmacia BioTech, Uppsala, Sweden; column dimension: 1000 x 16 mm; eluent: 0.9 % NaCI in water; flow rate: 2.5 ml_ min-1 ; abscissa: retention time (h); ordinate: UV-absorption at 280 nm (arbitrary units)). The arrow indicates a fraction which contained the unknown substance with the molecular weight of 1001 .5 Da.

FIGURE 1 B

Reversed phase chromatography of the size-exclusion fraction labelled by an arrow in Figure 1 .A using an analytical reversed-phase high performance liquid chromatographic column (conditions: ChromolithTM SpeedROD (100 x 4.6 mm I.D., Merck, Darmstadt, Germany; eluent A: 0.1 % trifluoroacetic acid (TFA) in water; eluent B: 0.1 % trifluoroacetic acid (TFA) in water-acetonitrile (20:80, v/v-%); gradient: 0-2 min: 0 % eluent B, 2-32 min: 0-75 % B, 32-32.5 min: 75-100 % eluent B; 32.5-33.5 min: 100 % B; flow rate: 2.0 ml_ min-1 ; abscissa: retention time (min); ordinate: UV-absorption at 280 nm (AU arbitrary units)).

FIGURE 1 C

MALDI-TOF mass spectrum of the substance isolated by RP-HPLC. The peak with an m/z ratio of 1001 .5 (calculated m/z 1001 .5) corresponds to peptide sequence Pro-Glu-Val-Tyr- lle-His-Pro-Phe.

FIGURE 1 D MALDI-TOF/TOF-MS/MS spectrum of the peak with m/z 1 001 .5 shown in Figure 1 .C. Relevant ions are labelled according to the accepted nomenclature. Both the b ions and the y ions confirm the amino acid sequence of Pro-Glu-Val-Tyr-lle-His-Pro-Phe.

FIGURE 1 E

Equimolar Angioprotectin concentrations reduced Ang-ll-induced constrictions by about 30 %. Contractions of rings of isolated rat aorta induced by Ang-ll in the absence (left bar) and presence of increasing concentrations of Angioprotectin (10-6, 10-7, 10-8, 10-9 mol L-1 ) (ordinate: constriction relative to 1 μιτιοΙ L-1 phenylephrine (%); means ± S.E.M., n=10).

FIGURE 1 F

Changes in Ca2+-influx of VSMC after exposure to 100 nmol L-1 Ang-ll in absence (grey line) and presence (black line) of 100 nmol L-1 Angioprotectin (each point represents mean ± S.E.M., n=8).

FIGURE 2

(A) Mass-spectrometry-based determination of Angioprotectin (left Y-axis) and Ang-ll (right Y-axis) levels in plasma of healthy control subjects (open bars) and chronic renal failure patients (CRF, black bars) (mean ± S.E.M., n=8, single values and means; p<0.05). FIGURE 3

Quantification of synthetic Angioprotectin and synthetic Ang-ll by enzyme immuno assay (concentrations used in the assay are given in pmol L-1 in the corresponding bars; each n=5).

Table 1

Clinical data of chronic renal failure (CRF) patients and healthy control subjects who were included into the study for analyses of angioprotectin and Ang-ll plasma level as presented by FIGURE 4A.

Table 2

Genes that are regulated in primary human dermal endothelial cells (DEC) in the human renal cell line H295R after exposure to angioprotectin

Regulated gene

up-regulated

down-regulated Mode of action

ANKRD20B

Ankyrin repeat domain 20B; the in vivo function of this gene is unknown so far.

BIVM, Isoform 1 of Basic immunoglobulin-like variable motif- containing protein; endodeoxyribonuclease activity, DNA binding, endonuclease activity, hydrolase activity in transcription-coupled nucleotide-excision repair.

Carbonic anhydrase-like sequence, lacks CA activity; one- carbon compound metabolism; metal ion binding protein; implicated in cell grows.

CMT1 A duplicated region transcript 1 , F-box protein family member, transcription factor activity, zinc/metal ion binding; implicated in cell grows; opposite regulation to Ang-ll.

Carboxyl ester lipase pseudogene; serine esterase activity; in vivo function is not known

CST1 cystatin SN; contributes to cell proliferation and cathepsin inhibition. ELFN2

ELFN2 extracellular leucine-rich repeat and fibronectin type III domain containing 2; physiological function not identified so far

LOC728748

Homo sapiens protein-kinase, interferon-inducible double stranded RNA dependent inhibitor, repressor of (P58 repressor) pseudogene (LOC728748); Upstream regulator of interferon- induced serine/threonine protein kinase R (PKR)

MEP1 A

MEP1 A meprin A, alpha (PABA peptide hydrolase);

metallopeptidase activity, implicated in inflammatory disease.

NR4A1

Nuclear receptor 4A1 , DNA dependent regulation of transcription, association to diseases like arteriosclerosis

NR4A3 Nuclear receptor 4A3, DNA dependent regulation of

transcription, association to

NT5CIII

NT5C3 5'-nucleotidase, cytosolic III ; catalyze the dephosphorylation of the pyrimidine 5'-monophosphates UMP and CMP to the corresponding nucleosides.

SLC9A4

SLC9A4 solute carrier family 9 (sodium/hydrogen exchanger), member 4; participates in processes like ion transport, regulation of pH, sodium ion transport. ZNF674

ZNF674 zinc finger family member 674, this protein may bind a zinc ion and may function as transcription factor via DNA binding.

Definition of terms

"Animal" term as used herein may be defined to include human, domestic (e.g., cats, dogs, etc.), agricultural (e.g., cows, horses, sheep, etc.) or test species (e.g., mouse, rat, rabbit, etc.).

"Biomarker" are measurable and quantifiable biological parameters (e.g. by presence of biological substances like peptides) which serve as indices for health - and physiology related assessments, such as disease risk, psychiatric disorders, environmental exposure and its effects, disease diagnosis, metabolic processes, substance abuse, pregnancy, cell line development, epidemiologic studies, etc.. Parameter that can be used to identify a toxic effect or a disease-related effect in an individual organism and can often be used in extrapolation between species. A biomarker may serve as an indicator signaling an event or condition in a biological system or sample and giving a measure of exposure, effect, or susceptibility.

Biological markers can reflect a variety of disease characteristics, including the level of exposure to an environmental or genetic trigger, an element of the disease process itself, an intermediate stage between exposure and disease onset, or an independent factor associated with the disease state but not causative of pathogenesis. Depending on the specific characteristic, biomarkers can be used to identify the risk of developing an illness (antecedent biomarkers), aid in identifying disease (diagnostic biomarkers), or predict future disease course, including response to therapy (prognostic biomarkers).

"Microarray" is used as a term for nucleic acid arrays that have been used in the present invention like those that are commercially available from Affymetrix (Santa Clara, Calif.) under the brand name GeneChip Human Genome U133 Plus 2.0 Array.® or Rat Genome U230 plus 2.0 Array, respectively. These microarrays represent the complete coverage of the Human Genome U133 Set plus 9921 probe sets which include approximately 6,500 new genes (with a total of approximately 56 000 transcripts) or the Rat Genome, respectively. The Affymetrix (Santa Clara, Calif.) GeneChip technology platform consists of high-density microarrays and tools to help process and analyze those arrays, including standardized assays and reagents, instrumentation, and data management and analysis tools. GeneChip microarrays consist of small DNA fragments (referred to as probes), chemically synthesized at specific locations on a coated quartz surface. By extracting and labeling nucleic acids from experimental samples, and then hybridizing those prepared samples to the array, the amount of label can be monitored enabling a measurement of gene regulation. The GeneChip human genome arrays include a set of human maintenance genes to facilitate the normalization and scaling of array experiments and to perform data comparison. This set of normalization genes shows consistent levels of expression over a diverse set of tissues.

Therapeutic Indications and Methods

It was found by the present applicant that ANGIOPROTECTIN is formed in human beings and it is a component of the blood.

Cardiovascular diseases, cardiovascular disorders

Heart failure is defined as a pathophysiological state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failures such as high output and low output, acute and chronic, right sided or left sided, systolic or diastolic, independent of the underlying cause.

Myocardial infarction (Ml) is generally caused by an abrupt decrease in coronary blood flow that follows a thrombotic occlusion of a coronary artery previously narrowed by arteriosclerosis. Ml prophylaxis (primary and secondary prevention) is included as well as the acute treatment of Ml and the prevention of complications. Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen. This group of diseases includes stable angina, unstable angina and asymptomatic ischemia. Arrhythmias include all forms of atrial and ventricular tachyarrhythmias, atrial tachycardia, atrial flutter, atrial fibrillation, atrio ventricular reentrant tachycardia, preexitation syndrome, ventricular tachycardia, ventricular flutter, ventricular fibrillation, as well as bradycardic forms of arrhythmias. Hypertensive vascular diseases include primary as well as all kinds of secondary arterial hypertension, renal, endocrine, neurogenic, others. The genes may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications arising from cardiovascular diseases.

Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon and venous disorders. Atherosclerosis is a cardiovascular disease in which the vessel wall is remodeled, compromising the lumen of the vessel. The atherosclerotic remodeling process involves accumulation of cells, both smooth muscle cells and monocyte/macrophage inflammatory cells, in the intima of the vessel wall. These cells take up lipid, likely from the circulation, to form a mature atherosclerotic lesion. Although the formation of these lesions is a chronic process, occurring over decades of an adult human life, the majority of the morbidity associated with atherosclerosis occurs when a lesion ruptures, releasing thrombogenic debris that rapidly occludes the artery. When such an acute event occurs in the coronary artery, myocardial infarction can ensue, and in the worst case, can result in death.

The formation of the atherosclerotic lesion can be considered to occur in five overlapping stages such as migration, lipid accumulation, recruitment of inflammatory cells, proliferation of vascular smooth muscle cells, and extracellular matrix deposition. Each of these processes can be shown to occur in man and in animal models of atherosclerosis, but the relative contribution of each to the pathology and clinical significance of the lesion is unclear. Thus, a need exists for therapeutic methods and agents to treat cardiovascular pathologies, such as atherosclerosis and other conditions related to coronary artery disease.

Cardiovascular diseases include but are not limited to disorders of the heart and the vascular system like congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular arrhythmias, hypertensive vascular diseases, peripheral vascular diseases, and atherosclerosis. To high or to low levels of fats in the bloodstream, especially cholesterol, can contribute to the development of cardiovascular diseases. Kidney disorders may lead to hyper or hypotension. Examples for kidney problems possibly leading to hypertension are renal artery stenosis, pyelonephritis, glomerulonephritis, kidney tumors, polycistic kidney disease, injury to the kidney, or radiation therapy affecting the kidney. Excessive urination may lead to hypotension. There is evidence that chronic kidney disease or affected kidney function contributes to the accelerated formation and progression of cardiovascular male functions and disease. Likewise, loss of pulmonary function effects certain pathologies of the cardiovascular system.

Disorders related renal failure, kidney disease and urological disorders

Genitourinary disorders comprise benign and malign disorders of the organs constituting the genitourinary system of female and male, renal diseases like acute or chronic renal failure, immunologically mediated renal diseases like renal transplant rejection, lupus nephritis, immune complex renal diseases, glomerulopathies, nephritis, toxic nephropathy, obstructive uropathies like benign prostatic hyperplasia (BPH), neurogenic bladder syndrome, urinary incontinence like urge-, stress-, or overflow incontinence, pelvic pain, and erectile dysfunction. Applications

The present invention provides ANGIOPROTECTIN or derivatives thereof for prophylactic, therapeutic and diagnostic methods for cardiovascular diseases, hematological diseases, neurological diseases, respiratory diseases, gastroenterological diseases and urological diseases.

The regulatory method of the invention involves contacting a cell with ANGIOPROTECTIN or derivatives thereof to modulate one or more activities of the cell. An agent that modulates activity can be an agent as described herein, such as a naturally-occurring peptide, an analog of the peptide, e.g. a peptidomimetic, or any small molecule. In one embodiment, the agent stimulates one or more of the biological activities of a cell or a tissue or an organ. Examples of such stimulatory agents include the regulation of gene expression by ANGIOPROTECTIN or derivatives thereof and peptides encoding a portion of ANGIOPROTECTIN. In another embodiment, the agent regulates the biological activities of cellular receptors thus influencing the metabolism or physiological status of a cell or tissue or organ. These regulatory modes of action can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g, by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by unwanted expression of genes or unwanted activity of receptor activities or cellular signal pathways.

The present invention discloses the use of ANGIOPROTECTIN or fragments of ANGIOPROTECTIN as a biomarker for cardiovascular diseases, hematological diseases, neurological diseases, respiratory diseases, gastroenterological diseases, renal disorders, and urological diseases. Methods of the detection and quantification of angioprotectin comprise mass spectrometry methods similar to those described herein, or other chromatographic methods or immunological methods like enzyme linked immuno-sorbent assays known in the art. This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

Pharmaceutical Compositions

This invention further pertains to novel agents identified by the above- described assays and uses of the agents thereof for treatments as described herein.

The peptides, and derivatives thereof (also referred to herein as "active compounds" or "regulators") of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the peptide molecule, or peptide derivatives and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

An additional embodiment of the invention relates to the administration of a pharmaceutical composition containing ANGIOPROTECTIN or derivatives thereof in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of ANGIOPROTECTIN, antibodies to ANGIOPROTECTIN, and mimetics, agonists, antagonists, or inhibitors of ANGIOPROTECTIN. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones. A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. 4,522,81 1 . It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. For pharmaceutical compositions which include ANGIOPROTECTIN or derivatives thereof, the instructions for administration will specify use of the composition for cardiovascular diseases, hematological diseases, neurological diseases, respiratory diseases, gastroenterological diseases and urological diseases.

Diagnostics

One embodiment of the invention describes ANGIOPROTECTIN as a biomarker for diagnostic use.

Use of ANGIOPROTECTIN as a biomarker in diagnostics is based by the comparison of ANGIOPROTECTIN level in a biological sample from a diseased mammal with the ANGIOPROTECTIN level in a control sample from a healthy or normal mammal. Does the ANGIOPROTECTIN level in the diseased mammal differs from the ANGIOPROTECTIN level in a normal or healthy mammal then the diseased mammal is diagnosed with a disease associated with an altered ANGIOPROTECTIN level. Furthermore, comparing ANGIOPROTECTIN levels of a biological sample from a diseased mammal with ANGIOPROTECTIN levels of control samples from mammals with a ANGIOPROTECTIN-associated disease already diagnosed with different stages or severity of said disease, allows the diagnose of a ANGIOPROTECTIN-associated disease of said first diseased mammal and specifying the severity of the ANGIOPROTECTIN-associated disease. The biological sample is taken from the analogue tissue or body fluid than the control sample.

Normal or standard values for ANGIOPROTECTIN level are established by using control samples from healthy or diseased mammalian subjects. A control sample can be obtained by collecting separate or combined body fluids or cell extracts taken from normal mammalian subjects, preferably human, achieving statistical relevant numbers. To obtain the normal or standard ANGIOPROTECTIN level of the control samples, the samples were subjected to suitable detection methods to detect ANGIOPROTECTIN peptide or activity. The determination of ANGIOPROTECTIN level in a mammal subjected to diagnosis is performed analogously by collecting a biological sample from said mammal. Quantities of ANGIOPROTECTIN levels in biological samples from a mammal subjected to diagnosis are compared with the standard or normal values measured from a control sample. Deviation between standard value (determined from control sample) and subject value (determined from biological sample) establishes the parameters for diagnosing disease. Absolute quantification of ANGIOPROTECTIN levels measured from biological or control samples may be achieved by comparing those values with values obtained from an experiment in which a known amount of a substantially purified polypeptide is used.

Antibodies which specifically bind ANGIOPROTECTIN may be used for the diagnosis of disorders characterized by the formation of the biomarker ANGIOPROTECTIN, or in diagnostic assays to monitor patients being treated achieving guidance for therapy for such a disease. Such a treatment includes medication suitable to treat such a disease, and treatment with ANGIOPROTECTIN or derivatives thereof. Antibodies useful for diagnostic purposes may be prepared in the a standard manner by immunizing animals with ANGIOPROTECTIN or suitable ANGIOPROTECTIN containing immunization products known in the art. Diagnostic assays for ANGIOPROTECTIN include methods which utilize the antibody and a label to detect ANGIOPROTECTIN in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent joining with a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.

A variety of protocols for measuring ANGIOPROTECTIN, including ELISAs, RIAs, Planar Waveguide technology, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of ANGIOPROTECTIN expression. Planar Waveguide Technology bioassays are designed to perform multiplexed nucleic acid hybridization assays, immunoaffinity reactions and membrane receptor based assays with high sensitivity and selectivity. The recognition elements specific for the analytes of interest are bound onto the surface in small discrete spots; the transfer of the recognition elements onto the surface is performed using an adequate spotting technology, which requires only minute amounts of recognition elements. Such an arrangement of different recognition elements in an array format allows the simultaneous detection and quantification of hundreds to thousands of different analytes per sample including replicates.

Reactions on microarrays usually follow a typical scheme:

Recognition elements (e.g. oligonucleotides, cDNAs, or antibodies) are spotted onto the chemically modified planar waveguide surface with typical spot diameters of 100 - 200 μιτι. The remaining free binding sites on the surface subsequently are being blocked to reduce or eliminate nonspecific binding. In a next step the sample (e.g. fluorescently labeled cDNA or pre- incubated analyte / fluorescently labeled antibody complex) is transferred onto the surface for incubation. The incubation time where a selective recognition and binding between recognition elements and corresponding target molecules ( e.g. DNA - DNA hybridization or antigen - antibody interaction) occurs depends on the affinity between the analytes and the immobilized recognition elements. The resulting fluorescing spots can then be detected during readout.

Due to the laterally resolved imaging of the fluorescence signals of the individual spots by a CCD-camera, a large variety of different analytes can be quantified simultaneously, requiring typically sample volumes in the range of 15 μΙ. Calibration and referencing spots allow for accurate quantification of analytes using just one chip and enable the establishment of dose response and time dependent activity profiles [Pawlak (2002), Duveneck (2002)]. Normal or standard values for ANGIOPROTECTIN formation are established by using control samples from healthy or diseased mammalian subjects. A control sample can be obtained by collecting separate or combined body fluids or cell extracts taken from normal mammalian subjects, preferably human, achieving statistical relevant numbers. To obtain normal or standard values the control samples are combined with an antibody to ANGIOPROTECTIN under conditions suitable for complex formation. The amount of standard complex formation may be quantified by various methods, preferably by photometric means. The determination of ANGIOPROTECTIN level in a mammal subjected to diagnosis is performed analogously by collecting a biological sample from said mammal, combining said sample with an antibody to ANGIOPROTECTIN and determination of complex formation. Quantities of ANGIOPROTECTIN formed in biological samples from a mammal subjected to diagnosis are compared with the standard or normal values measured from a control sample. Deviation between standard value (determined from control sample) and subject value (determined from biological sample) establishes the parameters for diagnosing disease. Absolute quantification of ANGIOPROTECTIN levels measured from biological or control samples may be achieved by comparing those values with values obtained from an experiment in which a known amount of a substantially purified ANGIOPROTECTIN is used.

Biomarker

Use of ANGIOPROTECTIN as a biomarker

One of ordinary skill in the art knows several methods and devices for the detection and analysis of the markers of the instant invention. With regard to polypeptides or proteins in patient test samples, immunoassay devices and methods are often used. These devices and methods can utilize labelled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of an analyte of interest. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labelled molecule.

Preferably the markers are analyzed using an immunoassay, although other methods are well known to those skilled in the art (for example, the measurement of marker RNA levels). The presence or amount of a marker is generally determined using antibodies specific for each marker and detecting specific binding. Any suitable immunoassay may be utilized, for example, enzyme- linked immunoassays (ELISA), radioimmunoassay (RIAs), competitive binding assays, planar waveguide technology, and the like. Specific immunological binding of the antibody to the marker can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase and the like. For an example of how this procedure is carried out on a machine, one can use the RAMP Biomedical device, called the Clinical Reader sup™, which uses the fluorescent tag method, though the skilled artisan will know of many different machines and manual protocols to perform the same assay. Diluted whole blood is applied to the sample well. The red blood cells are retained in the sample pad, and the separated plasma migrates along the strip. Fluorescent dyed latex particles bind to the analyte and are immobilized at the detection zone. Additional particles are immobilized at the internal control zone. The fluorescence of the detection and internal control zones are measured on the RAMP Clinical Reader sup™, and the ratio between these values is calculated. This ratio is used to determine the analyte concentration by interpolation from a lot-specific standard curve supplied by the manufacturer in each test kit for each assay. The use of immobilized antibodies specific for the markers is also contemplated by the present invention and is well known by one of ordinary skill in the art. The antibodies could be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay place (such as microtiter wells) , pieces of a solid substrate material (such as plastic, nylon, paper), and the like. An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a coloured spot.

The analysis of a plurality of markers may be carried out separately or simultaneously with one test sample. Several markers may be combined into one test for efficient processing of a multiple of samples. In addition, one skilled in the art would recognize the value of testing multiple samples (for example, at successive time points) from the same individual. Such testing of serial samples will allow the identification of changes in marker levels over time. Increases or decreases in marker levels, as well as the absence of change in marker levels, would provide useful information about the disease status that includes, but is not limited to identifying the approximate time from onset of the event, the presence and amount of salvagable tissue, the appropriateness of drug therapies, the effectiveness of various therapies, identification of the severity of the event, identification of the disease severity, and identification of the patient's outcome, including risk of future events.

An assay consisting of a combination of the markers referenced in the instant invention may be constructed to provide relevant information related to differential diagnosis. Such a panel may be constucted using 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more or individual markers. The analysis of a single marker or subsets of markers comprising a larger panel of markers could be carried out methods described within the instant invention to optimize clinical sensitivity or specificity in various clinical settings.

The analysis of markers could be carried out in a variety of physical formats as well. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion, for example, in ambulatory transport or emergency room settings. Particularly useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different analytes. Such formats include protein microarrays, or "protein chips" and capillary devices.

Cardiac markers serve an important role in the early detection and monitoring of cardiovascular disease. Markers of disease are typically substances found in a bodily sample that can be easily measured. The measured amount can correlate to underlying disease pathophysiology, presence or absence of a current or imminent cardiac event, probability of a cardiac event in the future. In patients receiving treatment for their condition the measured amount will also correlate with responsiveness to therapy. Markers can include elevated levels of blood pressure, cholesterol, blood sugar, homocysteine and C- reactive protein (CRP). However, current markers, even in combination with other measurements or risk factors, do not adequately identify patients at risk, accurately detect events (i.e., heart attacks), or correlate with therapy. For example, half of patients do not have elevated serum cholesterol or other traditional risk factors. Use of markers in diagnosis of cardiac conditions is described in, for example, Alpert et al. (2000); Newby et al. (2001 ); de Lemos et al.(2002); Boersma et al. (2002); Christenson et al. (2001 ), each of which is incorporated by reference in its entirety.

Biomarker classes

ANGIOPROTECTIN could be used as a biomarker for cardiovascular diseases, hematological diseases, neurological diseases, respiratory diseases, gastroenterological diseases and urological diseases in different classes :

Disease Biomarker: a biomarker that relates to a clinical outcome or measure of disease.

Efficacy Biomarker: a biomarker that reflects beneficial effect of a given treatment.

Staging Biomarker: a biomarker that distinguishes between different stages of a chronic disorder.

Surrogate Biomarker: a biomarker that is regarded as a valid substitute for a clinical outcomes measure.

Toxicity Biomarker: a biomarker that reports a toxicological effect of a drug on an in vitro or in vivo system.

Mechanism Biomarker: a biomarker that reports a downstream effect of a drug.

Target Biomarker: a biomarker that reports interaction of the drug with its target.

One embodiment of the invention is a method of use of ANGIOPROTECTIN as a biomarker for a disease comprising :

(a) obtaining a biological sample from a mammal,

(b) measuring the level of ANGIOPROTECTIN in the biological sample,

(c) obtaining a control sample from a mammal,

(d) measuring the level of ANGIOPROTECTIN in the control

sample,

(e) comparing the level of ANGIOPROTECTIN in the biological sample with the level of ANGIOPROTECTIN in a control sample, and (f) diagnosing a disease based upon the ANGIOPROTECTIN level of the biological sample in comparison to the control sample.

The biological sample in step (a) of the methods is in a preferred

embodiment a biological sample comprised in a group of samples consisting of a blood sample, a plasma sample, a serum sample, a tissue sample, a oral mucosa sample, a saliva sample, an interstitial fluid sample or an urine sample. The blood sample is for example a whole blood sample, a

fractionated blood sample, a platelet sample, a neutrophil sample, a leukocyte sample, a white blood cell sample, a monocyte sample, a red blood cell sample, a granulocyte sample, and a erythrocyte sample. A tissue sample is for example a sample collected from muscle, adipose, heart or skin. In a preferred embodiment ANGIOPROTECTIN is used as a biomarker diagnosing a disease which is associated with altered ANGIOPROTECTIN levels. Another preferred embodiment ANGIOPROTECTIN is used as a biomarker for identifying an individual risk for developing a disease, or for predicting an adverse outcome in a patient diagnosed with a disease,

Use of ANGIOPROTECTIN as a disease biomarker in diagnostics is based by the comparison of ANGIOPROTECTIN level in a biological sample from a diseased mammal with the ANGIOPROTECTIN level in a control sample from a healthy or normal mammal or a group of healthy or normal mammals. Does the ANGIOPROTECTIN level in the diseased mammal differs from the ANGIOPROTECTIN level in a normal or healthy mammal then the diseased mammal is diagnosed with a disease associated with altered

ANGIOPROTECTIN level.

Furthermore, using ANGIOPROTECTIN as a staging biomarker, the

ANGIOPROTECTIN levels of a diseased mammal are compared with ANGIOPROTECTIN levels of a mammal with a ANGIOPROTECTIN- associated disease already diagnosed with different stages or severity of said disease, allows the diagnose of said first diseased mammal specifying the severity of the ANGIOPROTECTIN-associated disease.

A control sample can be a sample taken from a mammal. A control sample can be a previously taken sample from a mammal, as a ANGIOPROTECTIN level in a control sample can be a predetermined level of

ANGIOPROTECTIN measured in a previously taken sample. The level of ANGIOPROTECTIN in a control sample or in a biological sample can be determined for example as a relative value and as an absolute value. A previously measured ANGIOPROTECTIN level from a control sample can be for example stored in a database, in an internet publication, in an

electronically accessible form, in a publication. Comparing the level of ANGIOPROTECTIN of a biological sample to a control sample may be comparing relative values or absolute quantified values.

Another embodiment is a method of use of ANGIOPROTECTIN as a biomarker for guiding a therapy of a disease comprising:

(a) obtaining a baseline level of ANGIOPROTECTIN in biological sample from a diseased mammal, (b) administering to the diseased mammal a treatment for the

disease,

(c) obtaining one or more subsequent biological samples from the diseased mammal

(d) measuring the level of ANGIOPROTECTIN in the one or more subsequent biological samples,

(e) comparing the level of ANGIOPROTECTIN in the one or more subsequent biological samples with the baseline sample, and

(f) determining whether increased dosages, additional or alternative treatments are necessary based on

ANGIOPROTECTIN levels obtained from one or more subsequent biological samples compared to the baseline ANGIOPROTECTIN level.

In a preferred embodiment ANGIOPROTECTIN is used as a biomarker for guiding a therapy in a disease which is associated with altered

ANGIOPROTECTIN levels.

Use of ANGIOPROTECTIN as a disease, efficacy or surrogate endpoint biomarker in diagnostics is based by the comparison of ANGIOPROTECTIN level in a biological sample from a diseased mammal before treatment (the baseline sample level) with the ANGIOPROTECTIN level in subsequent samples from said mammal receiving a treatment for the disease. Does the ANGIOPROTECTIN level in the baseline sample differs from the

ANGIOPROTECTIN level in the subsequent samples then the therapy can be considered as successful. Does the ANGIOPROTECTIN level in the baseline sample does not differ or differs only slightly from the

ANGIOPROTECTIN level in the subsequent samples then the therapy can be considered as not successful. If the therapy is considered not successful increased dosages of the same therapy, repeat of the same therapy or an alternative treatment which is different from the first therapy can be considered.

The biological sample in step (a) of the methods is in a preferred

embodiment a biological sample comprised in a group of samples consisting of a blood sample, a plasma sample, a serum sample, a tissue sample, a oral mucosa sample, a saliva sample, an interstitial fluid sample or an urine sample. The blood sample is for example a whole blood sample, a fractionated blood sample, a platelet sample, a neutrophil sample, a leukocyte sample, a white blood cell sample, a monocyte sample, a red blood cell sample, a granulocyte sample, and a erythrocyte sample. A tissue sample is for example a sample collected from muscle, adipose, heart, skin or a biopsy.

In another preferred embodiment the level of ANGIOPROTECTIN is determined by determining the level of ANGIOPROTECTIN peptide. In a further preferred embodiment the level of ANGIOPROTECTIN is determined by determining the level of ANGIOPROTECTIN activity.

In a preferred embodiment the disease associated with ANGIOPROTECTIN is comprised in a group of diseases consisting of cardiovascular diseases, hematological diseases, neurological diseases, respiratory diseases, gastroenterological diseases, and urological diseases. In a more preferred embodiment the cardiovascular disease associated with ANGIOPROTECTIN is comprised in a group of diseases consisting of congestive heart failure, pulmonary hypertension, left ventricular dysfunction, and right ventricular dysfunction, myocardial infarction, coronary occlusion, disease, ischemic heart disease, cardiac hypertrophy disorder, cardiac fibrosis disorders.

In a preferred embodiment of the invention the mammal is a human.

In a preferred embodiment of the invention the level of ANGIOPROTECTIN of the biological sample is elevated compared to the control sample.

Another embodiment of the present invention prefers the use of

ANGIOPROTECTIN in combination with the use of one or more biomarkers, more preferably with biomarkers used in diagnosing ANGIOPROTECTIN- associated diseases. In a preferred embodiment of the invention the use of ANGIOPROTECTIN is combined with the use of one or more biomarkers which are comprised in a group of biomarkers consisting of fibronectin, CTGF, BNP, ANP, Troponin, CRP, Myoglobin, CK-MB and metabolites, osteopontin, PAI-1 , TIMP-1 , NGAL (Lipocalin-2), KIM-1 , H-FABP, L-FABP, collagen or fragments thereof etc.

In a further preferred embodiment the use of ANGIOPROTECTIN is combined with the use of one or more clinical biomarkers which are comprised in a group of biomarkers consisting of blood pressure, heart rate, pulmonary artery pressure, or systemic vascular resistance. In a further preferred embodiment the use of ANGIOPROTECTIN is combined with the use of one or more diagnostic imaging methods which are comprised in a group of methods consisting of PET (Positron Emission Tomography), CT (Computed Tomography), ultrasonic, SPECT (Single Photon Emission Computed Tomography), Echocardiography, or Impedance Cardiography.

In a further preferred embodiment the use of ANGIOPROTECTIN is combined with the use of one or more diagnostic imaging methods which are comprised in a group of methods consisting of PET (Positron Emission Tomography), CT (Computed Tomography), ultrasonic, SPECT (Single Photon Emission Computed Tomography), Echocardiography, Impedance Cardiography, blood pressure, heart rate, pulmonary artery pressure, systemic vascular resistance, CRTAC, FN1 , NPR3, LTBP2, TGFB2, or CTGF, BNP, ANP, Troponin, CRP, Myoglobin, CK-MB, and metabolites.

In a further preferred embodiment is a kit for identifying an individual risk for developing a disease, for predicting a disease or an adverse outcome in a patient diagnosed with a disease, or for guiding a therapy in a patient with a disease, the kit comprising one ore more antibodies which specifically binds ANGIOPROTECTIN, detection means, one or more containers for collecting and or holding the biological sample, and an instruction for its use. Another preferred embodiment is a kit for identifying an individual risk for developing a disease, for predicting a disease or an adverse outcome in a patient diagnosed with a disease, or for guiding a therapy in a patient with a disease, the kit comprising one or more substrates for detecting

ANGIOPROTECTIN level, detection means, one or more containers for collecting and or holding the biological sample, and an instruction for its use.

Production of ANGIOPROTECTIN Specific Antibodies Two approaches are utilized to raise antibodies to ANGIOPROTECTIN, and each approach is useful for generating either polyclonal or monoclonal antibodies. In one approach, purified ANGIOPROTECTIN from reverse phase HPLC separation is obtained in quantities up to 75 mg. This denatured peptide is used to immunize mice or rabbits using standard protocols; about 100 g are adequate for immunization of a mouse, while up to 1 mg might be used to immunize a rabbit. For identifying mouse hybridomas, the denatured protein is radioiodinated and used to screen potential murine B-cell hybridomas for those which produce antibody. This procedure requires only small quantities of protein, such that 20 mg is sufficient for labeling and screening of several thousand clones.

In the second approach, the amino acid sequence of an appropriate ANGIOPROTECTIN fragment is being used for immunization. The optimal amino acid sequences for immunization are usually at the C-terminus, the N- terminus and those intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the external environment when the protein is in its natural conformation. Since ANGIOPROTECTIN is different to Ang-ll at the amino-terminus, fragments containing the amino-terminus comprising the Pro-Glu-Val residues have to be used for the immunization.

Typically, selected peptides, about 5-8 residues in length, are synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc- chemistry and coupled to keyhole limpet hemocyanin (KLH; Sigma, St. Louis, MO) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester, MBS. If necessary, a cysteine is introduced at the N-terminus of the peptide to permit coupling to KLH. Rabbits are immunized with the peptide- KLH complex in complete Freund's adjuvant. The resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1 % bovine serum albumin, reacting with antisera, washing and reacting with labeled (radioactive or fluorescent), affinity purified, specific goat anti-rabbit IgG. Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with labeled ANGIOPROTECTIN to identify those fusions producing the monoclonal antibody with the desired specificity. In a typical protocol, wells of plates (FAST; Becton-Dickinson, Palo Alto, CA) are coated during incubation with affinity purified, specific rabbit anti-mouse (or suitable antispecies 1 g) antibodies at 10 mg/ml. The coated wells are blocked with 1 % bovine serum albumin, (BSA), washed and incubated with supernatants from hybridomas. After washing the wells are incubated with labeled ANGIOPROTECTIN at 1 mg/ml. Supernatants with specific antibodies bind more labeled ANGIOPROTECTIN than is detectable in the background. Then clones producing specific antibodies are expanded and subjected to two cycles of cloning at limiting dilution. Cloned hybridomas are injected into pristane- treated mice to produce ascites, and monoclonal antibody is purified from mouse ascitic fluid by affinity chromatography on Protein A. Monoclonal antibodies with affinities of at least 10⁸ M^"1 , preferably 10⁹ to 10¹⁰ M^"1 or stronger, are typically made by standard procedures.

Diagnostic Test Using ANGIOPROTECTIN Specific Antibodies

Particular ANGIOPROTECTIN antibodies are useful for investigating signal transduction and the diagnosis of disease conditions which are characterized by differences in the amount or distribution of ANGIOPROTECTIN in body fluids.

Diagnostic tests for ANGIOPROTECTIN include methods utilizing antibody and a label to detect ANGIOPROTECTIN in human body fluids, membranes, cells, tissues or extracts of such. The polypeptides and antibodies of the present invention are used with or without modification. Frequently, the polypeptides and antibodies are labeled by joining them, either covalently or noncovalently, with a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, chromogenic agents, magnetic particles and the like. A variety of protocols for measuring soluble or membrane-bound ANGIOPROTECTIN, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on ANGIOPROTECTIN is preferred, but a competitive binding assay may be employed.

Purification of Native ANGIOPROTECTIN Using Specific Antibodies

Native ANGIOPROTECTIN can be purified by immunoaffinity chromatography using antibodies specific for ANGIOPROTECTIN apart from the chromatographic methods described for ANGIOPROTECTIN purification afore. In general, an immunoaffinity column is constructed by covalently coupling the anti-TRH antibody to an activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.

Such immunoaffinity columns are utilized in the purification of ANGIOPROTECTIN from body fluids. A soluble ANGIOPROTECTIN- containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of ANGIOPROTECTIN (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/protein binding (e.g., a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and ANGIOPROTECTIN is collected.

Rational Drug Design

The goal of rational drug design is to produce structural analogs of biologically active peptides that show superior activity and ideally oral active pharmacokinetic properties. Any of these examples are used to fashion drugs which are more active or stable forms of the peptide or which enhance or interfere or effectively with the function of a target of the drug in vivo.

In one approach, the three-dimensional structure of a protein of interest, or of a protein-inhibitor complex, is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of a polypeptide is gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design efficient inhibitors. Useful examples of rational drug design include molecules which have improved activity or stability or which act as inhibitors, agonists, or antagonists of native peptides.

It is also possible to isolate a target-specific antibody, selected by functional assay, as described above, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design is based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids is expected to be an analog of the original receptor. The anti-id is then used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides then act as the pharmacore. By virtue of the present invention, sufficient amount of peptide are made available to perform such analytical studies as X-ray crystallography. In addition, knowledge of the ANGIOPROTECTIN amino acid sequence provided herein provides guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.

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The invention provides ANGIOPROTECTIN which is associated with the cardiovascular diseases, hematological diseases, neurological diseases, respiratory diseases, gastroenterological diseases and urological diseases. The invention also provides assays for the identification of compounds useful in the treatment or prevention of cardiovascular diseases, hematological diseases, neurological diseases, respiratory diseases, gastroenterological diseases and urological diseases. The invention also features compounds which bind to and/or activate or inhibit the activity of ANGIOPROTECTIN as well as pharmaceutical compositions comprising such compounds. The invention also provides ANGIOPROTECTIN as a biomarker for diseases as cardiovascular diseases, hematological diseases, neurological diseases, respiratory diseases, gastroenterological diseases and urological diseases SEQ ID NO: 1 Pro-Glu-Val-Tyr-lle-His-Pro-Phe SEQ ID NO: 2 X_aa-X_bb-Val-Tyr-lle-His-Pro-Phe; X_aa = L-N-methyl-Ala or DL-N-methyl-Ala,

X_bb = L-Glu or DL-Glu or L-Asp or DL-Asp

SEQ ID NO: 3 X_Cc-Asp-Val-Tyr-lle-His-Pro-Phe; Xcc = L-alpha-methyl-proline or DL- alpha-methyl-proline,

Claims

Octapepide of SEQ I D No.1 having the structure X_aa-X_bb-Val-Tyr-lle- His-Pro-Phe composed of L-amino acids where X_aa is L-proline and Xbb is L-glutamate.

Octapepide of SEQ I D No.2 having the structure X_aa-X_bb -Val-Tyr-lle- His-Pro-Phe composed of L-amino acids where X_aa is L-alpha-N- methyl-alanine or DL-alpha-N-methyl-alanine and X_bb is L-aspartate or L-glutamate.

Octapepide of SEQ I D No.2 having the structure X_aa-X_bb -Val-Tyr-lle- His-Pro-Phe composed of L-amino acids where X_aa is L-alpha-methyl- proline or DL-alpha-methyl-proline and X_bb is L-aspartate or L- glutamate.

Octapepide of SEQ I D No.1 having the structure X_aa-X_bb -Val-Tyr-lle- His-Pro-Phe composed of L-amino acids where X_aa is an analog of proline and X_bb is an analog of glutamate.

Octapeptide or peptide analogs of claims 1 - 4 for therapeutic use to treat diseases.

Octapeptide or peptide analogs of claims 1 - 4 for therapy of diseases which are caused by affected blood vessel functions.

Octapeptide or peptide analogs of claims 1 - 4 for therapy of diseases which are caused by a dysfunctional renin-angiotensin-aldosterone system (RAAS) in patients.

Octapeptide of claim 1 for diagnostic use.

9. Octapeptide of claim 1 used as a component of an assay for quantitative measurement of the octapeptide of SEQ I D NO.1 in human samples obtained from blood, saliva, liquor, tears, sweat, urine or biopsies. l O. Octapeptide or peptide fragments of SEQ I D NO: 1 to generate antibodies for therapeutic or diagnostic use. 1 1 . Octapeptide or peptide analogs of claim 1 - 4 for use in screening assays and other test methods aimed at the identification and development of therapeutics or diagnostic products.