WO2019048851A1

WO2019048851A1 - Treatment of neurodegenerative diseases

Info

Publication number: WO2019048851A1
Application number: PCT/GB2018/052513
Authority: WO
Inventors: Peter Richardson; David Sheppard
Original assignee: BenevolentAI Bio Ltd
Current assignee: BenevolentAI Bio Ltd
Priority date: 2017-09-06
Filing date: 2018-09-05
Publication date: 2019-03-14
Anticipated expiration: 2020-03-06
Also published as: GB201714303D0

Abstract

L'invention concerne des méthodes pour la prévention et le traitement de maladies neurodégénératives, en particulier de maladies des motoneurones telles que la sclérose latérale amyotrophique (ALS), ainsi que des compositions et des préparations combinées destinées à une utilisation avec les méthodes. Les méthodes consistent à inhiber la CDK7 dans le système nerveux central d'un sujet nécessitant une telle prévention ou un tel traitement.The invention provides methods for the prevention and treatment of neurodegenerative diseases, particularly motor neuron diseases such as amyotrophic lateral sclerosis (ALS), as well as compositions and combined preparations for use with the methods. The methods involve inhibiting CDK7 in the central nervous system of a subject in need of such prevention or treatment.

Description

Treatment of Neurodegenerative Diseases

This invention relates to the treatment of neurodegenerative diseases, in particular motor neuron diseases such as amyotrophic lateral sclerosis, and to compositions or combined preparations for use in the methods.

The motor neuron diseases (MNDs) are a group of progressive neurological disorders that destroy motor neurons, the cells that control essential voluntary muscle activity such as speaking, walking, breathing, and swallowing. Normally, messages from nerve cells in the brain (upper motor neurons) are transmitted to nerve cells in the brain stem and spinal cord (lower motor neurons) and from them to particular muscles. Upper motor neurons direct the lower motor neurons to produce movements such as walking or chewing. Lower motor neurons control movement in the arms, legs, chest, face, throat, and tongue.

When there are disruptions in the signals between the lowest motor neurons and the muscle, the muscles do not work properly; the muscles gradually weaken and may begin to waste away and develop uncontrollable twitching (called fasciculations). When there are disruptions in the signals between the upper motor neurons and the lower motor neurons, the limb muscles develop stiffness (called spasticity), movements become slow and effortful, and tendon reflexes such as knee and ankle jerks become overactive. Over time, the ability to control voluntary movement can be lost.

MNDs are classified according to whether they are inherited (familial) or sporadic, and to whether degeneration affects upper motor neurons, lower motor neurons, or both. In adults, the most common MND is amyotrophic lateral sclerosis (ALS), which affects both upper and lower motor neurons. It has inherited and sporadic forms and can affect the arms, legs, or facial muscles. Primary lateral sclerosis (PLS) is a disease of the upper motor neurons, while progressive muscular atrophy (PMA) affects only lower motor neurons in the spinal cord. In progressive bulbar palsy (PBP), the lowest motor neurons of the brain stem are most affected, causing slurred speech and difficulty chewing and swallowing. There are almost always mildly abnormal signs in the arms and legs.

Table 1 . Classification of Motor Neuron Diseases

Primary lateral sclerosis (PLS) Yes No

Progressive muscular atrophy (PMA) No Yes

Progressive bulbar palsy (PBP) No

Yes, bulbar region

Pseudobulbar palsy Yes, bulbar region No

ALS is a progressive, ultimately fatal disorder that disrupts signals to all voluntary muscles. The terms motor neuron disease and ALS are often used interchangeably. ALS most commonly strikes people between 40 and 60 years of age, but younger and older individuals also can develop the disease. Men are affected more often than women. Familial forms of ALS account for 10 per cent or less of cases of ALS, with more than 10 genes identified to date. However, most of the gene mutations discovered account for a very small number of cases. The most common familial forms of ALS in adults are caused by mutations of the superoxide dismutase gene, or SOD1 , located on chromosome 21 .

There is no cure or standard treatment for ALS or the other MNDs. Riluzole (Rilutek®), the only prescribed drug approved by the U.S. Food and Drug Administration to treat ALS, prolongs life by 2-3 months but does not relieve symptoms, and has undesirable side effects such as nausea and fatigue. The drug reduces the body's natural production of the neurotransmitter glutamate, which carries signals to the motor neurons. It is believed that too much glutamate can harm motor neurons and inhibit nerve signalling. There remains, therefore, an urgent need for improved treatment of ALS, and other neurodegenerative diseases.

The mammalian central nervous system (CNS) is considered to be immunologically privileged, with relatively few resident immune cells and a highly specific blood-brain barrier (BBB). However, considerable evidence supports the presence of immune and inflammatory abnormalities in neurodegenerative diseases. Neuroinflammation is characterised by the activation and proliferation of microglia (microgliosis), astrogliosis, and infiltrating immune cells. It is a pathological characteristic of many neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and ALS. Neuroinflammatory responses can be beneficial or harmful to motor neuron survival. These distinct effects are elicited by the different activation states of microglia/macrophages and astrocytes, and are modulated by infiltrating T cells (Zhao et ai, J Neuroimmune Pharmacol. 2013; 8(4): 888-899). Microglia act as the first line of immune defence in the CNS, surveying the surrounding environment through their processes. Microglia are sensitive to pathological changes in the CNS and respond to danger signals from damaged tissue. During the early stage of motor neuron injury in ALS, it is believed that repair signals from motor neurons induce activation of microglial cells to an M2 phenotype. M2 microglia release neuroprotective factors (such as neurotrophic and anti-inflammatory factors) to repair motor neurons and protect against further injury. Astrocytes also participate in the neuroprotective process by secreting neurotrophic factors. As the disease progresses, injured motor neurons release danger signals that transform microglia to a cytotoxic M1 phenotype. M1 microglia release proinflammatory cytokines (such as tumor necrosis factor a, TNF-a, and interleukin-1 β, I L- 1 β , ) , and promote neurotoxicity by releasing reactive oxygen species. These pro-inflammatory cytokines further activate microglia leading to excessive neurotoxicity. M1 microglia also promote astrocyte activation. Activated astrocytes acquire deleterious inflammatory phenotypes with release of reactive oxygen species and pro-inflammatory cytokines, which in turn further induce microglial activation and enhance motor neuron degeneration. The activated glial cells also recruit peripheral monocytes/macrophages and T cells into the CNS, which further exacerbate motor neuron degeneration. The neuroinflammatory response in ALS is reviewed by Zhao et al., supra, and by Lewis et al. (Neurology Research International, 2012, Article ID 803701 ).

Cyclin dependent kinase 7 (CDK7) is a serine/threonine kinase involved in ceil cycle control, RNA polymerase !i-mediated RNA transcription and p53 activation (and thus apoptosis). CDK7 is the catalytic subunit of the CDK-activating kinase (CAK) complex. Cyclin- dependent kinases (CDKs) are activated by the binding to a cyclin and mediate the progression through the ceil cycle. CAK activates the cyclin-associated kinases CDK1 , CDK2, CDK4 and CDK8 by threonine phosphorylation, thus regulating cell cycle progression. CAK also activates RNA polymerase H by serine phosphorylation of the repetitive C-terminal domain (CTD) of its large subunit (POLR2A), allowing elongation of transcripts. Upon DNA damage, CDK7 triggers p53/TP53 activation by phosphorylation, but is inactivated in turn by p53/TP53; this feedback loop may lead to an arrest of the ceil cycle and of the transcription, helping in cell recovery, or to apoptosis. Recently, CDK7 has been strongly implicated in the stimulation of the transcription of short-lived mRNAs and proteins, particularly those to which cancer cells are "addicted" (Wang et a/., Cell 2015 163, 174- 186).

In a search for alternative methods of treating ALS we have surprisingly found that inhibition of the expression of CDK7 in ALS patient derived iAstrocytes protected motor neurons against the toxic effects of these astrocytes in vitro.

According to the invention there is provided a method of preventing or treating a neurodegenerative disease, which comprises: inhibiting CDK7 in the central nervous system (CNS) of a subject in need of such prevention or treatment. As used herein, the terms "inhibiting", "inhibition", "inhibitor" and the like, may include at least the inhibition of the expression of CDK7; or any inhibition which relates to inhibition of the action of the enzyme itself.

There is also provided according to the invention an inhibitor of CDK7, for use in the prevention or treatment of a neurodegenerative disease.

The invention also provides use of an inhibitor of CDK7 in the manufacture of a medicament for the prevention or treatment of a neurodegenerative disease.

Within the CNS, CDK7 may be inhibited in endothelial cells, microglia, astrocytes, or neurons, in microglia and astrocytes, in microglia and neurons, in astrocytes and neurons, or in microglia, astrocytes, and neurons.

According to the invention there is provided a pharmaceutical composition, which comprises an inhibitor of CDK7.

There is further provided according to the invention a pharmaceutical composition, or a preparation, of the invention, which further comprises a P-glycoprotein inhibitor.

There is also provided according to the invention a composition, which comprises an inhibitor of CDK7, and a P-glycoprotein inhibitor.

There is also provided according to the invention a pharmaceutical composition, which comprises an inhibitor of CDK7, a P-glycoprotein inhibitor, and a pharmaceutically acceptable carrier, excipient, or diluent.

According to the invention there is also provided a combined preparation, which comprises: (a) an inhibitor of CDK7 and a P-glycoprotein inhibitor.

The inhibitor of CDK7 and the P-glycoprotein inhibitor may be administered together (i.e. co-administered) or sequentially, in any order. If preferred, the P-glycoprotein inhibitor may be administered before the inhibitor of CDK7.

The CDK7 inhibitor may be selected from the group consisting of R547, AT-7519, foretinib, AST-487, alvocidib, BMS-387032, staurosporine, A-674563, lestaurtinib, fedratinib, PHA- 665752, sorafenib, KW-2449, NVP-TAE684, crizotinib, sunitinib, Gefitinib and BMS-345541 . Combinations of any two or more of these compounds may also be used.

The P-glycoprotein inhibitor may be selected from the group consisting of cyclosporine A, ketoconazole, quinidine, ritonavir, verapamil, everolimus, or elacridar (GF120918), or quinidine. In a preferred aspect, the P-glycoprotein inhibitor is elacridar. Combinations of any two or more of these compounds may also be used.

There is further provided according to the invention a composition, a pharmaceutical composition, or a combined preparation of the invention for use in the prevention or treatment of a neurodegenerative disease.

There is also provided according to the invention use of a composition, a pharmaceutical composition, or a combined preparation of the invention in the manufacture of a medicament for the prevention or treatment of a neurodegenerative disease.

There is also provided according to the invention a method of preventing or treating a neurodegenerative disease, which comprises administering effective amounts of CDK7 inhibitor and a P-glycoprotein inhibitor to a subject in need of such prevention or treatment.

The CDK7 and the P-glycoprotein inhibitors may be co-administered, or administered sequentially.

The neurodegenerative disease may be a motor neuron disease, such as amyotrophic lateral sclerosis (ALS).

The neurodegenerative disease may be a familial or sporadic neurodegenerative disease. In a preferred aspect of the invention, the neurodegenerative disease (in particular motor neurone disease, such as ALS) is a familial neurodegenerative disease. In another preferred aspect of the invention, the neurodegenerative disease (in particular motor neurone disease, such as ALS) is a sporadic neurodegenerative disease.

The components of a combined preparation of the invention may be for simultaneous, separate, or sequential use.

The term "combined preparation" as used herein refers to a "kit of parts" in the sense that the combination components (a) and (b) can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination components (a) and (b). The components can be administered simultaneously or one after the other. If the components are administered one after the other, preferably the time interval between administration is chosen such that the effect on the treated disorder or disease in the combined use of the components is greater than the effect which would be obtained by use of only any one of the combination components (a) and (b).

The components of the combined preparation may be present in one combined unit dosage form, or as a first unit dosage form of component (a) and a separate, second unit dosage form of component (b). The ratio of the total amounts of the combination component (a) to the combination component (b) to be administered in the combined preparation can be varied, for example in order to cope with the needs of a patient sub-population to be treated, or the needs of the single patient, which can be due, for example, to the particular disease, age, sex, or body weight of the patients.

Preferably, there is at least one beneficial effect, for example an enhancing of the effect of one of the components, or a mutual enhancing of the effect of the combination components (a) and (b), for example a more than additive effect, additional advantageous effects, fewer side effects, less toxicity, or a combined therapeutic effect compared with a non-effective dosage of one or both of the combination components (a) and (b), and very preferably a synergism of the combination components (a) and (b).

In an aspect of the invention, the neurodegenerative disease is a motor neuron disease, such as ALS, PLS, PMA, PBP, or Pseudobulbar palsy, or Alzheimer's disease, or Parkinson's disease, or fronto temporal dementia (FTD).

As used herein, the terms "treatment", "treating", "treat" and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or can be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting or slowing its development; and (c) relieving the disease, i.e., causing regression of the disease.

The term "subject" used herein includes any human or nonhuman animal. The term "nonhuman animal" includes all mammals, such as nonhuman primates, sheep, dogs, cats, cows, horses.

It will be appreciated that, in methods of the invention, the subject should be administered with a therapeutically effective amount of an inhibitor of CDK7 (and a P-glycoprotein inhibitor, where appropriate).

A "therapeutically effective amount" refers to the amount of an inhibitor of CDK7 that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the inhibitor(s) used, the disease and its severity and the age, weight, etc., of the subject to be treated.

An inhibitor of CDK7 may be administered to a subject using any available method and route suitable for drug delivery to the CNS, including systemic or localized routes. In general, routes of administration contemplated by the invention include, but are not necessarily limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intrathecal, and intravenous routes, i.e., any route of administration other than through the alimentary canal. Parenteral administration can be carried to effect systemic or local delivery. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations. Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g., using a suppository) delivery.

Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intra-tracheal, intrathecal, intracranial, subcutaneous, intradermal, topical, intravenous, intraperitoneal, intra-arterial (for example, via the carotid artery), spinal or brain delivery, rectal, nasal, oral, and other enteral and parenteral routes of administration.

If desired, an inhibitor of CDK7 is administered by injection and/or delivery, for example, to a site in a brain artery or directly into brain tissue.

In an aspect of the invention, an inhibitor of CDK7 is administered by direct delivery to the CNS, in particular in to the spinal cord or brain, such as by intracerebroventricular (ICV) administration. Direct administration in to the brain can be undertaken in combination with a controlled delivery device, such as an in-dwelling cannula or pump (for example, implanted subcutaneously at a suitable location). Suitable methods of ICV administration to human subjects are described, for example, in Paul et al., J Clin Invest. 2015; 125(3): 1339-1346.

A composition of the invention may be provided in a formulation suitable for, or adapted for, administration directly to the CNS, in particular in to the spinal cord or brain, for example of a human subject. If desired, the formulation comprises one or more electrolytes present in endogenous CSF. In a preferred aspect, the one or more electrolytes are selected from sodium, potassium, calcium, magnesium, phosphorous, and chloride ions. A formulation comprising all of the above electrolytes may be used. In a preferred aspect of the invention, the formulation comprises a solution that closely matches the electrolyte concentrations of endogenous CSF of the subject to be treated, for example a human subject. For example, the formulation may comprise a solution comprising any (or each) of: 100-200mM sodium ion; 1 -5mM potassium ion; 1 -2mM calcium ion; 0.5-1 .5mM magnesium ion; 0.5-1 .5mM phosphorous ion; and 100-200mM chloride ion. For example, in one preferred composition of the invention, the formulation comprises a solution comprising 150mM sodium ion, 3mM potassium ion, 1 .4mM calcium ion, 0.8mM magnesium ion, 1 .0mM phosphorous ion, and 155mM chloride ion.

In one aspect of the invention, a composition of the invention suitable for, or adapted for, administration directly to the CNS, in particular in to the spinal cord or brain, for example of a human subject, does not include a P-glycoprotein inhibitor.

The inhibitor(s) may be administered in a single dose or in multiple doses. A suitable frequency of administration may be at least once per day, every other day, once per week, once every two, three, or four weeks, once every month, two months, or once every three to six months. The inhibitor(s) may be administered over a period of at least a week, at least a month, at least three to six months, at least one, two, three, four, or five years, or over the course of the disease, or the lifetime of the subject.

Where a P-gp inhibitor is used, this may be co-administered with the inhibitor CDK7, or the P-gp inhibitor and the inhibitor of CDK7 may be administered sequentially, for example within 96, 72, 48, 36, 24, 12, 6, 5, 4, 3, 2, or 1 hours of each other (i.e. the P-gp inhibitor may be administered before, or after the inhibitor(s) of CDK7).

The amount of P-gp inhibitor that is co-administered, or administered sequentially with the inhibitor of CDK7 is likely to depend on the particular inhibitors used. However, a person of ordinary skill in the art can readily determine the appropriate amount of each inhibitor to administer to ensure that a therapeutically effective amount of the inhibitor of CDK7 penetrates the CNS of the subject to be treated.

Methods of preparing pharmaceutical compositions are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985.

Compositions of the invention can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers, pharmaceutically acceptable diluents, or pharmaceutically acceptable excipients, and can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, solutions, injections, inhalants and aerosols.

Pharmaceutically acceptable carriers, excipients, or diluents may include, for example: water, saline, dextrose, glycerol, ethanol, a salt, e.g., NaCI, MgCl2, KCI, MgS04, etc.; a buffering agent, e.g., a phosphate buffer, a citrate buffer, a Tris buffer, N-(2- Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-

Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3- aminopropanesulfonic acid (TAPS), etc. ; a solubilizing agent; a detergent, e.g. , a non-ionic detergent such as Tween-20, etc.; glycerol; and the like.

Pharmaceutically acceptable carriers, excipients and diluents are nontoxic to recipients at the dosages and concentrations employed, and can for example include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

For oral preparations, a pharmaceutical composition of the invention may include appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

Pharmaceutical compositions for injection can be formulated by dissolving, suspending or emulsifying the active ingredients in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, propylene glycol, synthetic aliphatic acid glycerides, injectable organic esters (e.g., ethyl oleate), esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishes (such as those based on Ringer's dextrose), and the like. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.

The pharmaceutical composition can be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however solutions comprising antibacterial agents can be used for the production of pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 131 1 -54.

A tonicity agent can be included in the formulation to modulate the tonicity of the formulation. Exemplary tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions can be suitable. The term "isotonic" denotes a solution having the same tonicity as some other solution with which it is compared, such as a physiological salt solution or serum.

Embodiments of the invention are described below, by way of example only, with reference to the accompanying drawings in which:

Figure 1 shows the effect of CDK7 shRNA in an in vitro model in which iAstrocytes derived from fibroblasts of two ALS patients (ALS1 , and ALS2) were co-cultured with mouse motor neurons. ALS iAstrocytes were pre-treated with shRNA for 3 days prior to murine Hb9-GFP motor neuron seeding in co-culture. The number of viable motor neurons was then measured 48 hours after motor neuron seeding, and the percentage motor neuron survival was calculated, and then normalised to the untreated control. *P<0.05, **P<0.01 , ***P<0.001 . One-way ANOVA with Dunnett's post-hoc test. Data are mean ± SD. n=5-6.

Example 1 - Effect of CDK7 shRNA in an in vitro model of ALS

This example describes the effect of CDK7 shRNA on motor neuron survival in an in vitro model of ALS. This model uses human fibroblast-derived astrocytes and mouse Hb9-GFP+ motor neurons in co-culture (Meyer et al., 2014, Proceedings of the National Academy of Sciences (PNAS) 1 1 1 , 829-832). The fibroblasts were reprogrammed to induced neural progenitor cells (iNPCs), which were differentiated into Astrocytes. Astrocytes derived from ALS patients cause death of the wild-type Hb9-GFP+ mouse motor neurons in co-culture, a property not seen in astrocytes derived from normal (non-ALS) patients.

Materials and Methods iNPCs were derived from ALS patient fibroblasts as previously described (Meyer et al. 5 2014, PNAS 1 1 1 , 829-832), and were differentiated into iAstrocytes by culturing in supplemented DMEM (Sigma) (10% (v/v) FBS (Sigma), 50units/ml penicillin/streptomycin (Lonza), 1X N-2 supplement (Thermo-Fisher Scientific) for at least 5 days. Murine Hb9- GFP+ motor neurons were differentiated from murine Hb9-GFP+ embryonic stem cells via embryoid bodies, as previously described (Haidet-Phillips et al. 201 1 , Nature Biotechnology 29, 824-828; Wichterle et al. 2002, Cell 1 10, 385-397).

3,000 human iAstrocytes were seeded per well on fibronectin-coated 384-well plates. 24 hours later, virus containing shRNA was added. Plates were centrifuged at 1 ,760 x g for 60 s. 72 hours later, 2,000 murine Hb9-GFP+ motor neurons were seeded per well in motor neuron media (KnockOut DMEM (45% v/v), F12 medium (45% v/v), KO Serum Replacement (10% v/v), 50units/ml penicillin/streptomycin (Lonza), 1 mM L- glutamine, 1X N-2 supplement (Thermo-Fisher Scientific), 0.15% filtered glucose, 0.0008% (v/v) 2- mercaptoethanol, 20 ng/ml GDNF, 20 ng/ml BDNF, 20 ng/ml CNTF) and co-cultured on top of the pre-treated iAstrocytes. Plates were centrifuged at 1 ,760 x g for 60 s. Hb9- GFP+ motor neurons were imaged after 24 and 72 hours using an INCELL analyser 2000 (GE Healthcare), and the number of viable motor neurons was counted using the INCELL analyser software (GE Healthcare).

The number of viable motor neurons (defined as GFP+ motor neurons with at least 1 axon) that survived after 48 hours in co-culture was calculated as a percentage of the number of viable motor neurons after 24 hours in co-culture. One-way ANOVA with Dunnett's post hoc test was performed.

Results

The results, plotted in Figure 1 , show that CDK7 shRNA promotes an increase in motor neuron survival in iAstrocyte co-cultures, suggesting that c-kit inhibitors will have beneficial effects in patients with ALS.

Conclusions

It was concluded from these results that the toxic nature of ALS patient-derived astrocytes, as revealed by decreased motor neuron survival in ALS astrocyte/motor neuron coll cultures, is reduced by reducing the expression of CDK7 in astrocytes.

Claims

1 . A method of preventing or treating a neurodegenerative disease, which comprises inhibiting CDK7 in the central nervous system of a subject in need of such prevention or treatment.

2. A method according to claim 1 , wherein CDK7 is inhibited in endothelial cells, microglial cells, astrocytes, or neurons of the subject.

3. A method according to any preceding claim, wherein CDK7 is inhibited by administering an inhibitor of CDK7 to the subject.

4. A method according to claim 3, wherein the CDK7 inhibitor is co-administered, or administered sequentially, to the subject with a P-glycoprotein inhibitor.

5. A method according to claim 3, wherein the CDK7 inhibitor is selected from the group consisting of R547, AT-7519, foretinib, AST-487, alvocidib, BMS-387032, staurosporine, A- 674563, lestaurtinib, fedratinib, PHA-665752, sorafenib, KW-2449, NVP-TAE684, crizotinib, sunitinib, Gefitinib and BMS-345541 , and combinations of any two or more of the above.

6. A method according to any preceding claim, wherein the neurodegenerative disease is a motor neuron disease.

7. A method according to claim 6, wherein the motor neuron disease is amyotrophic lateral sclerosis (ALS).

8. A method according to any preceding claim, wherein the neurodegenerative disease is a familial neurodegenerative disease.

9. An inhibitor of CDK7 for use in the prevention or treatment of a neurodegenerative disease.

10. Use of an inhibitor of CDK7 in the manufacture of a medicament for the prevention or treatment of a neurodegenerative disease.

1 1 . An inhibitor for use according to claim 9, or use according to claim 10, wherein the CDK7 inhibitor inhibits astrocyte activation.

12. An inhibitor for use according to claim 9 or 1 1 , or use according to claim 10 or 1 1 , or a method according to any one of claims 1 to 8, wherein the inhibitor of CDK7 are administered directly to the brain or spinal cord.

13. An inhibitor for use according to any one of claims 9, 1 1 or 12, or use according to any one of claims 10-12, which further includes a P-glycoprotein inhibitor.

14. An inhibitor for use according to any one of claims 9, 1 1 , 12, or 13, or use according to any one of claims 10-13, wherein the neurodegenerative disease is a motor neuron disease.

15. An inhibitor for use, or use, according to claim 14, wherein the motor neuron disease is ALS.

16. An inhibitor for use, or use, according to any of claims 13 to 15, wherein the neurodegenerative disease is a familial neurodegenerative disease.

17. A pharmaceutical composition, which comprises an inhibitor of CDK7 and a pharmaceutically acceptable carrier, excipient, or diluent.

18. A pharmaceutical composition according to claim 17 which further comprises a P- glycoprotein inhibitor.

19. An inhibitor for use, or use, according to any one of claims 13 to 16, or a pharmaceutical composition according to claim 17 or 18, wherein the P-glycoprotein inhibitor is selected from the group consisting of cyclosporine A, ketoconazole, quinidine, ritonavir, verapamil, everolimus, and elacridar, and combinations of any two or more of the above.

20. A composition, which comprises a CDK7 inhibitor, and a P-glycoprotein inhibitor.

21 . A pharmaceutical composition, which comprises a CDK7 inhibitor, and a P-glycoprotein inhibitor, and a pharmaceutically acceptable carrier, excipient, or diluent.

22. A combined preparation, which comprises: (a) a CDK7 inhibitor; and (b) a P- glycoprotein inhibitor.

23. A composition according to claim 20, a pharmaceutical composition according to claim 21 , or a combined preparation according to claim 22, wherein the P-glycoprotein inhibitor is selected from the group consisting of cyclosporine A, ketoconazole, quinidine, ritonavir, verapamil, everolimus, and elacridar, and combinations of any two or more of the above.

24. A composition according to claim 20, a pharmaceutical composition according to claim 21 , or a combined preparation according to claim 22, wherein the P-glycoprotein inhibitor is quinidine.

25. A pharmaceutical composition, which comprises an inhibitor of CDK7 and a pharmaceutically acceptable carrier, excipient, or diluent, wherein the pharmaceutical composition is suitable for, or adapted for, administration directly to the CNS.

26. A pharmaceutical composition according to claim 25, which comprises one or more electrolytes present in endogenous CSF, wherein the one or more electrolytes is selected from sodium, potassium, calcium, magnesium, phosphorous, and chloride ions.

27. A pharmaceutical composition according to claim 26, which comprises a solution comprising 150mM sodium ion, 3mM potassium ion, 1 .4mM calcium ion, 0.8mM magnesium ion, 1 .0mM phosphorous ion, and 155mM chloride ion.

28. A composition, a pharmaceutical composition, or a combined preparation according to any of claims 17-27 for use in the prevention or treatment of a neurodegenerative disease.

29. Use of a composition, a pharmaceutical composition, or a combined preparation according to any of claims 20 to 28 in the manufacture of a medicament for the prevention or treatment of a neurodegenerative disease.

30. Use according to claim 29, wherein the neurodegenerative disease is a motor neuron disease.

31 . Use according to claim 30, wherein the motor neuron disease is amyotrophic lateral sclerosis (ALS).

32. Use according to any of claims 29 to 31 , wherein the neurodegenerative disease is a familial neurodegenerative disease.