WO2012139732A1

WO2012139732A1 - Preparation containing disc1 and/or at least one disc1 fragment for use in the therapeutic treatment of the human or animal body

Info

Publication number: WO2012139732A1
Application number: PCT/EP2012/001511
Authority: WO
Inventors: Carsten Korth; Joseph P. HUSTON; Martin PUM
Original assignee: Carsten Korth; Huston Joseph P; Pum Martin
Priority date: 2011-04-13
Filing date: 2012-04-05
Publication date: 2012-10-18
Also published as: DE102011016875A1

Abstract

The invention relates to a preparation containing DISC1 and/or at least one DISC1 fragment for use in the therapeutic treatment of the human or animal body, wherein preferably at least one immunostimulating substance is additionally contained and preferably the at least one DISC1 fragment is DISC1-598-785.

Description

Preparation containing DISCl and / or at least one DISCl fragment for use in the therapeutic treatment of the human or animal body

The invention relates to a novel preparation for use in the therapeutic treatment of the human or animal body, in particular for the modulation of neurochemical processes and behavior, wherein in one embodiment the preparation has at least one DISC fragment in combination with an immunostimulating substance.

The DISC1 protein is thought to play a key role in genetic engineering studies (Chubb et al., 2008, Molecular Psychiatry 13:36) and reverse genetic studies on animal models (Shen et al., 2008, J. Neuroscience 48: 10893) Attributed to control of behavioral control and mental homeostasis. Inter alia, animals that modeled dysfunctionality of DISCI also had learning or memory disorders as well as disorders of sensory gating or depression-like symptoms (reviewed in Shen et al., 2008, J. Neuroscience 48: 10893).

DISCl is a protein whose function is not yet conclusively known. It plays a role in neuronal development both in the proliferation of neuronal progenitor cells and in neuronal differentiation, neuronal migration and the formation of a functional dopamine metabolism (Niwa et al., 2010, Neuw. ron 65: 480). DISC1 is a cytosolic-expressed protein lacking typical signal sequences that allow export to the endoplasmic reticulum. In this respect, according to the state of the art, it is not to be expected that it will be present extracellularly in biologically-functionally relevant quantities and that it can be used as a target occurring outside the cell, for example.

From US 2005/0210536 is known to investigate neurological disorders in animal models, wherein a gene construct containing the DISCl gene is injected into the brain of the animals.

From WO 02/058637 it is known to use DISC1 as a marker in clinical studies to determine the efficacy of drugs for DISC-correlated diseases.

WO 2009/154697 describes a method for the treatment of neurological disorders in which a DISCl activator is administered. The activator may be e.g. to act DISCl. However, in WO 2009/154697, no distinction is made between monomeric and multimeric DISC, which both activate differently, eg. with antagonistic effects. In addition, immunogenic effects, in particular of multimeric DISC, are not discussed.

In the publication by Leliveld et.al. - Biochemistry 2009, 48, 7746-7755 - describes the study of different DISCI fragments. The DISCI fragments are designated in the publication with numbers indicating the position of the first and last amino acid of the fragment in the sequence of the DISC1 protein. This designation of DISCI fragments is also used in the present application.

In the cited publication it was shown that a C-terminal fragment of DISCl also occurs in vivo in the cytosol. In the same publication it is assumed that further C-terminal DISCI fragments are present in vivo and a hitherto unknown role in the modulation of DISC-dependent intracellular Functions can play. However, this publication also shows that DISC1 fragments are present in the cytosol and not in secreted form.

The object of the invention is to provide new applications for DISC1 or DISC1 fragments.

The problem is solved according to claim 1.

According to the invention, it is provided to use a preparation containing DISC1 and / or at least one DISC1 fragment as a therapeutic for the treatment or modulation of neurochemical processes in the human or animal body or for influencing the proliferation or migration of cells.

Preferably, a multimeric DISC1 fragment is used.

It was surprisingly found that the exogenous, subcutaneous administration of a preparation containing a DISC1 fragment, a modulation of the behavior in rats in open-field tests and the inoculation of a preparation containing a DISCl fragmentation , caused a palliative effect on the anxiety behavior of rats in the bright field / dark field preference test. These effects were not to be expected since, according to the state of research, the DISC1 protein should not be extracellular as a cytosolic protein and should therefore be inaccessible to direct modulation. However, the effect was DISC1-dependent since no results different from the completely untreated control were obtained in a DISC1-free control (see Figure 1, Example 1a; Figure 4B, Example 1b).

Open-field tests are animal experiments in which test animals are placed in open-top, mostly bright containers, usually with a footprint of 1 meter x 1 meter. During the experiment the conditions (lighting, temperature etc.) are kept constant. After a rat or a test animal is placed in the container, the location changes of the animal over a certain period of time observed and quantified. The tests are based on the fact that animals want to avoid the stay on an unknown, brightly lit open space as far as possible. If they are nevertheless brought into such an environment, they show a movement behavior that is dictated by timidity, readiness to flee and curiosity.

In a similar direction is the bright field / dark field preference test, where rats are placed in a bright field (61 x 38 x 32 cm) connected to an adjacent dark field. While e.g. For 5 minutes, the time spent by the rat in brightfield or darkfield is measured.

In some experiments, a preparation consisting of the rat DISC1 fragment 598-785 and the immunostimulating substance ABM-ZK was used (Linaris, Germany); ABM-ZK consists of 0.5 mg trehalose dimycolate (TDM), 0.5 mg monophosphoryl lipid A (MPL), 0.5 mg Cell Wall Skeleton (CWS), 2% oil (squalene) and an unspecified amount of Tween 80th

It can be assumed that in addition to the inserted DISC1 fragment, other DISC1 fragments, e.g. DISCI 765-854, 598-854, DISCI 1-854, or fragments other than those mentioned in the above-cited publication have a similar effect. It is conceivable to use as a single protein or peptide or as a fusion protein.

In a first experiment, it was shown that after administration of the DISCl fragment in healthy rats, these showed a lower locomotor in open-field tests than an untreated control group. It can be deduced from this that the fragment has a calming effect on such rats (FIG. 1).

In contrast, rats previously subjected to amphetamine sensitization by chronic administration of D-amphetamine, after administration of the DISCl fragment, exhibited a marked increase in locomotion over an amphetamine-sensitized control group to which no DISC1 fragment was administered (Figure 2). Amphetamine sensitizations are used in animal studies to elicit neurochemical balance disorders similar to those in neuropsychiatric disorders with schizophrenia-typical symptoms.

As a rule, amphetamine sensitization results in e.g. Rats in open-field experiments show that they show increased locomotion compared to non-sensitized rats. The fact that a further increase in locomotion was observed after administration of DISC1 fragments suggests that the DISC1 fragment has modulated the disordered neurochemical processes, specifically the dopamine metabolism, which is known to undergo dopamine metabolism at amphetamine sensitization (Featherstone et al., 2007, Progress in Neuropsychopharmacology & Biological Psychiatry 31: 1556).

In another experiment (bright field / dark field preference test), it was shown that an anxiolytic effect in rats could be achieved by inoculating the DISC fragment (598-785) into the brain.

The experiments indicate that the administration of DISC1 fragments may induce immunogenic and / or psychotropic effects, both of which are e.g. cause a modulation of anxiety behavior.

In experiments conducted by the Applicant it was further shown (by Elisa) that rats immunized with a preparation of the rat DISC fragment 598-785 and the immunostimulating substance ABM-ZK all had antibodies to DISC.

In general, the ratios of rats can be transferred to humans, so that it is readily conceivable, for example drugs prepared from DISCI fragments as tranquilizers or for the therapy of neuropsychiatric disorders, in particular those disorders which interfere with the DISCI metabolism or the Dopaminstoffwechsels are correlated to use. In the sense of a DISC-dependent regulation of the dopamine metabolism which is modulated by the preparation according to the invention, it is conceivable, for example, for use in schizophrenia, (recurrent) depression, bipolar disorder, autism, addictions, Huntington's disease, Tourette syndrome, attention deficit hyperactivity disorder. Syndrome, restless legs syndrome, as well as indirectly synucleinopathies such as Lewy body dementia or Parkinson's disease; in Parkinson's disease, for example, as adjuvant therapy for "dopamine dysregulation syndrome". Due to the existing memory disorders in animal models with DISC1 mutations, the preparation of the invention is also applicable to transient or chronic memory disorders such as mild cognitive impairment (MCI), or diseases such as Alzheimer's disease.

In addition, the preparation according to the invention could be used for the treatment of side effects of certain drugs or substances which affect the dopamine metabolism, by favorably influencing them, including acute poisoning.

Furthermore, the preparation according to the invention can be used for the treatment of chronic pain conditions in which dopamine metabolism plays a role, including but not limited to chronic fatigue syndrome, fibromyalgia, "burning mouth syndrome".

Dopamine also plays a role in cognitive peculiarities or disorders such as anhedonia, disturbances of social interaction or overall "incentive salience" and the inventive preparation can therefore also be used as a cognitive enhancer in non-ill individuals.

Moreover, the preparation according to the invention is conceivable for the treatment of the following non-brain-specific diseases in which the peripheral dopamine metabolism plays a role, for example cardiovascular diseases, arrhythmias, blood disorders. high pressure, obesity, diabetes, immune regulation, lactation disorders, ammenorrhea, libido and sexual dysfunction.

The preparation according to the invention can also influence the proliferation and migration of cells, since this is a core function of DISC1. Thus, it is also possible to use in cancers or use to control the proliferation of immune cells, e.g. in autoimmune diseases such as multiple sclerosis.

The various influences of the preparation according to the invention on DISO-dependent functions, such as neurochemical processes, cell proliferation and migration, allow the preparation according to the invention to have an overall life-prolonging effect, so that the preparation according to the invention can also generally be used for regeneration.

The surprising finding that DISC1 or DISC1 fragments are in secreted form suggests that DISC1 may act as a signaling molecule, possibly triggered by certain circumstances such as cellular stress, and may be delivered into the extracellular space by unknown transport mechanisms independent of the endoplasmic reticulum and then binds to specific molecules or receptors. These molecular mechanisms were not expected in the current state of the art because secretion of DISC1 or DISC1 fragments was unknown and considered impossible because DISC1 was classified as a cytosolic cytoskeleton-associated molecule. In this respect, the preparation of the invention justifies a new teaching.

The preparation according to the invention may be formulated for subcutaneous, intramuscular, intravenous, intracerebral, intranasal or intraperitoneal administration. On the basis of the examples given below, which describe the subcutaneous and intracerebral administration, it is no problem for the person skilled in the art to also provide suitable formulations for the other stated administration possibilities. In the following, the invention will be explained in more detail by means of examples and figures:

FIG. 1 shows in a bar chart the locomotion behavior of healthy ones

Rats in an open-field test in which untreated rats (adjuvant only) were compared to rats injected with the DISC 1 fragment (together with adjuvant).

FIG. 2 shows, similar to FIG. 1, the effect of (subcutaneous) immunization of

DISC 1 (598-785) on the basal behavior of rats in the open field.

FIG. 3 shows the locomotion behavior of amphetamine-sensitized rats determined under similar conditions as in FIG.

FIG. 4 shows a (e)

A) fluorescence microscopy of a brain section of the nucleus accumbens of a rat stereotactically inoculated with Dylight-labeled (red), recombinant DISC1 598-785,

B) Diagram of the results of the light-dark preference test two weeks after bilateral inoculation of DyLight-labeled recombinant DISC 1 (598-785) or control (sham) in the nucleus accumbens of a wild-type rat, and

C) Diagram of the results of an open field experiment following a single, intraperitoneal amphetamine injection two weeks after bilateral inoculation of DyLight-labeled recombinant DISC 1 (598-785) or control (sham) in the nucleus accumbens of a wild-type rat.

Figure 5 shows diagrams A and B showing the effect of DISC 1 immunization on the behavior in the bright field / dark field preference test (black bars, dark field, white bars, bright field). Figure 6 shows a Western blot of cell lysates and the accompanying supernatant expressing DISC 1 and secreting full length DISC1 or a DISC1 fragment into the supernatant

Example 1: Examination of rats in an open-field experiment a) Influence of DISC1 on the locomotion behavior in healthy rats

In this experiment, the behavior of one group of untreated rats was compared to another group of rats given a D IS Cl fragment.

Administration was by injection (subcutaneously) of a solution containing 150 μg of the rat DISC fragment 598 to 785 and in 150 x L adjuvant ABM-ZK (Linaris, Germany, consisting of 0.5 mg trehalose dimycolate (TDM), 0 , 5 mg monophosphoryl lipid A (MPL), 0.5 mg Cell Wall Skeleton (CWS), 2% oil (squalene) and an unspecified amount of Tween 80).

There were a total of four injections of the same amount at intervals of three weeks.

The rats were placed in the open-field environment 14 days after the last injection. The rats were observed over a period of 20 minutes and their movement measured by either measuring the total distance covered (Example 1) or dividing the open-field into nine parts (sectors) and the number of crosses from one sector into one adjacent was counted (Example 2). The two measurement methods are equivalent and allow the same statements. Ten rats were examined per group.

The results of the experiment are summarized in FIG. The behavior of untreated rats (adjuvant only) is shown in the left bar. The right panel shows the locomotion behavior of the rats injected with the DISC1 fragment (together with the adjuvant).

It can be seen that the treated rats show a significantly lower tendency to move than the untreated rats. Since novelty-induced locomotion can be linked to the dopamine system, this finding suggests differences in the dopamine system of the two treatment groups

It can be deduced from this that the peripheral introduction of DISC1 into the body of the rat has a clear effect on the behavior in terms of a reduction of the movement. b) Influence of (subcutaneous) immunization of DISCI (598-785) with adjuvant (ABM-ZK, Linaris, as described) on the basal behavior of older rats in the open field

As in Example la, the effect of (subcutaneous) immunization of DISC 1 (598-785) together with adjuvant (ABM-ZK, Linaris, as described) or adjuvant alone as control or only application of PBS as further control, examined for the basal behavior in the open field. The experimental conditions of the open field measurement were as in Example 1a, but the immunized rats were about one year old and the time was determined for the rats to remain in the center of the open field after 15 minutes.

The results are shown in FIG. 2, wherein the results are shown as black for control rats immunized with DISC 1 (n = 10 per group) and as white rats for control immunized rats (adjuvant only) and as white bars for PBS immunized rats , The results were statistically evaluated and rated as significant (univariate ANOVA F (2, 13) = 3,931; p = 0.05. Post hoc LSD for 15 min: DISC vs. pbs: p = 0.041, DISC vs. adjuvant: p = 0.030 ). Consistent with the results of Example 1a, the DISC1 -immunized rats are significantly less frequently in the center, which in turn demonstrates a behavioral effect of DISC1 immunization. c) Influence of DISC1 on the locomotion behavior of amphetamine-sensitized rats

The experiment was carried out essentially as described under a). The only difference was that the rats were additionally sensitized with amphetamine before conducting the experiment. Sensitization was performed 2 weeks prior to insertion into the Open-Field container by intraperitoneal (i.p.) administration of 2 mg / kg / rat D-amphetamine daily for five days (dissolved in saline, 1 ml / kg injection volume). After a treatment-free interval of 2 weeks, the open-field testing was then carried out 24 hours after a single injection of 0.5 mg / kg / rat D-amphetamine.

The results are shown in the bar graph shown in FIG. Here it can be seen that the amphetamine-sensitized rats without DISC1 treatment showed a significantly lower tendency to move than the rats that were given DISC1. In a more detailed analysis, the "center time" of each group was determined, i. the time the rats spend in the middle of the Open Field Box, which is a measure of anxiety-induced effects. This was identical for all groups, so it could be ruled out that the increased locomotion after immunization with DISC1 (598-785) is a fear-induced locomotion.

It has also been found that the repeated introduction of the preparation according to the invention had no toxic effect, since no side effects in the form of neurological symptoms, behavioral symptoms, disorders of grooming behavior, weight loss or the like were observed over the entire observation period (about 10 months). The application is therefore safe to call.

From these results it can be deduced that DISC1 effects a modulation of the neurochemical processes disturbed by amphetamine sensitization. It is of it assume that such a modulation can be exploited in the treatment of DISC-associated diseases. In particular, for example, depressions associated with a significantly decreasing tendency to activities or movements, a positive effect can be postulated.

Example 2: Influence of exogenous DISCI application on the anxiety behavior in healthy rats

Wistar rats were stereotactically inoculated with approximately 5 μg of recombinant DISCI (598-785) protein in the nucleus accumbens (stereotactic coordinates in the head frame (AP: 1.2 mm, ML: ± 0.8 mm, DV: -5.2 mm) Inoculation was used to study the bright field / dark field preference test and the open field test to measure anxiety-induced behavior In the bright field / dark field preference test, the rat is placed in a bright field (61 x 38 x 32 cm) connected to a neighboring dark field measured, which the rat spends in brightfield or darkfield.

Furthermore, amphetamine, the sensitivity to amphetamine was investigated in the open field experiment after a single intrperitoneal administration of 0.5 mg / kg body weight. Each study period was 15 minutes. The horizontal movement is measured automatically with the TruScan light barrier measuring system (Coulbourn Instruments, Allentown, USA).

The results are shown in Fig. 4B + C annotated below.

Figure 4A shows fluorescence microscopy of a brain section of the rat nucleus accumbens stereotactically inoculated with Dylight-labeled (red) recombinant DISCL 598-785. Counterstaining with anti-NeuN antibody, a neuron-specific marker protein. The picture shows that recombinant DISC1 protein was taken up in the cytosol of neurons. Bar 20 μι ^η ,

Fig. 4B shows a graph of the results of the light-dark preference test two weeks after bilateral inoculation of DyLight-labeled recombinant DISCI (598-785). or control (sham) in the nucleus accumbens of a wild-type rat. The diagram shows that inoculation with DISCI (598-785) has an anxiolytic effect because the residence preference is changed towards the bright field. F (I, 17) = 4,671; * p = 0.045 (ANOVA).

Fig. 4C shows a graph of the results of an open field experiment following a single, intraperitoneal amphetamine injection two weeks after bilateral inoculation of DyLight-labeled recombinant DISCI (598-785) or control (sham) into the nucleus accumbens of a wild-type rat. Inoculation with multimeric recombinant DISCL resulted in increased amphetamine sensitivity as hyperactivity was observed in these rats compared to the control. Y-axis: relative locomotion in relation to baseline before amphetamine in%. F (1, 17) = 4.5; * p = 0.049 (ANOVA).

Example 3: Examination of DISCl / 598-785 treated rats in

Bright field / dark field preference test

After immunization with DISCL / 598-785, or the controls (adjuvant or PBS phosphate buffered saline only), the rats were screened in with the bright field / darkfield preference test to measure anxiety-induced behavior. In the bright field / dark field preference test, the rat is placed in a bright field (61 x 38 x 32 cm), which is connected to an adjacent dark field. During 5 minutes, the time spent by the rat in the bright field or in the dark field is measured.

Fig. 5 shows diagrams showing the effect of DISCl immunization on the behavior in the bright field / dark field preference test (black bars, dark field, white bars, bright field).

A. It can be seen that the DISCl immunization causes a change in the relative preference of stay and duration of stay from the dark field to the bright field.

B. This effect (from A.) is clarified when from the same experiment the length of stay in the bright field is divided by those in the dark field. Example 4: Studies demonstrating the secretion of DISCI

A human neuroblastoma cell line (SHSY-5Y) was retrovirally transfected with the full-length human DISC1 gene (1-854) or a C-terminal human DISC1 fragment fused to the greenfluorescent protein (GFP) can be triggered by the addition of tetrycycline 1 ug / ml ("tet-on") in the cell culture medium (retroviral inducible transfection system pTRE-TIGHT from Clontech / Invitrogen, USA); Tetracycline-induced expression has the advantage over other transient expression systems that the cells do not die as a result of the transfection and thus cytosolic constituents pass conditionally into the cell culture supernatant through cell death. The cells were then induced for 2 days in serum-free medium with 1 .mu.l / .eta.1 tetracycline and the entire cell culture medium supernatant was finally centrifuged after two days to separate any existing dead cells precipitated with 7g ammonium sulfate / 10 ml of supernatant and separated on an SDS-PAGE gel and displayed in Western blot with standard methods and the antibody 14F2 against human DISC1 protein. As a control, the cell lysates were also examined.

Figure 6 shows a Western blot of supernatants (lanes 1-4) or cell lysates (lanes 5-8). The even-numbered lanes 2, 4, 6 and 8 show tetracycline-induced, non-even-numbered lanes 1, 3, 5, 7 uninduced human SHSY-5Y cells. Lanes 2 and 6 show full length human DISC1 protein or human DISC1 (598-854) fragment (lanes 4 and 8), c-terminally fused to green-fluorescent protein (GFP).

It can be seen that large amounts of DISCl immunoreactivity are found in the supernatant (compare corresponding lanes "induced" DISCl full length (VL) or GFP DISCl (598-854) vs. "not induced"). Since no dead cells were observed during the gentle induction with tetracycline, and the amount of immunoreactivity can not be explained by a few dead cells, it is concluded that DISCl is surprisingly secreted from the cells.

Claims

claims:

1. A preparation containing DISC1 and / or at least one DISC1 fragment for use in the therapeutic treatment or modulation of neurochemical processes of the human or animal body or in the therapeutic treatment for influencing the proliferation or migration of cells in the human or animal body.

2. A preparation according to claim 1, characterized in that it is formulated for subcutaneous, intramuscular, intravenous, intracerebral, intranasal or intraperitoneal administration.

3. Preparation according to claim 1 or 2, characterized in that it additionally contains at least one immunostimulating substance.

4. Preparation according to one of claims 1-3, characterized in that it is the at least one fragment to DISC1 -598-785.

5. Preparation according to one of claims 1-4, for use as a sedative in animals and humans.

6. Preparation according to any one of claims 1-5 for the treatment of animals and humans whose neurochemical processes are disturbed.

7. A preparation according to claim 6 for use in the treatment of disorders of Dopaminstoffwechsels or in the modulation of Dopaminstoffwechsels.

8. A preparation according to any one of claims 1-4 for use in the treatment of addictions, disorders of indulgence or anhedonia.

9. A preparation according to claim 7, for use in on the peripheral Dopaminstoffwechsel declining diseases such as in particular cardiovascular diseases, obesity or sexual dysfunction.

10. A preparation according to any one of claims 1 -7 for use in the treatment of impaired or impaired memory, in particular of mild cognitive impairment or Alzheimer's disease.

11. A preparation according to any one of claims 1-7 for use in the treatment of side effects of drugs or substances that affect the dopamine metabolism, or in acute poisoning by substances.

12. A preparation according to any one of claims 1-4 for use in the treatment of cancer.

13. A composition according to any one of claims 1-4 for use in the treatment of autoimmune diseases such as multiple sclerosis.

14. A composition according to any one of claims 1-4 for use in influencing or delaying effects of biological aging.