WO1999038516A1

WO1999038516A1 - Neurotrophic properties of ipgs and ipg analogues

Info

Publication number: WO1999038516A1
Application number: PCT/GB1998/003847
Authority: WO
Inventors: Thomas William Rademacher; Hugo Norberto Caro; Manuel Martin-Lomas; Isabel Varela Nieto; Yolanda Leon Alvarez
Original assignee: Rademacher Group Limited
Priority date: 1998-01-29
Filing date: 1998-12-21
Publication date: 1999-08-05
Also published as: AU1770899A; JP2002501899A; GB9801899D0; EP1064003A1; CA2318584A1

Abstract

Inositolphosphoglycans (IPGs) or inositol-containing IPG analogues can be used to specifically cause neuron proliferation or neuron differentiation, and in particular neurite outgrowth. P-type IPGs or chiro-inositol containing analogues cause neurite outgrowth and A-type IPGs or myo-inositol containing analogues cause neuron proliferation. Compositions comprising these agents and their medical uses are also disclosed.

Description

Neurotrophic Properties of IPGs and IPG Analogues

Field of the Invention

The present invention relates to the neurotrophic properties of inositolphosphoglycans (IPGs) and IPG analogues, and in particular to the findings that P-type IPGs or chiro-inositol containing IPG analogues promote neurite growth, and A- type IPGs or myo-inositol containing IPG analogues promote neuron proliferation.

Background of the Invention

Many of the actions of growth factors on cells are thought to be mediated by a family of inositol phosphoglycan (IPG) second messengers (Rademacher et al, 1994) . It is thought that the source of IPGs is a "free" form of glycosyl phosphatidylinositol (GPI) situated in cell membranes. IPGs are thought to be released by the action of phosphatidylinositol -specific phospholipases following binding of growth factors to receptors on the cell surface. There is evidence that IPGs mediate the action of a large number of growth factors including insulin, nerve growth factor, hepatocyte growth factor, insulin-like growth factor I (IGF-I) , fibroblast growth factor, transforming growth factor β, the action of IL-2 on B-cells and T-cells, ACTH signalling of adrenocortical cells, IgE, FSH and hCG stimulation of granulosa cells, thyrotropin stimulation of thyroid cells, cell proliferation in the early developing ear and rat mammary gland.

Soluble IPG fractions have been obtained from a variety of animal tissues including rat tissues (liver, kidney, muscle brain, adipose, heart) and bovine liver. IPG biological activity has also been detected in malaria parasitized RBC and mycobacteria. We have divided the family of IPG second messengers into distinct A and P- type subfamilies on the basis of their biological activities. In the rat, release of the A- and P-type mediators has been shown to be tissue-specific (Kunjara et al , 1995 ) .

W098/11116 and W098/11117 disclose the purification, isolation and characterisation of P and A- type IPGs from human tissue. Prior to these applications, it had not been possible to isolate single components from the tissue derived IPG fractions, much less in sufficient quantities to allow structural characterisation. Accordingly, while some prior art studies describe the biological activities of the IPG containing fractions, speculation as to the identity of the active components from non-human sources of the fractions were based on indirect evidence from metabolic labelling and cleavage techniques .

IGF-I together with the nerve growth factor family of neurotrophins have been shown to control growth and differentiation in the developing inner ear. It has also been shown that fractions containing A-type mediators can stimulate cell proliferation in chick cochleovestibular ganglia (CVG) . The vertebrate inner ear develops from the embryonic otic vesicle. This is a transient structure that undergoes a distinct period of cell proliferation that precedes the differentiation of the various cell types that populate the adult ear. The cochleovestibular ganglion develops pari pasu with the formation of the otic vesicle. It originates from the otic placode and contains the afferent neurones that connect the sensory epithelium of the inner ear to the central nervous system.

Summary of the Invention

The present invention is based on the finding that inositolphosphoglycans (IPGs) or inositol -containing IPG analogues can be used to specifically cause neuron proliferation or neuron differentiation, and in particular neurite outgrowth. Neurite outgrowth is the phenomenon by which neurons develop processes (axons or dendrites) from their cell bodies. In contrast, neuron proliferation is the process by which neurons increase in number by cell division.

The experiments leading to the present invention investigated the neurotrophic effects of two types of inositol phosphoglycan preparations (type-A and type-P) from rat liver, and their biological effects in comparison with those of insulin- like growth factor- I in organotypic cultures of chicken embryo cochleovestibular ganglion (CVG) . Proliferative biological effects were stimulated by either an inositol phosphoglycan type-A or a myo- inositol -containing inositol phosphoglycan analogue, as measured by thymidine incorporation and proliferating cell nuclear antigen expression (PCNA) . In contrast, differentiation was observed after treatment with either the inositol phosphoglycan type-P or a chiro- inositol -containing inositol phosphoglycan analogue, as measured by neurite outgrowth and expression of the neuron specific antigen G4. In addition, the effects of the compounds were also investigated in neuron cell culture, which unlike the CVG cultures, are substantially pure cultures of neurons, i.e other cell types are not present.

Accordingly, in a first aspect, the present invention provides the use of a P-type inositolphosphoglycan (IPG) or a chiro- inositol containing IPG analogue in the preparation of a medicament for promoting neurite growth.

The P-type IPGs can be obtained using the methods described in Caro et al, 1997. The chiro- inositol containing IPG analogue used in the experiments below (referred to as compound C4) can be made according to the synthesis described in Jaramillo et al, 1994. In a further aspect, the present invention provides a method for promoting neurite growth, the method comprising exposing neurons to a P-type IPG or a chiro- inositol containing IPG analogue. The method is applicable both in vitro or in vivo.

In a further aspect, the present invention provides the use of an A- type inositolphosphoglycan (IPG) or a myo- inositol containing IPG analogue in the preparation of a medicament for promoting neuron proliferation.

The A-type IPGs can be obtained using the methods described in Caro et al, 1997. The myo- inositol containing IPG analogue used in the experiments below (referred to as compound C3) can be made according to the synthesis described in Zapata et al, 1994, and Jaramillo et al, 1994.

In a further aspect, the present invention provides a method for promoting neuron proliferation, the method comprising exposing neurons to an A-type IPG or a myo- inositol containing IPG analogue. The method is applicable both in vitro or in vivo.

While the prior art describes experiments in which fractions containing A-type IPGs and compound C3 are tested in a CVG assay, the results of these experiments do not unambiguously show that these compounds cause neuron proliferation. As mentioned above, the CVG contains a variety of different cell types, and so it is not possible to distinguish the proliferative response of neurons from that of any other cell type present it. Thus, until the experiments on pure neuron cultures described below, the role of A-type IPGs and compound C3 in causing neuron proliferation was uncertain. In contrast, as neurons are the only cell type present in a CVG which can undergo neurite outgrowth, the results described herein unambiguously demonstrate these properties of the P-type IPGs or chiro-inositol containing IPG analogues.

The above compounds have applications in the prophylactic or therapeutic treatment of any conditions where neurite growth or neuron proliferation are required. The neurons may be central (brain and spinal cord) neurons, peripheral (sympathetic, parasympathetic, sensory and enteric) neurons, or motor neurons. Compositions comprising one or more of the above compounds can be used in the treatment of damage to the nervous system, motor neuron disease, neurodegenerative disorders or neuropathy. The pharmaceutical uses and formulation of the compounds is discussed in more detail below.

The present invention will now be described by way of example and not by limitation with reference to the accompanying drawings .

Brief Description of the Drawings

Figure 1: Effect of purified and chemically synthesised inositol phosphoglycans on CVG proliferation.

A. Photomicrographs showing CVG cellular proliferation under the following conditions: control medium with no foetal calf serum (OS), C3 (10 μ ) C4 (10 μm) , IGF-I (1 nM) , IPG type-A (1/100) and IPG type-P (1/100) . Calibration bar, 150 um.

B. Acid-precipitable [³H] thymidine incorporation into CVG cultured in the presence of increasing concentrations of C3 (closed circles) and C4 (open circles) from 0.1 to 10 μm. The bars to the right indicate the [³H] thymidine uptake in CVGs cultured in parallel experiments with IPG type-A (1/100) and IPG type-P (1/100) . Data are representative of at least 4 different experiments with 6 an average of 5 CVGs per condition.

C. Expression of PCNA within the CVG after treatment with OS, IGF-I (1 nM) , C3 (10 μm) , C4 (10 μm) , IPG-A (1/100) and IPG-P (1/100) . The resulting autoradiogram was quantitated by densitometry and the fold change in PCNA protein is indicated below each lane. Data are representative of at least 4 blots per data point with 2 CVGs per point.

Figure 2: Stimulation of neurite outgrowth by chiro- inositol phosphoglycans in CVG.

A. Appearance of CVG cultures in multidishes coated with a substrate of Collagen G for 24 hours in control medium (OS) or in the presence of C3 (10 μm) , C4 (10 μm) , IPG-A (1/100) , IPG-P (1/100) and IGF-I (1 nM) . Calibration bar, 225 μm.

B. Quantification of neurite extension by analysis of G4 expression within CVGs after treatment with OS, IPG-A (1/100), IPG-P (1/100), C3 (10 μm) , C4 (10 μm) and IGF-I (1 nM) . The histograms represent the densitometrical analysis of the induction of G4 expression in CVGs cultured under the same treatments as indicated above. Data are representative for at least 6 different blots with 2 CVGs per point. The inserts show a typical Western blot developed with the antibody anti-G4.

Detailed Description

IPGs and IPG Analogues

Studies have shown that A-type mediators modulate the activity of a number of insulin-dependent enzymes such as cAMP dependent protein kinase (inhibits) , adenylate cyclase (inhibits) and cAMP phospho-diesterases

(stimulates) . In contrast, P-type mediators modulate the activity of insulin-dependent enzymes such as pyruvate 7 dehydrogenase phosphatase (stimulates) and glycogen synthase phosphatase (stimulates) and cAMP dependent protein kinase (inhibits). The A-type mediators mimic the lipogenic activity of insulin on adipocytes, whereas the P-type mediators mimic the glycogenic activity of insulin on muscle. Both A-and P-type mediators are mitogenic when added to fibroblasts in serum free media. The ability of the mediators to stimulate fibroblast proliferation is enhanced if the cells are transfected with the EGF -receptor. A-type mediators can stimulate cell proliferation in the chick cochleovestibular ganglia.

Soluble IPG fractions having A-type and P-type activity have been obtained from a variety of animal tissues including rat tissues (liver, kidney, muscle brain, adipose, heart) and bovine liver. A- and P-type IPG biological activity has also been detected in human liver and placenta, malaria parasitized RBC and mycobacteria. The ability of an anti-inositolglycan antibody to inhibit insulin action on human placental cytotrophoblasts and BC3H1 myocytes or bovine-derived IPG action on rat diaphragm and chick cochleovestibular ganglia suggests cross -species conservation of many structural features. However, it is important to note that although the prior art includes these reports of A- and P-type IPG activity in some biological fractions, the purification or characterisation of the agents responsible for the activity is not disclosed.

A-type substances are cyclitol-containing carbohydrates, also containing Zn²⁺ ion and optionally phosphate and having the properties of regulating lipogenic activity and inhibiting cAMP dependent protein kinase. They may also inhibit adenylate cyclase, be mitogenic when added to EGF-transfeeted fibroblasts in serum free medium, and stimulate lipogenesis in adipocytes. P-type substances are cyclitol -containing carbohydrates, also containing Mn²⁺ and/or Zn²⁺ ions and optionally phosphate and having the properties of regulating glycogen metabolism and activating pyruvate dehydrogenase phosphatase. They may also stimulate the activity of glycogen synthase phosphatase, be mitogenic when added to fibroblasts in serum free medium, and stimulate pyruvate dehydrogenase phosphatase.

Methods for obtaining A-type and P-type IPGs are set out in Caro et al, 1997, and in W098/11116 and W098/11117.

The present invention also relates to inositol -containing IPG analogues, prepared using synthetic organic chemistry methods. Thus, by way of example, compound C3 , ID- 6-0- (2-amino-2-deoxy-α-D-glucopyranosyl) -myo- inositol 1,2- (cyclic phosphate) , has been prepared previously, see Zapata et al, 1994. Compound C4, 1D-6 -0- (2 -amino-2 - deoxy- -D-glucopyranosyl) -chiro-inositol 1-phosphate can be synthesised as described in Jaramillo et al, 1994.

Pharmaceutical Compositions

The mediators and analogues of the invention can be formulated in pharmaceutical compositions. These compositions may comprise, in addition to one or more of the mediators, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non- toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes .

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Preferably, the pharmaceutically useful compound according to the present invention is given to an individual in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. Typically, this will be to cause a therapeutically useful amount of neurite growth or neuron proliferation, or the prevention of a useful amount of neuron damage. The actual amount of the compounds administered, and rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of 10 administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. (ed) , 1980.

As mentioned above, the IPGs and IPG analogues can be used in the treatment of all conditions where neurite growth or neuron proliferation are required. The neurons may be central (brain and spinal cord) neurons, peripheral (sympathetic, parasympathetic, sensory and enteric) neurons, or motor neurons. Compositions comprising one or more of the above compounds can be used in the treatment of damage to the nervous system, motor neuron disease, neurodegenerative disorders or neuropathy. Damage to the nervous system includes the result of trauma, stroke, surgery, infection (e.g. by viral agents), ischemia, metabolic disease, toxic agents, or a combination of these or similar causes. Motor neuron conditions include conditions involving spinal muscular atrophy, paralysis or amyotrophic lateral sclerosis. Neurodegenerative disorders include Parkinson's disease, Alzheimer's disease, epilepsy, multiple sclerosis, Huntingdon's chorea and Meniere's disease.

Methods of Screening Candidate Compounds

Candidate synthetic compounds can tested using the assays disclosed herein, or similar assays, to determine whether the analogues have the property of causing neurite outgrowth or neuron proliferation. Such testing may include the use of IGF-I or compounds C3 or C4 as positive controls and/or an unrelated factor as a negative control. In one embodiment, the method involves exposing a culture including neurons to the candidate compound and determining whether neuron proliferation or differentiation occurs. The results of such assays can then be used to select candidate compounds with the 11 desired property, and optionally that property at a magnitude indicating that it could be suitable for further testing as a lead compound.

The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a lead compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, eg peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property.

There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its pharmacophore .

Once the pharmacophore has been found, its structure is modelled to according its physical properties, eg stereochemistry, bonding, size and/or charge, using data from a range of sources, eg spectroscopic techniques, X- ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process . 12

In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.

A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

Examples

The experiments described below evaluate the biological effects of both families of purified rat liver IPG and those of the IPG analogues 1D-6-0- (2-amino-2-deoxy-α-D- glucopyranosyl) -myo-inositol 1,2-cyclic phosphate (termed C3) and ID- 6 -0- (2 -amino-2 -deoxy-α-D-glucopyranosyl) - chiro- inositol 1 -phosphate (termed C4) on CVG growth and differentiation. The effects of the IPGs on neuron cells in culture is also evaluated.

Materials

Human insulin-like growth factor-I (IGF-I) was purchased from Boehringer Mannheim (Mannheim, Germany) .

[³H] thymidine (specific activity 20-30 Ci/mmol) was obtained from Amersham International (Aylesbury, UK) . A mouse monoclonal anti-proliferative cell nuclear antigen

(PCNA) antibody, used at a 1:2000 dilution, was obtained 13 from ATOM (Barcelona, Spain) . The mouse monoclonal anti- G4 -antibody, used at a 1:5000 dilution, was a generous gift from Dr Enrique J de la Rosa (CIB-CSIC, Madrid, Spain) . Secondary antibodies conjugated with peroxidase were purchased from Bio-Rad (CA, USA) . Cell culture vessels were obtained from NUNC (Roskilde, Denmark) . All other reagents were of analytical grade or better.

Organotypic culture of CVG and estimation of neurite outgrowth

Chicken embryos were obtained from fertilised eggs (Granja Rodriguez Serrano, Salamanca, Spain) that were incubated at 38°C in a humidified atmosphere. The embryos were staged according to Hamburger and Hamilton criteria, see Hamburger et al, 1951. Chicken embryos

(from stage 19-20) were removed from eggs and placed in 35 mm Petri dishes containing phosphate buffered saline (PBS) . CVGs were aseptically dissected as previously described in Bernd et al, (1989). Explanted CVGs were cultured in four-well multidishes coated with a substrate of collagen G (Biochrom, Berlin, Germany) prepared according to the manufacturer's procedure. Typically, culture medium was Fl2/Dulbecco' s modified Eagle medium (Biochrom, Berlin, Germany) containing lOOμg/ml transferrin, 16μg/ml putrescine, 6 ng/ml progesterone, 5.2 ng/ml sodium selenite (all from Sigma, 50 IU/ml penicillin and 50μg/ml streptomycin (Biochrom, Berlin, Germany) . CVG explants were cultured in control medium or medium containing test substances for 24 hours before photographs were taken using a Nikon TMS microscope equipped with a Nikon HFX-DX camera.

Determination of DNA synthesis

CVG explants were cultured in medium containing [³H] thymidine (0.2 mM, 10 μCi/ml) during 24 hours.

Incubations were carried out at 37°C in a water-saturated atmosphere containing 5% C0₂. The explants were then 14 individually rinsed three times with ice-cold PBS. Explants were extracted with 10% trichloroacetic acid and radioactivity determined by liquid scintillation counting (OptiPhase "HiSafe" liquid scintillation cocktail, Wallac, Turku, Finland) .

Synthesis of IPG analogues and purification of IPG subtypes

C3 and C4 were prepared as previously described in Zapata et al, 1994, and Jaramillo et al, 1994. Aqueous stock solutions were stored at -20°C until used. IPG types -A and -P were prepared from rat liver according to Nestler et al, 1991, with minor modifications Caro et al, 1997. IPG type-A was further purified using C18 reverse-phase chromatography. Briefly, IPG type-A was resuspended in 200μl distilled water before loading onto a C18 reverse - phase cartridge (Waters, MA, USA) which had been previously washed with methanol (5 ml) and distilled water (10 ml) . The run through and a 5 ml distilled water wash were collected together and subsequently lyophilised twice. IPG type-P was also subject to an additional purification step. This involved resuspending in 200μl of butanol/ethanol water (4/1/1,v/v/v) before loading onto a cellulose column (1 ml bed volume) equilibrated with the above solvent mixture. The run through was followed by a 4 ml wash with the same solvent mixture. After washing with methanol (4 mis) , the column was eluted with distilled water (4 mis) . This latter fraction was lyophilised twice. Both types of IPG were stored at -70°C until used. For analyses, aliquots of each type of mediator were made by dissolving the amount of material obtained from 18g (wet weight) of tissue in 200μl of Hank's solution (stock) and adjusted to pH 7.0 with 1 M KOH.

Western blotting

CVG explants were homogenised in sodium dodecyl sulphate- 15 polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer with 1 mM phenylmethylsulfonylfluoride and then frozen immediately. Gels were loaded with solutions containing equal amounts of proteins, typically two CVGs per condition. CVG proteins were resolved using 6% SDS- PAGE before being electrophoretically transferred onto PVDF membranes (Dupont-NEN,MA, USA) . Filters were blocked with Tris -buffered saline containing 5% (wt/vol) non-fat dried milk and incubated with the primary specific antibody. Filters were subsequently washed and incubated with the corresponding secondary antibody conjugated with peroxidase. Bound peroxidase activity was visualised by chemiluminescence (Dupont-NEN,MA,USA) and quantified by densitometric scanning. The mouse monoclonal anti-G4 antibody recognises an axonal glycoprotein expressed by neurones, Rathjen et al, 1987.

Neuron cell culture

Freshly dissociated cell populations enriched in small cerebellar neurons (-99% pure) were prepared from 5- to 7 -day-old ICR mice (Schnitzer and Schachner, 1981; Keilhauer et al, 1985) . Cells were plated at a concentration of 2 x 10⁶ cells/ml onto glass coverslips in serum- free, hormone -supplemented medium (Fischer et al, 1986) containing 25 mM Hepes (50 μl per 16 -mm diameter coverslip) . Coverslips were coated either with poly-L- lysine (10 μg/ml H₂0) or laminin from Engelbreth-Holm- Swarm sarcoma (Boehringer Mannheim Corp.; 20 μg/ml in basal medium Eagle's). Three coverslips were then placed in 35 -mm diameter tissue culture plastic Petri dishes (Nunc) . The medium was removed by gentle suction 3-4h after plating of cells on laminin or 20h after plating of cells on poly-L-lysine. The IPG or IPG analogues were then added in 50 μl medium per coverslip. Cultures were examined 24 and 48h later by phase contrast microscopy. 16

Results

During the early stages of inner ear development cell proliferation, differentiation and apoptosis are modulated by neurotrophic factors and members of the insulin family of hormones and growth factors.

Organotypic cultures of CVG explants have proved to be a good experimental model to dissect, in defined medium, of the biological actions of these growth factors and their putative second messengers. These investigations examined the differences in the biological activity between the two families of IPG (type-A and type-P) , in conjunction with related chemically synthesised IPG analogues C3 and C4 (containing myo- and chiro-inositol, respectively) , in cultured explants of chicken embryo CVG and in a pure culture of neurons.

In the first instance, experiments were designed to determine the growth promoting effects of the various agents under test. Figure 1, panel A shows that IPG type-A and the myo-inositol-containing compound C3 were able to induce growth of the CVG resulting in a observable size increase. IGF-I effects are shown for comparison. This was not the case with either IPG type-P or the chiro- inositol -containing compound C4. Cell proliferation of the CVG was quantified by measuring thymidine incorporation into DNA. These results are shown in panel B, both IPG type-A and C3 but not IPG type-P nor C4 were able to increase [³H] thymidine uptake by the CVG. These results indicate that IPG type-A and C3 mimic IGF-I effects on cell growth. Furthermore, IGF- I promotes a fast hydrolysis of a GPI lipidic precursor and IGF-I effects are blocked by an anti-IPG antibody.

Additional data supporting the growth promoting activity of C3 and IPG A-type is shown in Figure 1, panel C. The proliferating-cell nuclear antigen (PCNA) is a nuclear protein of 36 kD association with the cell cycle and is 17 essential for DNA synthesis. Hence, the level of expression of PCNA was used as a marker for cell proliferation within the CVG after the different treatments. IGF-I, as expected, increased the level of the expression of PCNA by over four-fold. IPG type-A and C3 also augmented the levels of PCNA about two-fold when compared to control cultures of CVG. Both IPG type-P and C4 caused no appreciable change in the expression of PCNA, in agreement to the results in panels A and B.

Ganglia were isolated at stage 19-20 and cultured on a collagen substrate under different conditions. Figure 2A illustrates one of these experiments in which ganglia were incubated in control medium (OS) or with IGF-I. Although a low rate of spontaneous neurite outgrowth was present in control ganglia, the number of neurites was far more abundant in IGF-I- treated ganglia.

Effects of IPGs and the synthetic analogues were also studied in CVG neurones to test for their ability to induce neuronal differentiation. Figure 2 illustrates the differential effects of IGF-I and IPG type A and C3 on G4 expression. IGF-I strongly induced G4 whereas IPG type-A and C3 did not (Figure 2A) . In contrast, both IPG type-P and C4 promoted extensive neurite outgrowth

(Figure 2A) . Figure 2B illustrates the immunodetection by Western blotting of the G4 epitope in explanted ganglia incubated with medium only (OS) or with InM IGF-I and revealed that IGF- I was the strongest agent to stimulate the expression of G4 (almost a three- fold increase over control levels) . This increase in G4 expression was mimicked, albeit to a lesser degree, by IPG type-P and by C4. No significant increases were observed using IPG type-A nor by using C3 , consistent with the results in panel A. Average values from different experiments are shown in the histogram presented in Figure 2B. The CVG is formed by the migration of neuroblasts from the otic vesicle. The process is completed by stage 20 and parallelled by an intense mitogenic activity that generates terminal neuroblasts. Proliferation of the CVG is under the control of neurotrophic factors and is strongly stimulated by phospholipase C-generated IPG (i.e. containing a 1,2 cyclic phosphate) and IGF-I. In addition to its mitogenic effect, IGF-I displays a distinct differentiative effect. It promotes neurite outgrowth and expression of the neuronal differentiation marker G4. The chicken G4 antigen is a calcium- independent cell -cell adhesion molecule that is related to the mouse Ll group of antigens which are functionally involved in axon bundle formation. An increase in G4 expression is associated with the differentiation of neural precursors in the chicken retina. IGF-I, but not other mitogens such as IPG type-A or NGF, was able to strongly induce G4.

As the CVG comprises a variety of cell types, it was important to further investigate the effects of the IPGs and IPG analogues in pure neuron cell culture. In the absence of insulin, the neurons show significantly reduced levels of neurite outgrowth. A-type IPG (or a myo-inositol containing IPG analogue) showed proliferation effects after 2 days in culture. When P- type IPG (or a chiro-inositol containing IPG analogue) was added once after plating the cells, it was found to be effective in stimulating neurite outgrowth as effectively as an insulin control after 3 days in culture, and better at causing neurite outgrowth than the A-type IPG.

In summary, these experiments investigating the biological effects of IPGs and IPG analogues on neurons in comparison to IGF-I and insulin demonstrate for the first time that proliferative biological effects are 99/38516

19 stimulated by A-type IPGs or a myo -inositol containing IPG analogue, whereas differentiation was observed after treatment with either P-type IPGs or a chiro- inositol containing IPG analogue.

20

References :

The references mentioned herein are all expressly incorporated by reference.

Rademacher et al, Brazilian J. Med. Biol. Res., 27:327- 341, 1994.

Caro et al, Biochem. Molec. Med., 61:214-228, 1997.

Kunjara et al, In: Biopolymers and Bioproducts:

Structure, Function and Applications, Ed Svati et al, 301-305, 1995.

Zapata et al, Carbohydrate Res., 264; 21 -31, 1994.

Jaramillo et al, J. Org. Chem, 59:3135-3141, 1994.

Hamburger and Hamilton, J. Morphol., 88:49-92, 1951.

Bernd and Represa, Dev. Biol., 134:11-20, 1989.

Nestler et al, Endocrinology, 129:2951-2956, 1991.

Rathjen et al, J. Cell. Biol., 104:343-353, 1987.

Hernandez -Sanchez et al, P.N.A.S. USA, 92:9834-9838, 1995.

Schnitzer and Schnacher, J. Neuroimmunol . , 1:429-456, 1981.

Keilhauser et al, Nature, 316:728-730, 1985.

Fischer et al, J. Neurosci. , 6 :605-612, 1986.

Claims

21Claims :

1. Use of a P-type inositolphosphoglycan (IPG) or a chiro- inositol containing IPG analogue in the preparation of a medicament for promoting neurite growth of neurons.

2. The use of claim 1 wherein the chiro- inositol containing IPG analogue is 1D-6 -0- (2 -amino-2 -deoxy-╬▒-D- glucopyranosyl) -chiro-inositol 1-phosphate.

3. Use of an A-type inositolphosphoglycan (IPG) or a myo-inositol containing IPG analogue in the preparation of a medicament for promoting neuron proliferation.

4. The use of claim 3 wherein the myo-inositol containing IPG analogue is 1D-6 -0- (2 -amino-2 -deoxy-╬▒-D- glucopyranosyl) -myo-inositol 1,2- (cyclic phosphate).

5. The use of any one of the preceding claims wherein the medicament is for the treatment of damage to the nervous system, motor neuron disease, neurodegenerative disorders or neuropathy.

6. A method for promoting neurite growth in vi tro, the method comprising exposing neurons to a P-type IPG or a chiro-inositol containing IPG analogue.

7. A method for promoting neuron proliferation in vi tro, the method comprising exposing neurons to an A- type IPG or a myo-inositol containing IPG analogue.

8. A pharmaceutical composition comprising lD-6-0-(2- amino-2 -deoxy-╬▒-D-glucopyranosyl) -chiro-inositol 1- phosphate or ID-6 -0- (2 -amino-2 -deoxy-╬▒-D-glucopyranosyl) - myo-inositol 1,2- (cyclic phosphate), in combination with a pharmaceutically acceptable carrier.

9. A method of testing a candidate chiro-inositol 22 containing compound for the property of promoting neurite growth, the method comprising exposing a cell culture including neurons to the candidate chiro-inositol containing analogue and determining whether neurite growth occurs .

10. A method of testing a candidate myo-inositol containing compound for the property of promoting neuron proliferation, the method comprising exposing a cell culture including neurons to a candidate myo-inositol containing analogue and determining whether neuron proliferation occurs.