Patent Description:
Schizophrenia is a syndrome which encompasses a variety of mental symptoms like auditory hallucinations, paranoia, delusions, catatonia, bizarre behavior and emotional withdrawal. Schizophrenia affects about <NUM>% of the total population and its economical as well as social burdens on society are enormous. The onset of the disease occurs in early age and, thus, patients typically need life-long medical and psychiatric supervision. Schizophrenia is, therefore, rated as one of the most costly diseases in the industrial world.

Schizophrenia has been shown to involve an autoimmune process and lately autoantibodies and cytotoxic T-cells against platelets were demonstrated in schizophrenic patients. The cytotoxic T-cell reaction in schizophrenic patients was evaluated by a skin test in which most schizophrenic patients reacted positively against their autologous platelets whereas only a very minor number of non-schizophrenic tested individuals reacted positively in this test [<NUM>].

In addition, elevated levels of autoantibodies against platelets were observed in schizophrenic patients but not in patients suffering from manic-depressive disorder, depression, personality disorders and schizoaffective disorder [<NUM>, <NUM>].

Previous studies demonstrated several proteins which bind autoantibodies that are found in elevated levels in body fluids of schizophrenic patients [<NUM>]. These proteins reacted with purified platelet-derived autoantibodies (PAA) from schizophrenic patients but could not differentiate between plasma or blood samples of schizophrenic and non-schizophrenic individuals. Enzymatic digestion of one of these proteins, the enzyme Enolase, resulted in a fragment which bound substantially higher to plasma samples of schizophrenic patients compared to plasma samples of non-schizophrenic individuals. On the basis of this fragment several additional peptides were synthesized and such having a high binding activity to PAAs of schizophrenic individuals were isolated. These synthesized peptides are each comprised of at least <NUM> amino acids.

The effect of clozapine and other antipsychotics on the level of PAAs in children with schizophrenia was also studied [<NUM>].

Reference [<NUM>] relates to a specific peptide which is reported to bind to a body fluid sample obtained from a schizophrenic patient at a higher level than its binding to a body fluid sample obtained from a.

The present invention provides a composition comprising 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. The particular combinations enable autoantibodies to bind to the peptide-based materials, thereby rendering the compositions of the invention most useful in the diagnosis of schizophrenia. Thus, although peptides of the prior art [<NUM>,<NUM>] were able to bind to autoantibodies present at higher levels in schizophrenic patients as compared to non-schizophrenic individuals, the peptide combinations of the present invention demonstrate a vastly improved affinity and unique characteristics which enable a more efficient and early detection of schizophrenia in children (i.e. under the age of <NUM>, as per NIH policy [<NUM>] and young adults, being <NUM>-<NUM> years old).

As demonstrated herein, assays of the art exhibited a sensitivity which is at best about <NUM>%, while the combination of the present invention demonstrated a sensitivity of about <NUM>%.

Thus, the invention contemplates a diagnostic tool in the form of a combination comprising two diagnostically active materials: at least one peptide and at least one dimer of said peptide. As further shown below, the at least one peptide comprises an amino acid having at least one sulfur atom, e.g., cysteine, and the dimer of said at least one peptide is formed by forming a covalent bond between a sulfur atom present on each of said at least one peptide. Thus, the dimer comprises a -S-S- bond.

By a first aspect, the present invention provides a composition comprising a compound of the general formula (I) in combination with a compound of the general Formula (Ia) and/or (Ib):.

X<NUM>-X-X<NUM>-S-S-X<NUM>-X'-X<NUM>     (I),.

As stated, the invention concerns a composition of at least two active peptide-based materials. The first being a compound of the general Formula (I), being a dimer containing a disulfide bond; and the other a peptide of the general Formula (Ia) and/or (Ib). The dimer utilized in a composition of the invention is typically selected to be a dimer of the peptide component of Formula (Ia) and/or (Ib). For example, where the peptide is of the general Formula (Ia): X<NUM>-X-X<NUM>-S-H, the dimer of Formula (I) is X<NUM>-X-X<NUM>-S-S-X<NUM>-X-X<NUM>. Similarly, where the peptide is of the general Formula (Ib): H-S-X<NUM>-X'-X<NUM>, the dimer of Formula (I) is X<NUM>-X'-X<NUM>-S-S-X<NUM>-X'-X<NUM>.

Putting it differently, where selecting a compound of Formula (I) and a compound of Formula (Ia) and/or (Ib), each of X, X<NUM>, X<NUM>, X<NUM>, X<NUM> and X' are selected identically, such that the compound of Formula (I) is a dimer of at least one peptide of Formula (Ia) or Formula (Ib) present in the composition.

In some embodiments, where the combination is of a compound of Formula (I), a compound of Formula (Ia) and a compound of Formula (Ib), the dimer being a compound of Formula (I) is a dimer of the compound of Formula (Ia) or the compound of Formula (Ib).

The weight ratio between the at least two components of the composition, i.e., (<NUM>) a compound of the general Formula (I), and (<NUM>) a peptide of the general Formula (Ia) and/or (Ib) is at least <NUM>:<NUM>. In some embodiments, the compound of the general Formula (I) is in excess. In some embodiments, the compound of the general Formula (I) exits in an amount which is at least twice as the amount of a peptide of the general Formula (Ia) and/or (Ib). In some embodiments, the amount of the compound of the general Formula (I) is <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> times as much as a peptide of the general Formula (Ia) and/or (Ib).

In some embodiments, in a composition of the invention, the compound of the general Formula (I) constitutes at least <NUM>% of the combination. In some embodiments, the compound of the general Formula (I) constitutes at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or at least <NUM>% of the active combination.

In other embodiments, in a composition of the invention, the compound of the general Formula (Ia) and/or (Ib) constitutes at most <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or at most <NUM>% of the active combination.

In some embodiments, the dimer of Formula (I) is asymmetric, namely it is a dimer of two different peptides, the peptide of the Formula (Ia) and/or (Ib) may be any of the two peptides making up the dimer.

Thus, when selecting a compound of Formula (I) for a composition of the invention, the identity of a peptide of Formula (Ia) or (Ib) may be easily determined.

As used herein an "amino acid" is any amino acid as known in the art. In some embodiments, the amino acid is any one of the <NUM> naturally occurring amino acids. In other embodiments, the amino acid is any of the <NUM> naturally occurring amino acids which is chemically modified. In further embodiments, the amino acid is a synthetic non-naturally occurring amino acid.

When referring to any one amino acid making up the peptides used in accordance with the invention, the amino acids may be referred to by their acceptable nomenclature or by their one-letter designation, as acceptable in the art. For example, the amino acid referred to by the letter L is Leucine. The amino acid referred to by the letter V is Valine. The amino acid referred to by the letter G is Glycine. The amino acid referred to by the letter K is Lysine. The amino acid referred to by the letter M is Methionine and the amino acid referred to by the letter C is Cysteine. Thus, in the exemplary peptide motif "LLVVG" is the peptide Leucine-Leucine-Valine-Valine-Glycine. Unless specifically indicated, the directionality of the peptide moiety or any peptide group depicted, may be either C→N or N→C.

In a compound of Formula (I), each of X and X', independently, is a pentapeptide comprising five amino acids, each of the amino acids being connected to each other via a peptide bond.

More specifically, each of X and X', independently, is a pentapeptide consisting of the amino acids L, V and G, with a sequence as recited in claim <NUM>.

In other embodiments, each of X and X' may or may not be the same. In some another embodiments, X and X' are different from each other.

In reference compositions the composition comprises a compound of Formula (I) which is selected amongst compounds having the structure:.

wherein in each of the above formulae, X<NUM>, X<NUM>, X<NUM> and X<NUM> are as defined.

In reference compositions, the compound of Formula (I) is a compound wherein X is absent and X' is as defined above, or a compound wherein both X and X' are absent or a compound wherein X is as defined above and X' is absent.

In the above formulae, each of X<NUM>, X<NUM>, X<NUM> and X<NUM>, independently of the other, may be absent or is an amino acid, a di-amino acid or a tri-amino acid as defined in claim <NUM>.

As used herein, a di-amino acid is a dipeptide, namely two amino acids connected to each other via a peptide bond; a tri-amino acid is a tripeptide, namely three amino acids connected to each other via peptide bonds; wherein the amino acid in each of "amino acid", "di-amino acid" and "tri-amino acid" is as defined herein.

In reference compositions, each of the amino acids indicated in the general Formula (I) may be substituted by at least one amino acid group. The "at least one amino acid group " is a single amino acid or a chain of amino acids which are connected to each other, in some embodiments, via peptide bond(s); or a group comprising one or more amino acids.

As a reference, the substituting amino acid group is or comprises an amino acid selected from C, K and M. The at least one amino acid group may be C, K or M. The at least one amino acid group may comprise one or more of C, K and M.

For example the amino acid group is C. For example the amino acid group is K. For example the amino acid group is M.

In some embodiments, the disulfide group is derived from cystine. In some embodiments, the cystine is substituted by at least one amino acid.

Each of X<NUM>, X<NUM>, X<NUM> and X<NUM>, independently, is an amino acid, a di-amino acid or a tri-amino acid comprising an amino acid selected from K, C and M. In some embodiments, the amino acid is K.

More specifically each of X<NUM> and X<NUM>, independently, is an amino acid, a di-amino acid or a tri-amino acid comprising an amino acid selected from K, C and M and X<NUM> and X<NUM> are absent.

Disclosed for reference is a composition which comprises a compound having the general Formula (I), wherein each of X<NUM>, X<NUM>, X<NUM> and X<NUM>, is absent; the compound being of the general Formula (II):.

wherein each of X and X' are as defined above.

In some embodiments, the disulfide group in the compound of Formula (II) is a cystine; In disclosed examples the compound is of general Formula (III) :.

X-C(O)-CHY-CH<NUM>-S-S-CH<NUM>-CHY'-C(O)-X'     (III).

wherein each of X and X' are as defined above, -C(O)- designates a carbonyl group (-C=O), and each of Y and Y', independently of the other, is at least one amino acid, as defined herein. For example, each of Y and Y', independently, is selected from K, C and M.

Disclosed for reference is a composition which comprises a compound of general Formula (I) having a general Formula (IV):.

Disclosed for reference is a composition which comprises a compound of Formula (I) having the structure of Formula (V):.

wherein each of X and X' are as defined above, and Y' is at least one amino acid.

As required in claim <NUM>, each of X and X', independently of the other, is a pentapeptide comprising at least one amino acid L, at least one amino acid V and at least one amino acid G. In particular each of X and X', independently of the other, is a pentapeptide comprising at least two amino acids L. In particular, each of X and X', independently of the other, is a pentapeptide comprising at least two amino acids V. More specifically, each of X and X', independently of the other, is a pentapeptide selected from LGVVL and LVVGL. These are included in the following Table <NUM>, along with a number of further pentapeptides that are provided merely for reference.

The peptide connectivity may be read from right to left or from left to right.

In disclosed examples, each X and X' independently of the other, is selected from the amino acid residues: LLVVG (SEQ ID NO: <NUM>), LLVGV (SEQ ID NO: <NUM>), LLGVV (SEQ ID NO: <NUM>), LVLVG (SEQ ID NO: <NUM>), LVLGV (SEQ ID NO: <NUM>), LGLVV (SEQ ID NO: <NUM>), LVVLG (SEQ ID NO: <NUM>), LVGLV (SEQ ID NO: <NUM>), LGVLV (SEQ ID NO: <NUM>), LVGVL (SEQ ID NO: <NUM>), VLLVG (SEQ ID NO: <NUM>), VLLGV (SEQ ID NO: <NUM>), GLLVV (SEQ ID NO: <NUM>), VLVLG (SEQ ID NO: <NUM>), VLGLV (SEQ ID NO: <NUM>), GLVLV (SEQ ID NO: <NUM>), VLVGL (SEQ ID NO: <NUM>), VLGVL (SEQ ID NO: <NUM>), GLVVL (SEQ ID NO: <NUM>), VVLLG (SEQ ID NO: <NUM>), VGLLV (SEQ ID NO: <NUM>), GVLLV (SEQ ID NO: <NUM>), VVLGL (SEQ ID NO: <NUM>), VGLVL (SEQ ID NO: <NUM>), GVLVL (SEQ ID NO: <NUM>), VVGLL (SEQ ID NO: <NUM>), VGVLL (SEQ ID NO: <NUM>) and GVVLL (SEQ ID NO: <NUM>).

In disclosed examples, each X and X' independently of the other, is selected from LLVVG, VLLVG, VLLGV, VVLLG, VGLLV and GVLLV.

In disclosed examples, each X and X' independently of the other, is selected from VLVGL, VLGVL, LVVLG, LVGLV, LGVLV, and GVLLV.

In disclosed examples, each X and X' independently of the other, is selected from VLLGV, GLLVV, VVLGL, VGLVL, LLVVG and LLVGV.

In the present invention, each X and X' independently of the other, is selected from LGVVL and LVVGL.

In some embodiments, one or both of X and X' in any one of the Formulae (I)-(V) is -LGVVL.

Disclosed for reference is a composition which comprises a compound of Formula (I) having the general Formula (VI) or (VII):.

X-C(O)-CHY-CH<NUM>-S-S-CH<NUM>-CHY'-C(O)- LGVVL     (VI).

LVVGL-C(O)-CHY-CH<NUM>-S-S-CH<NUM>-CHY'-C(O)-X'     (VII).

wherein each of X, X', Y and Y', independently of the other, is as defined above.

For example, one or both Y and Y' in the Formulae (III)-(VII) is the amino acid K. For example, Y = Y' in the Formulae (III)-(VII) and is the amino acid K.

In particular X and X' in any one of the formulae disclosed for reference, independently, is a pentapeptide selected from LLVVG, LLVGV, LLGVV, LVLVG, LVLGV, LGLVV, LVVLG, LVGLV, LGVLV, LVGVL, VLLVG, VLLGV, GLLVV, VLVLG, VLGLV, GLVLV, VLVGL, VLGVL, GLVVL, VVLLG, VGLLV, GVLLV, VVLGL, VGLVL, GVLVL, VVGLL, VGVLL and GVVLL.

In disclosed examples, X and X' of any one of the formulae of the invention, independently, is a pentapeptide selected from VVGLL, VGLLV, GLLVV, LVGVL, GVLVL. In the present invention, X and X', independently of each other, is selected from LGVVL and LVVGL. In some compositions disclosed for reference, the composition comprises a compound (VI), as defined herein, in combination with LVVGL-C(O)-CHY-CH<NUM>-S-H or H-S-CH<NUM>-CHY'-C(O)-X'.

In other compositions disclosed for reference, the composition comprises a compound (VII), as defined herein, in combination with X-C(O)-CHY-CH<NUM>-S-H or H-S-CH<NUM>-CHY'-C(O)- LGVVL.

In some embodiments, the composition of the invention comprises the compound of Formula (VIII):.

LVVGL-CO-CH(NH-K)-CH<NUM>-S-S-CH<NUM>-CH(NH-K)-CO-LGVVL     (VIII).

In the compound of Formula (VIII), the group (NH-K) is pendant and covalently bonded to the carbon atom of the -CH- moiety. The amino acid K is bonded only to the -NH- group which is linking the amino acid K and -CH-.

In some embodiments, the composition comprises the compound of Formula (VIII) in combination with the peptide LVVGL-CO-CH(NH-K)-CH2-SH.

The compound of any of Formulae (I) to (VIII) may be associated to biotin.

For example, the compounds of Formulae (Ia) and (Ib) may be associated to biotin.

In particular, the compound of Formula (I) may be a compound of Formula (VIII) when associated with biotin:
Biotin-LVVGL-CO-CH(NH-K)-CH<NUM>-S-S-CH<NUM>-CH(NH-K)-CO-LGVVL-Biotin.

In another aspect, the invention provides use of at least one compound of Formula (I) and at least one compound of Formula (Ia) and/or (Ib), for the preparation of a diagnostic composition.

In a further aspect, there is provided a combination of at least one compound of Formula (I) and at least one compound of Formula (Ia) and/or (Ib) for use in a method of diagnosis.

In some embodiments, the composition is for the diagnosis of schizophrenia in an individual being, in some embodiments, a child under the age of <NUM>.

Disclosed herein for reference is a method for the diagnosis of schizophrenia in a subject, the method comprising:.

As readily realized by the person of skill in the art, the herein defined diagnosis, i.e. the determination of the level of binding of the peptide components present in the composition to said sample, may be achieved by a number of (e.g. chemical, biological) detection methods common to the pertinent field of the art that employ binding of peptides to generate a measurable signal which, based thereupon, diagnosis of schizophrenia is achieved. Some non-limiting examples of detection methods which can be used include enzyme-linked immunosorbent assay (ELISA), bimolecular fluorescence complementation (BiFC), chemical cross-linking followed by high mass matrix-assisted laser desorption/ionization mass spectrometry, proximity ligation assay (PLA), dual polarization interferometry (DPI), static light scattering (SLS), surface plasmon resonance, fluorescence polarization/anisotropy, fluorescence correlation spectroscopy, fluorescence resonance energy transfer (FRET), protein-protein docking, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST).

For example, the method is for use in confirming a high probability of Schizophrenia in an individual determined by at least one other diagnostic assay.

In some embodiments, the detection method is ELISA.

In another aspect, the invention provides a kit for use in the diagnosis of Schizophrenia, the kit comprising a support comprising one or more peptide-based materials defined herein, immobilized onto an anti-human immunoglobulin (hIg) antibody or fragment thereof, reagents for carrying out a detection assay comprising e.g. an anti-human immunoglobin (hIg) antibody or fragment thereof bound to a chemiluminescent tag or an enzyme such as alkaline phosphatase or horseradish peroxidase, and instructions for use.

As readily recognized by the skilled artesian, the kit and diagnostic composition of the present invention may also contain additional reagents and components suitable for using said kit and diagnostic composition in the herein described diagnosis of Schizophrenia. Some non-limiting examples of such reagents and components include a buffer, a diluent, a carrier, a chemical stabilizer, a preservative, a salt, an enzyme, a detection reagent, a pretreatment reagent/solution, a substrate (e.g., as a solution) and a stop solution.

The kit of the present invention may also contain reagent reservoirs and any additional component (e.g. disposable pipettes) required for using said kit in the herein described diagnosis of Schizophrenia.

In some embodiments, the anti-hIg antibody is complexed to a detectable marker. In some embodiments, instead of said anti-hIg antibody, the kit comprises one or more non-bound peptide-based materials which bind to PAA present in a tested sample, said peptide-based materials being complexed to a detectable marker.

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

As can be seen from Table <NUM>, using the <NUM>% DMSO/<NUM>% DDW solvent system, after <NUM> hours, about <NUM>-<NUM>% of the peptide was in the dimer form.

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.

The data in Table <NUM> were obtained by using high capacity Streptavidin (SA) coated tubes of. Under these improved experimental conditions, the OD values were raised from <NUM>-<NUM> to more than <NUM>. The spacer arm between biotin-peptide was modified. Instead of "standard LC" (aminohexanoic acid), the stretch -SGSG- (Serine-Glycine-Serine-Glycine) has been used.

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=<NUM>) (aged <NUM>-<NUM> years) diagnosed with schizophrenia and controls (n=<NUM>) (aged <NUM>-<NUM> years). The blood samples of all participants were tested anonymously and were scored under a code number. A test recording above <NUM> OD units was defined as positive.

Tables <NUM>-<NUM> show results of screening tests for both pediatric/young adult (age <NUM>-<NUM> years) patients and controls.

As may be evident from the above Tables, PAA titers of young schizophrenia patients, aged <NUM>-<NUM> years, were significantly higher than those of the control group (<NUM> ± <NUM> OD units vs. <NUM> ± <NUM>; P < <NUM> x <NUM>-<NUM>).

Claim 1:
A composition comprising a compound having the general formula (I):

        X<NUM>-X-X<NUM>-S-S-X<NUM>-X'-X<NUM>     (I)

and at least one compound of the general Formula (Ia) and/or (Ib):

        X<NUM>-X-X<NUM>-S-H     (Ia)

        H-S-X<NUM>-X'-X<NUM>     (Ib)

wherein:
each of X<NUM> and X<NUM> independently, is an amino acid, a di-peptide or a tripeptide comprising an amino acid selected from K, C and M and X<NUM> and X<NUM> are absent;
S designates sulfur, H designates hydrogen and -S-S- designates a disulfide bond or a group comprising a disulfide bond,
X and X', independently of each other, is selected from LGVVL and LVVGL;
each of "-" is a bond,
wherein in the Formulae (Ia) and (Ib), each of X, X<NUM>, X<NUM>, X<NUM>, X<NUM> and X' are selected as in formula (I);
wherein the ratio between the compound of Formula (I) and the compound of Formula (Ia) or (Ib) is between <NUM>:<NUM> to <NUM>:<NUM>,
and the at least one compound of the general Formula (Ia) and/or (Ib) comprises cysteine, and the compound having the general formula (I) is formed by forming a covalent bond between a sulfur atom present on each of said at least one compound of the general Formula (Ia) and/or (Ib).