PEPTIDE COMBINATIONS FOR USE IN THE DIAGNOSIS OF SCHIZOPHRENIA

Abstract

Provided is a composition including a combination of at least one short peptide and at least one peptide dimer, in defined ratio, to which autoantibodies found in elevated levels in schizophrenic patients bind.

Claims

1. A composition comprising a compound having the general formula (I):
X.sub.3XX.sub.1SSX.sub.2XX.sub.4(I) and at least one compound of the general Formula (Ia) and/or (Ib):
X.sub.3XX.sub.1SH(Ia)
HSX.sub.2XX.sub.4(Ib) wherein: SS designates a disulfide bond or a group comprising a disulfide bond; X and X, independently of each other, may be absent or is each a pentapeptide comprising at least one amino acid selected from L, V and G; each of X.sub.1, X.sub.2, X.sub.3 and X.sub.4, independently of the other, may be absent or is an amino acid, a di-amino acid or a tri-amino acid; each of X, X, X.sub.1, X.sub.2, X.sub.3 and X.sub.4, independently of each other, is optionally substituted by at least one amino acid group; each of is a bond, wherein in the Formulae (Ia) and (Ib), each of X, X.sub.1, X.sub.2, X.sub.3, X.sub.4 and X are selected as in formula (I); and wherein the ratio between the compound of Formula (I) and the compound of Formula (Ia) or (Ib) is between about 1:1 to about 10:1.

2. The composition according to claim 1, wherein the compound of Formula (I) is selected from:
X.sub.3X.sub.1SSX.sub.2XX.sub.4;
X.sub.3X.sub.1SSX.sub.2X.sub.4; and
X.sub.3XX.sub.1SSX.sub.2X.sub.4; wherein each of X.sub.3, X.sub.1, X.sub.2, X, X and X.sub.4 is as defined in claim 1.

3. The composition according to claim 1, wherein the compound of Formula (I) is of general Formula (II):
XSSX wherein each of X and X is as defined in claim 1.

4. The compound composition to claim 1, wherein the compound of Formula (I) is of general formula (III):
XC(O)CHYCH.sub.2SSCH.sub.2CHYC(O)X wherein each of X and X are as defined in claim 1, C(O) designates a carbonyl group, and each of Y and Y, independently of the other, is at least one amino acid.

5. The composition according to claim 1, wherein the compound of Formula (I) is of general formula (IV):
XC(O)CHYCH.sub.2SSX wherein each of Y and X, independently of the other, is at least one amino acid.

6. The composition according to claim 1, wherein the compound of Formula (I) is of general formula (V):
XSSCH.sub.2CHYC(O)X wherein Y is at least one amino acid.

7. The composition according to claim 1, wherein the compound of Formula (I) and (Ia) and (Ib) each X and X independently of the other, is selected from the group consisting of LLVVG, LLVGV, LLGVV, LVLVG, LVLGV, LGLVV, LVVLG, LVGLV, LGVLV, LWGL, LVGVL, LGWL, VLLVG, VLLGV, GLLW, VLVLG, VLGLV, GLVLV, VLVGL, VLGVL, GLWL, WLLG, VGLLV, GVLLV, WLGL, VGLVL, GVLVL, WGLL, VGVLL and GWLL.

8. The composition according to claim 7, wherein in a compound of Formula (I) and (Ia) and (Ib) one or both of X and X is -LGVVL.

9. The composition according to claim 8, wherein the compound of Formula (I) is LVVGL-C(O)CHYCH.sub.2SSCH.sub.2CHYC(O)-LGVVL and the compound of Formula (Ia) is LWGL-C(O)CHYCH.sub.2SH or HSCH.sub.2CHYC(O)-LGVVL.

10. The composition according to claim 1, comprising a compound of the general formula (VI) or (VII):
XC(O)CHYCH.sub.2SSCH.sub.2CHYC(O)-LGVVL(VI)
LVVGL-C(O)CHYCH.sub.2SSCH.sub.2CHYLC(O)X(VII); wherein each of X, X, Y and Y, independently of the other, is as defined in claim 1.

11. The composition according to claim 10, comprising a compound of general Formula (VI) in combination with LVVGL-C(O)CHYCH.sub.2S-H or HSCH.sub.2CHYC(O)X.

12. The composition according to claim 10, comprising a compound of general Formula (VII) in combination with XC(O)CHYCH.sub.2SH or HSCH.sub.2CHYC(O)-LGVVL.

13. A composition according to claim 1, for use in the diagnosis of schizophrenia.

14. A method for the diagnosis of schizophrenia in a subject, the method comprising: a) contacting a blood sample obtained from a subject suspected of developing Schizophrenia with at least one compound of Formula (I) and at least one compound of Formula (Ia) and/or (Ib), in combination; b) determining the level of binding of said compounds of Formula (I), (la) and/or (Ib) to said sample; c) a level higher than the binding level of said compounds to a sample from non-schizophrenic individuals indicating that said individual has a high likelihood of having schizophrenia.

15. The method according to claim 14, for use in confirming a high probability of Schizophrenia in an individual determined by at least one other diagnostic assay.

16. A kit for use in the diagnosis of Schizophrenia, the kit comprising: a) a support comprising at least one compound of Formula (I) and at least one compound of Formula (Ia) and/or (Ib), in combination; b) reagents for carrying out a detection assay, whereby said compounds bind to PAA present in a tested sample; and c) instructions for use.

Description

EXAMPLES

Example 1: Dimerization of the Peptide Vs. Sensitivity of the Assay

[0090] Portions of 10 l of each of peptide samples I-X, were used for MALDI-MS analysis, to estimate the ratio of mono-versus dimer formed.

[0091] As can be seen from Table 2, using the 20% DMSO/80% DDW solvent system, after 8 hours, about 80-90% of the peptide was in the dimer form.

[0092] It should be noted that slightly basic conditions expedited the dimerization/oxidation of cysteine residues. For this purpose, ammonia solution was added before the reaction occurred. Diluted ammonia was useful in pushing the formation of the dimer to completeness. All samples were analyzes by MALDI-MS technique.

TABLE-US-00002 TABLE 2 Dimerization of the peptide vs. sensitivity of the assay (ACN = Acetonitrile; DDW = Double Distilled Water; TFA = Trifluoroacetic Acid; TFE = Trifluoroethanol; DMSO = Dimethyl Sulfoxide). The data in Table 2 were obtained by using low capacity Streptavidin (SA) coated tubes. Peptide Concentration Percent of dimer Sensitivity of mg/ml (stock Dimerization formed the assay Solvent solution) time (%) (%) ACN/DDW 0.35 4 hr RT 20-25 14-22 ACN/DDW 0.35 8 hr RT 15-20 19-24 ACN/DDW 0.35 18 hr RT 15-20 19-24 ACN/TFA/TFE 0.4 8 hr RT 0-5 17-19 ACN/NaHCO.sub.3 0.12 4 hr RT 40-45 45-51 ACN/NaHCO.sub.3 0.12 18 hr RT 55-60 44-55 20% DMSO 0.4 4 hr RT 75 55-61 80% DDW 20% DMSO 0.4 8 hr RT 80-90 58-61 80% DDW 20% DMSO 0.4 18 hr RT 80-90 58-61 80% DDW 20% DMSO 0.4 24 hr RT 80-90 58-61 80% DDW 28% DMSO 1.5 8-18 hr RT 80-90 61-84 72% DDW
The data in Table 2 were obtained by using high capacity Streptavidin (SA) coated tubes of Under these improved experimental conditions, the OD values were raised from 1.6-1.7 to more than 2.5. The spacer arm between biotin-peptide was modified. Instead of standard LC (aminohexanoic acid), the stretch -SGSG- (Serine-Glycine-Serine-Glycine) has been used.

Example 2: Use of a Mixture Comprising 80-90% of the Herein Described Dimer in a Screening Blood Test for the Diagnosis of Schizophrenia Via Measurement of PAA Levels in Young Schizophrenic Patients at Onset of Disease

[0093] Blood titers of PAA were evaluated blindly using an optimized ELISA test, wherein the results were expressed using a linear optical density (OD) scale. OD readings were scored for blood samples from pediatric/young adult patients (n=28) (aged 13-19 years) diagnosed with schizophrenia and controls (n=37) (aged 13-19 years). The blood samples of all participants were tested anonymously and were scored under a code number. A test recording above 1.4 OD units was defined as positive.

[0094] Tables 3-5 show results of screening tests for both pediatric/young adult (age 13-19 years) patients and controls.

TABLE-US-00003 TABLE 3 Screening results for pediatric/young adult patients (13-19 years) Young Patients (13-19 years) Number of Gender Patient Age (F/M) OD Conclusion Remarks 1 18 M 1.84 Positive 2 17 F 1.86 Positive Before treatment by Leponex 3 16 M 0.87 Negative Before treatment by Leponex 4 13 F 2.42 High Positive Before treatment by Leponex 5 19 M 2.48 High Positive Before treatment by Leponex 6 19 F 1.88 Positive Before treatment by Leponex 7 17 M 0.95 Negative 8 18 M 2.24 High Positive Before treatment by Leponex 9 14 F 1.36 Negative 10 16 M 1.36 Negative 11 18 M 0.88 Negative 12 13 M 1.23 Negative 13 18 M 2.36 High Positive 14 16 M 1.08 Negative 15 14 M 0.98 Negative 16 16 M 2.36 High Positive Before treatment by Leponex 17 18 F 2.28 High Positive Before treatment by Leponex 18 16 M 1.18 Negative 19 18 F 1.75 Positive 20 19 M 1.81 Positive 21 17 M 1.06 Negative 22 16 M 2.02 High Positive 23 14 M 2.21 High Positive 24 13 M 2.32 High Positive 25 19 M 1.72 Positive 26 17 M 1.93 High Positive 27 15 M 1.24 Negative 28 17 F 2.35 High Positive Mean 1.72 SD 0.55

TABLE-US-00004 TABLE 4 Screening results for pediatric/young adult controls (13-19 years) Pediatric/Young Adult Controls (13-19 years) Number of Gender Control Age (F/M) OD Conclusion 1 15 M 0.64 2 17 F 0.73 3 17 M 1.86 Positive 4 16 M 0.53 5 16 M 0.97 6 13 M 2.04 High Positive 7 13 M 0.41 8 16 M 1.12 9 18 M 0.86 10 14 F 1.27 11 13 M 0.64 12 16 M 0.7 13 13 M 0.95 14 13 M 0.93 15 16 F 1.69 Positive 16 15 F 0.78 17 16 F 0.53 18 17 F 1.91 High Positive 19 14 F 0.99 20 16 F 0.62 21 16 F 0.97 22 13 M 1.24 23 14 M 0.78 24 13 M 1.39 25 15 F 1.72 Positive 26 13 F 1.18 27 13 M 1.04 28 17 F 1.36 29 16 M 0.95 30 17 M 0.6 31 14 F 1.21 32 19 M 1.81 Positive 33 18 M 0.87 34 17 M 1.09 35 17 F 0.92 36 18 M 1.33 37 17 F 0.59 Mean 1.06 SD 0.43

TABLE-US-00005 TABLE 5A-B provide summaries of the pediatric/young adult patient and control screening study. A. Summary of the results Pediatric/Young Adult Pediatric/Young Adult Patients Controls N=28 N=37 P value Positive 17 Positive 6 P < 1.05 10.sup.6 Negative 11 Negative 31 Sensitivity % 60.7 Specificity % 83.8 B. OD Range Negative 1.4 Low Positive 1.4-1.6 (Grey-Zone) Positive 1.6-1.9 High Positive 1.9

[0095] As may be evident from the above Tables, PAA titers of young schizophrenia patients, aged 13-19 years, were significantly higher than those of the control group (1.720.55 OD units vs. 1.060.43; P<1.0510.sup.6).

Example 3: Data on Non-Schizophrenic Pediatric/Young Adult Patients

[0096]

TABLE-US-00006 TABLE 6 Non-schizophrenic pediatric and young adult patients. Diagnosis Number of Gender (as assessed by Patient (F/M) Age OD Conclusion clinical team) 1 M 15 0.682 Negative Mental retardation 2 M 17 1.03 Negative Mental retardation 3 M 16 1.08 Negative Mental retardation 4 F 16 1.03 Negative Mental retardation 5 F 16 0.548 Negative Mental retardation 6 F 15 0.807 Negative Mental retardation 7 F 14 0.681 Negative Pervasive Developmental Disorder (PDD) 8 M 18 0.89 Negative PDD 9 F 15 1.176 Negative Bipolar 10 M 17 0.597 Negative Bipolar 11 M 19 0.53 Negative Bipolar 12 F 17 0.982 Negative Bipolar 13 F 16 0.527 Negative Bipolar 14 F 16 0.442 Negative Bipolar 15 18 18 1.09 Negative Depression 16 M 14 0.742 Negative Depression 17 F 19 0.662 Negative Depression 18 M 18 0.712 Negative Depression 19 M 15 0.964 Negative Obsessive-Compulsive Disorder (OCD) 20 F 16 1.015 Negative OCD 21 M 15 0.443 Negative OCD 22 M 15 0.583 Negative OCD 23 F 18 0.864 Negative OCD 24 M 19 1.12 Negative OCD 25 M 17 1.78 Positive Borderline 26 F 16 0.889 Negative Borderline 27 F 17.5 0.541 Negative Borderline 28 F 14 0.892 Negative Borderline 29 F 16 1.09 Negative Borderline 30 F 15 0.301 Negative Borderline 31 F 17 1.206 Negative Borderline 32 F 15 0.891 Negative Borderline 33 F 15 0.432 Negative Borderline 34 M 17 0.41 Negative Borderline 35 F 19 0.621 Negative Borderline 36 F 17 0.584 Negative Borderline 37 M 14 0.507 Negative Conduct disorder.sup.a 38 F 16 0.386 Negative Personal disorder.sup.b 39 F 17 1.18 Negative Personal disorder 40 F 14 0.873 Negative Conduct disorder 41 M 13 1.89 Positive Conduct disorder 42 M 13 1.14 Negative Conduct disorder 43 F 15.5 0.286 Negative Conduct disorder 44 M 14 0.432 Negative Conduct disorder 45 F 16 0.943 Negative Conduct disorder 46 M 14 0.296 Negative Conduct disorder 47 M 16 0.712 Negative Personal disorder 48 M 16 0.445 Negative Conduct disorder 49 M 12 0.478 Negative Conduct disorder 50 F 14 1.112 Negative Conduct disorder 51 F 14 0.501 Negative Conduct disorder 52 F 16 0.964 Negative Conduct disorder 53 F 16 0.621 Negative Conduct disorder 54 M 14 0.851 Negative Conduct disorder 55 F 15 0.329 Negative Conduct disorder 56 F 18 0.815 Negative Personal disorder 57 M 15 1.922 Positive Conduct disorder 58 F 18 0.644 Negative Personal disorder 59 M 18 0.543 Negative Personal disorder 60 F 18 0.602 Negative Personal disorder 61 M 15 0.453 Negative Conduct disorder 62 M 14 0.369 Negative Conduct disorder Mean 0.77 SD 0.36 .sup.aConduct disorder (CD) is a psychological disorder diagnosed in childhood or adolescence (ages 0-17) that presents itself through a repetitive and persistent pattern of behavior in which the basic rights of others or major age-appropriate norms are violated. These behaviors are often referred to as antisocial behaviors. It is often seen as the precursor to antisocial personality disorder, which is not diagnosed until the individual is 18 years old. .sup.bPersonal (Personality) disorders are a class of mental disorders characterized by enduring maladaptive patterns of behavior, cognition, and inner experience, exhibited across many contexts and deviating markedly from those accepted by the individual's culture (ages 18 upwards).

[0097] As may be evident from Table 6, only 3/62 cases of non-schizophrenic pediatric and young adult patients between the ages of 12-19 tested positive using this schizophrenic diagnostic test showing that the herein described method for diagnosing schizophrenia constitutes a reliable assay (with a low, approximately 5%, of false positives) for differentiating between schizophrenia and other non-schizophrenic disorders (such as personal and conduct disorder).