Patent Application
20200085812
The present invention relates to a bioactive agent that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, for use in the treatment of frontotemporal disorders such as frontotemporal dementia.
Heat shock proteins are found in all compartments of a cell where conformational rearrangements of proteins occur. Heat shock proteins are also commonly known as molecular chaperones, as they serve to keep their client proteins in a proper, folded state. Protein synthesis is the major source of unfolded peptides in the cell but a challenge to the cell by high temperature or other stressful stimuli that render proteins structurally labile and hence prone to unfolding and aggregation is met with a specific cellular response involving the increased production of Heat shock proteins. This response is a phenomenon observed in every cell type ranging from prokaryotes to eukaryotes and is referred to as the heat-shock- or stress-response. The proteins induced by this response are known as the heat shock proteins (HSPs), of which there exist several families.
A primary example of a family of HSPs is the Hsp70 proteins. This family has recently been implicated in other aspects of cellular homeostasis besides serving as a molecular chaperone—most markedly through its anti-apoptotic features, its functions in immunity, and the apparent dependence of cancer cells on the upregulation of Hsp70. Furthermore, Hsp70 can serve a role in safeguarding lysosomal integrity.
HSP gene expression and protein expression can be amplified by HSP inducers. Examples of small molecule inducers of the heat shock response, including Hsp70, include bimoclomol, arimoclomol, iroxanadine and BGP-15.
The term frontotemporal disorder refers to changes in behavior and thinking that are caused by underlying brain diseases collectively called frontotemporal lobar degeneration (FTLD). FTLD is not a single brain disease but rather a family of neurodegenerative diseases, any one of which can cause a frontotemporal disorder. Frontotemporal dementia (FTD) on the other hand is one of several possible variations and is sometimes more precisely called behavioral variant frontotemporal dementia, or bvFTD.
Dementia results in severe loss of thinking abilities that interferes with a person's ability to perform daily activities. An estimated 10% of all cases of dementia are caused by FTLD and may be as common as Alzheimer's among people younger than age 65.
A main histological subtype of FTLD is FTLD-TDP (or FTLD-U) characterized by ubiquitin and TDP-43 positive, tau negative, FUS (fused in sarcoma/translocated in sarcoma) negative inclusions.
Mutations in valosin-containing protein (VCP) cause a multisystem disorder that includes inclusion body myopathy (IBM) associated with Paget's disease of the bone (PDB) and fronto-temporal dementia (FTD); or IBMPFD. Although IBMPFD is a multisystem disorder, muscle weakness is the presenting symptom in greater than half of patients and an isolated symptom in 30%. Patients with the full spectrum of the disease make up an estimated 12% of those affected; therefore it is important to consider and recognize IBMPFD in a neuromuscular clinic. In addition to myopathic features; vacuolar changes and tubulofilamentous inclusions are found in a subset of patients. The most consistent findings are VCP, ubiquitin and TAR DNA-binding protein 43 (TDP-43) positive inclusions.
Mutations in the VCP gene are also reported to be the cause of 1%-2% of familial amyotrophic lateral sclerosis (fALS) cases, potentially causing sporadic ALS-FTD.
RNA granules are microscopically visible cellular structures that aggregate by protein-protein and protein-RNA interactions. RNA granule formation relies on the multivalency of RNA and multi-domain proteins as well as low-affinity interactions between proteins with prion-like/low-complexity domains (e.g. FUS and TDP-43). Classes of these structures include nucleoli, Cajal bodies, nuclear speckles and paraspeckles in the nucleus, as well as P-bodies and stress granules in the cytoplasm.
Unlike other RNA granules, cytoplasmic stress granules are not constitutively present; instead, their formation is induced by cellular stress, such as heat shock or oxidative stress, and they disassemble once the perturbation subsides. Notably, morphologically similar cytoplasmic inclusions are observed in neurons of patients with amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and other age-related neurodegenerative disease, which often exhibit compositional overlap with endogenous stress granules (15,16).
Symptoms of frontotemporal dementia progress at a rapid, steady rate. There are currently no treatments available to prevent, stop or reverse frontotemporal dementia.
WO 2009/155936 discloses Hsp70 and inducers thereof for treating lysosomal storage diseases. WO 2005/041965 discloses use of the heat shock protein inducer arimoclomol for treating neurodegenerative diseases, including ALS.
The present inventors now find that mutant VCP (mVCP) mice not only show degenerative muscle pathology but also CNS pathology with motoneuron (motor neuron) loss in the spinal cord (ALS phenotype) and abnormal TDP-43, ubiquitin, p-tau, p62 and LC3 in the brain (FTD phenotype). As shown herein, all these features are seen to be attenuated in mVCP mice treated with an inducer of the heat shock proteins, including Hsp70 and co-chaperones. Furthermore, it is shown herein that mVCP mouse brains display stress granule protein markers and that treatment with an inducer of the heat shock proteins, including Hsp70 and co-chaperones, attenuate the appearance of said stress granule protein markers.
It is thus an aspect to provide a bioactive agent that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, for use in the treatment of frontotemporal disorders.
In one embodiment said bioactive agent increases the intracellular concentration and/or activity of Hsp70, i.e. is an inducer of Hsp70, such as a small molecule inducer of Hsp70, such as an inducer selected from the group consisting of arimoclomol, iroxanadine, bimoclomol, BGP-15, their stereoisomers and the acid addition salts thereof.
In one embodiment the frontotemporal disorder is selected from the group consisting of frontotemporal lobar degeneration (FTLD), frontotemporal dementia (FTD), inclusion body myopathy (IBM) with FTD, Paget's disease of bone (PDB) with FTD, IBM with early-onset PDB and FTD (IBMPFD), FTD with amyotrophic lateral sclerosis (ALS) (ALS-FTD), and IBM with FTD, PDB and ALS (IBMPFD-ALS).
The present inventors have identified TDP-43 mislocalisation, ubiquitin aggregation, p-tau lesions, p62 and LC3 expression and stress granule formation in mutant VCP mice as well as port-mortem human brain cortex from patients with Frontotemporal Dementia (FTD).
The effect of inducing the heat shock response, including the effect on heat shock proteins, such as Hsp70 and co-chaperones, observed herewith on abnormal TDP-43, ubiquitin, p-tau, p62, LC3 and stress granule markers in the brain has potential in therapies involving frontotemporal disorders and FTD-like pathologies associated with one or more of TDP-43 mislocalisation, ubiquitin aggregation, p-tau lesions, p62 and LC3 expression and stress granule formation; such as for example TDP-43 mislocalisation, ubiquitin aggregation, p-tau lesions, p62 and LC3 expression and stress granule formation caused by a VCP mutation.
It is thus an aspect of the present disclosure to provide a bioactive agent as defined herein that increases the intracellular concentration (or levels) and/or activity of one or more heat shock proteins, including Hsp70, for use in the treatment of a frontotemporal disorder.
In one embodiment said frontotemporal disorder is associated with frontotemporal dementia.
In one embodiment there is provided use of a bioactive agent as defined herein that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, for the manufacture of a medicament for the treatment of a frontotemporal disorder.
In one embodiment there is provided a method of treating a frontotemporal disorder, said method comprising one or more steps of administering a bioactive agent as defined herein that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, to an individual in need thereof.
The term “Individual” or “subject” refers to vertebrates, in particular a member of a mammalian species, preferably primates including humans. In a preferred embodiment, an individual as used herein is a human being, male or female, of any age.
An “individual in need thereof” refers to an individual who may benefit from the present treatment. In one embodiment, said individual in need thereof is a diseased individual, wherein said disease is associated with one or more of TDP-43 mislocalisation, ubiquitin aggregation p-tau lesions, p62 and LC3 expression or aggregation and stress granule formation, and/or associated with a VCP mutation, such as frontotemporal disorders as defined herein.
In one embodiment, said treatment may be prophylactic, curative or ameliorating. In one particular embodiment, said treatment is prophylactic. In another embodiment, said treatment is curative. In a further embodiment, said treatment is ameliorating.
The bioactive agents that increase the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, are defined in detail herein below, and encompass inducers of heat shock proteins including Hsp70.
The diseases associated with TDP-43 mislocalisation, ubiquitin aggregation, p-tau lesions, p62 and LC3 expression (or aggregation) and/or stress granule formation and/or a VCP mutation are defined in detail herein below, and encompass frontotemporal lobar degeneration (FTLD) or FTLD-TDP, frontotemporal dementia (FTD) including FTD-MND, FTD-U, FTD-TDPA and FTD-tau, inclusion body myopathy (IBM) with FTD, Paget's disease of bone (PDB) with FTD, IBM with early-onset PDB and FTD (IBMPFD), FTD with amyotrophic lateral sclerosis (ALS) (ALS-FTD), and IBM with FTD, PDB and ALS (IBMPFD-ALS).
Frontotemporal Disorders
Frontotemporal disorders are the result of damage to neurons in the frontal and temporal lobes of the brain. Frontotemporal disorders refer to changes in behaviour and thinking that are caused by underlying brain diseases collectively called frontotemporal lobar degeneration (FTLD). FTLD is not a single brain disease but rather a family of neurodegenerative diseases, any one of which can cause a frontotemporal disorder. FTLD encompasses the subgroups frontotemporal dementia (FTD), progressive nonfluent aphasia (PFNA), and semantic dementia (SD).
A main histological subtype of FTLD is FTLD-TDP (or FTLD-U) characterized by ubiquitin and TDP-43 positive, tau negative, FUS (fused in sarcoma/translocated in sarcoma) negative inclusions.
Frontotemporal disorders thus comprise frontotemporal lobar degeneration (FTLD), FTLD-TDP, frontotemporal dementia (FTD) including FTD-MND, FTD-U, FTD-TDPA and FTD-tau, inclusion body myopathy (IBM) with FTD, Paget's disease of bone (PDB) with FTD, IBM with early-onset PDB and FTD (IBMPFD), FTD with amyotrophic lateral sclerosis (ALS) (ALS-FTD), and IBM with FTD, PDB and ALS (IBMPFD-ALS).
In one embodiment of the present disclosure there is provided a bioactive agent as defined herein for use in the treatment of a frontotemporal disorder. In one embodiment the frontotemporal disorder is selected from the group consisting of frontotemporal lobar degeneration (FTLD) and FTLD-TDP, frontotemporal dementia (FTD), inclusion body myopathy (IBM) with FTD, Paget's disease of bone (PDB) with FTD, IBM with early-onset PDB and FTD (IBMPFD), FTD with amyotrophic lateral sclerosis (ALS) (ALS-FTD), and IBM with FTD, PDB and ALS (IBMPFD-ALS).
In one embodiment of the present disclosure there is provided a bioactive agent as defined herein for use in the treatment of a frontotemporal disorder selected from the group consisting of frontotemporal lobar degeneration (FTLD) and FTLD-TDP, frontotemporal dementia (FTD), inclusion body myopathy (IBM) with FTD, Paget's disease of bone (PDB) with FTD, IBM with early-onset PDB and FTD (IBMPFD), FTD with amyotrophic lateral sclerosis (ALS) (ALS-FTD), and IBM with FTD, PDB and ALS (IBMPFD-ALS).
In one embodiment said frontotemporal disorder or frontotemporal lobar degeneration (FTLD) is associated with (or displays or show symptoms of) frontotemporal dementia (FTD).
In one embodiment the frontotemporal dementia (FTD) is selected from the group consisting of frontotemporal Dementia (FTD) associated with motor neuron disease (FTD-MND), frontotemporal Dementia (FTD) associated with ubiquitin-positive inclusions (FTD-U), frontotemporal Dementia (FTD) associated with mutant TDP-43 (FTD-TDPA) and frontotemporal Dementia (FTD) associated with tau-positive inclusions (FTD-tau).
In one embodiment said frontotemporal disorder is associated with a mutation in the VCP gene (mVCP), or displays a mutation in the VCP gene (mVCP). In one embodiment said frontotemporal disorder comprising frontotemporal lobar degeneration (FTLD), FTLD-TDP, frontotemporal dementia (FTD) including FTD-MND, FTD-U, FTD-TDPA and FTD-tau, inclusion body myopathy (IBM) with FTD, Paget's disease of bone (PDB) with FTD, IBM with early-onset PDB and FTD (IBMPFD), FTD with amyotrophic lateral sclerosis (ALS) (ALS-FTD), and IBM with FTD, PDB and ALS (IBMPFD-ALS) is associated with a mutation in the VCP gene (mVCP), or displays a mutation in the VCP gene (mVCP).
“Associated with a mutation in the VCP gene” in the present context means that the patient presenting with the given disease is identified as having a mutation in the VCP gene.
Hence in one embodiment of the present disclosure there is provided a bioactive agent as defined herein for use in the treatment of a frontotemporal disorder, wherein said patient having a frontotemporal disorder has a mutation in the VCP gene (mVCP).
In one embodiment said frontotemporal disorder is associated with a mutation in the VCP gene causing TDP-43 mislocalisation and/or ubiquitin aggregation and/or p-tau lesions, and/or p62 and LC3 expression and/or stress granule formation. In one embodiment said frontotemporal disorder is associated with TDP-43 mislocalisation and/or ubiquitin aggregation and/or p-tau lesions, and/or p62 and LC3 expression or aggregation and/or stress granule formation.
In one embodiment said frontotemporal disorder comprising frontotemporal lobar degeneration (FTLD), FTLD-TDP, frontotemporal dementia (FTD) including FTD-MND, FTD-U, FTD-TDPA and FTD-tau, inclusion body myopathy (IBM) with FTD, Paget's disease of bone (PDB) with FTD, IBM with early-onset PDB and FTD (IBMPFD), FTD with amyotrophic lateral sclerosis (ALS) (ALS-FTD), and IBM with FTD, PDB and ALS (IBMPFD-ALS) is associated with TDP-43 mislocalisation and/or ubiquitin aggregation and/or p-tau lesions, and/or p62 and LC3 expression or aggregation and/or stress granule formation.
“Associated with TDP-43 mislocalisation and/or ubiquitin aggregation and/or p-tau lesions, and/or p62 and LC3 expression and/or stress granule formation” in the present context means that the patient presenting with the given disease is identified as having TDP-43 mislocalisation and/or ubiquitin aggregation and/or p-tau lesions, and/or p62 and LC3 expression and/or stress granule formation; such as TDP-43 cytoplasmic mislocalisation and/or cytoplasmic ubiquitin aggregation and/or p-tau lesion formation, and/or p62 expression or cytoplasmic aggregation and/or LC3 expression or cytoplasmic aggregation and/or stress granule formation.
In one embodiment said frontotemporal disorder is associated with stress granule formation. In one embodiment said frontotemporal disorder is associated with stress granule formation including one or more of the stress granule markers Tia1, FMRP (Fragile X Mental Retardation protein) and G3BP (RasGAP SH3 domain Binding Protein).
In one embodiment of the present disclosure there is provided a bioactive agent as defined herein for use in the treatment of a frontotemporal disorder selected from the group consisting of frontotemporal lobar degeneration (FTLD), frontotemporal dementia (FTD), inclusion body myopathy (IBM) with FTD, Paget's disease of bone (PDB) with FTD, IBM with early-onset PDB and FTD (IBMPFD), FTD with amyotrophic lateral sclerosis (ALS) (ALS-FTD), and IBM with FTD, PDB and ALS (IBMPFD-ALS); wherein said frontotemporal disorder is associated with a mutation in the VCP gene, and/or wherein said frontotemporal disorder is associated with one or more of TDP-43 mislocalisation, ubiquitin aggregation, p-tau lesions, p62 or LC3 expression (or aggregation) or stress granule formation.
VCP (Uniprot-P55072 (TERA_HUMAN)), or Transitional endoplasmic reticulum ATPase (TER ATPase), is an enzyme that in humans is encoded by the VCP gene. The main function of VCP is to segregate protein molecules from large cellular structures such as protein assemblies, organelle membranes and chromatin, and thus facilitate the degradation of released polypeptides by the multi-subunit protease proteasome. VCP gene codes for the protein VCP, which is a member of the AAA-ATPase (ATPases associated with diverse cellular activities) superfamily, and is involved in cell cycle control, membrane fusion, and the ubiquitin-proteasome degradation pathway.
In one embodiment of the present disclosure, the frontotemporal disorder as defined herein is associated with a mutation of the VCP gene selected from the group consisting of R93C, R95G, R95C, R95H, I126F, P137L, R155S, R155C, R155H, R155P, R155L, G157R, R159C, R159H, R159G, R191Q, L198W, A232E, T262A, N387H, A439P, A439S and D592N.
In one embodiment of the present disclosure there is provided a bioactive agent as defined herein that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, for use in the treatment of frontotemporal lobar degeneration (FTLD).
Frontotemporal dementia (FTD) is a term for a diverse group of uncommon disorders that primarily affect the frontal and temporal lobes of the brain. It is characterized by progressive neuronal loss and typical loss of over 70% of spindle neurons, while other neuron types remain intact. Although FTDs are clinically, genetically and neuropathologically heterogeneous, more than 95% of cases are TDP-43 proteinopathies or taupathies. FTD was originally called “Pick's disease”, a term now reserved for Pick disease, one specific type of FTD.
Some people with FDT undergo dramatic changes in their personality and become socially inappropriate, impulsive or emotionally indifferent, while others lose the ability to use language. Currently, there is no cure for FTD; only treatments that help alleviate symptoms are available.
Subtypes of FTD are identified clinically according to the symptoms that appear first and most prominently. Clinical diagnoses include behavioral variant FTD (bvFTD), primary progressive aphasia (PPA) which affects language, and the movement disorders progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD).
In one embodiment of the present disclosure there is provided a bioactive agent as defined herein that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, for use in the treatment of frontotemporal dementia (FTD).
In one embodiment of the present disclosure there is provided a bioactive agent as defined herein that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, for use in the treatment of frontotemporal dementia (FTD) selected from the group consisting of frontotemporal Dementia (FTD) associated with motor neuron disease (FTD-MND), frontotemporal Dementia (FTD) associated with ubiquitin-positive inclusions (FTD-U), frontotemporal Dementia (FTD) associated with mutant TDP-43 (FTD-TDPA) and frontotemporal Dementia (FTD) associated with tau-positive inclusions (FTD-tau).
The symptoms and pathology of FTD vary depending on the specific mutation. The majority of FTD patients with a genetic cause have a mutation occurring in one of the following genes: C9orf72; Microtubule-associated protein tau (MAPT, often referred to as “tau”); Progranulin (GRN or PGRN) and Valosin-Containing Protein (VCP). Three additional genes that have been associated with very rare FTD cases: Charged multivesicular body protein 2B (CHMP2B), TAR DNA-binding protein (TARDBP) and Fused in sarcoma (FUS).
In one embodiment, the frontotemporal disorder is Pick disease (PiD).
In one embodiment of the present disclosure there is provided a bioactive agent as defined herein that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, for use in the treatment of frontotemporal dementia (FTD) associated with a mutation in the VCP gene.
In another embodiment, the frontotemporal disorder is IBM with early-onset PDB and FTD (IBMPFD) (also termed IBM associated with PDB and FTD).
In one embodiment of the present disclosure there is provided a bioactive agent as defined herein that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, for use in the treatment of IBM with early-onset PDB and FTD (IBMPFD).
IBMPFD is a multisystem degenerative disorder that is characterized by inclusion body myopathy (IBM) which results in muscle weakness that sets in during adulthood, early-onset Paget's disease of bone (PDB), and premature FTD. It spreads to other systems and results in respiratory or cardiac failure.
PDB is caused by the excessive breakdown and formation of bone, followed by disorganized bone remodeling. This causes bones to grow larger and weaker than normal, resulting in pain, misshapen bones, fractures and arthritis in the joints near the affected bones. PDB can co-occur with FTD.
In one embodiment, the frontotemporal disorder is inclusion body myopathy (IBM) with FTD (IBM-FTD).
In one embodiment, the frontotemporal disorder is Paget's disease of bone (PDB) with FTD (PDB-FTD).
IBMPFD is a rare disorder in which affected individuals may have muscle weakness, Paget's disease of bone and/or dementia. Muscle weakness in this disorder has typically been attributed to a disease of muscle known as inclusion body myopathy (IBM). The major genetic cause of IBMPFD is mutation of the VCP (valosin-containing protein) gene. Mutations in VCP have also been reported to cause familial ALS (amyotrophic lateral sclerosis) and ALS sometimes occurs in families with IBMPFD. Thus, a condition comprising both IBMPFD and ALS is also identified and may be denoted IBMPFD-ALS (IBM with FTD, PDB and ALS). This condition has also been called multisystem proteinopathy (MSP).
In one embodiment, the frontotemporal disorder is IBMPFD-ALS.
In one embodiment of the present disclosure there is provided a bioactive agent as defined herein that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, for use in the treatment of IBMPFD-ALS.
Amyotrophic lateral sclerosis (ALS) has mainly been described as a neurological disorder that affects the motor system, but is now recognized as a multisystem neurodegenerative disease due to the fact that other than motor areas of the brain undergo degeneration. Both FTD and ALS are heterogeneous at the clinical, neuropathological and genetic levels and, even though they come across as distinct progressive disorders, there is increasing evidence of the fact that they share some clinical, neuropathological and genetic features.
ALS can co-occur with any of the FTLD clinical variants, but is most commonly associated with FTD (otherwise known as behavioral variant FTD or bvFTD).
In one embodiment, the frontotemporal disorder is FTD with amyotrophic lateral sclerosis (ALS) (ALS-FTD).
In one embodiment, the frontotemporal disorder is bvFTD with amyotrophic lateral sclerosis (ALS) (ALS-bvFTD).
In one embodiment of the present disclosure there is provided a bioactive agent as defined herein that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, for use in the treatment of ALS-FTD.
In one embodiment, the frontotemporal disorder is fALS associated with mVCP (VCP-fALS).
In one embodiment, the frontotemporal disorder is sporadic ALS-FTD.
Bioactive Agent
A “Bioactive agent” (i.e., biologically active substance/agent) is any agent, drug, substance, compound, composition of matter or mixture which provides some pharmacologic, often beneficial, effect that can be demonstrated in vivo or in vitro. As used herein, this term further includes any physiologically or pharmacologically active substance that produces a localized or systemic effect in an individual. Further examples of bioactive agents include, but are not limited to, agents comprising or consisting of an oligosaccharide, a polysaccharide, an optionally glycosylated peptide, an optionally glycosylated polypeptide, a nucleic acid, an oligonucleotide, a polynucleotide, a lipid, a fatty acid, a fatty acid ester and secondary metabolites.
A bioactive agent as defined herein increases the intracellular concentration (or levels) and/or activity of one or more heat shock proteins, in one embodiment including Hsp70 and co-chaperones. In one embodiment said bioactive agent is selected from:
A bioactive agent as defined herein is thus any agent, chemical or compound that increases the intracellular concentration and/or activity of one or more heat shock proteins, in one embodiment including Hsp70 and co-chaperones; and includes Hsp70 itself, or a functional fragment or variant thereof, any heat shock protein includes and any Hsp70 inducer known to the skilled person.
A bioactive agent that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, and a bioactive agent that increases the intracellular concentration and/or activity of Hsp70, can be used interchangeably with ‘Hsp70 inducer’ herein.
An Hsp70 inducer can amplify Hsp70 gene expression and protein expression with or without a concomitant stress. A direct Hsp70 inducer is a compound that can by itself amplify Hsp70 gene expression and protein expression without a concomitant stress. An indirect Hsp70 inducer, or an Hsp70 co-inducer, is a compound that cannot amplify Hsp70 gene expression and protein expression without a concomitant (mild) stress, but the stress-induced increase in Hsp70 levels is further elevated or enhanced by their presence.
It follows that a bioactive agent may increase the intracellular concentration and/or activity of heat shock proteins, such as Hsp70, either directly or indirectly.
In one embodiment, the bioactive agent is Hsp70, or a functional fragment or variant thereof.
In another embodiment, the bioactive agent is an inducer of heat shock proteins, including Hsp70.
In one embodiment the inducer of heat shock proteins, including Hsp70, is an inducer of one or more of Hsp70, Hsp40, Hsp72 and Hsp90, and co-chaperones.
In one embodiment the inducer of heat shock proteins is an inducer of at least Hsp70.
In one embodiment the inducer of heat shock proteins is an inducer of Hsp70.
Reference to an inducer of Hsp70, or inducing Hsp70, implies that at least Hsp70 is induced, and does not exclude co-induction of other proteins and effectors such as other heat shock proteins. An inducer of Hsp70 refers equally to Hsp70 inducers and co-inducers, and direct and indirect Hsp70 inducers.
In one embodiment, the bioactive agent comprises a combination of Hsp70, or a functional fragment or variant thereof, and an inducer of heat shock proteins including Hsp70.
In one embodiment, the bioactive agent reduces cytoplasmic ubiquitin aggregation. In another embodiment, the bioactive agent reduces Transactive response DNA binding protein 43 kDa (TDP-43) cellular mislocalisation. In yet another embodiment, the bioactive agent reduces motor unit loss. In one embodiment, the bioactive agent reduces stress granule formation, such as reduces stress granule markers including Tia1, FMRP and G3BP. In one embodiment, the bioactive agent reduces p-tau positive lesions. In one embodiment, the bioactive agent reduces P62 and/or LC3 expression or cytoplasmic aggregation.
Inducers of Heat Shock Proteins, Including Hsp70
In one embodiment the bioactive agent activates the heat shock response. In one embodiment the bioactive agent increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70. In one embodiment the bioactive agent increases the intracellular concentration (or level) and/or activity of Hsp70. In one embodiment the bioactive agent increases the intracellular concentration (or level) of Hsp70. In one embodiment the bioactive agent is an inducer of one or more heat shock proteins, including Hsp70. In one embodiment the bioactive agent is an inducer of Hsp70.
It is an aspect of the present disclosure to provide an inducer of one or more heat shock proteins, including Hsp70, for use in treating frontotemporal disorders.
In one embodiment there is provided use of an inducer of one or more heat shock proteins, including Hsp70, for the manufacture of a medicament for the treatment of a frontotemporal disorder.
In one embodiment there is provided a method of treating a frontotemporal disorder, said method comprising one or more steps of administering an inducer of one or more heat shock proteins, including Hsp70, to an individual in need thereof.
Small Molecule Inducers of Heat Shock Proteins
In one embodiment the bioactive agent is an inducer of one or more heat shock proteins, including Hsp70. In one embodiment the bioactive agent is a small molecule inducer of heat shock proteins, including Hsp70, such as a small molecule inducer of Hsp70.
In one embodiment an inducer of Hsp70; or a small molecule inducer of one or more heat shock proteins, including Hsp70; is a compound capable of increasing the intracellular concentration (or level) of inter alia Hsp70, such as by amplifying Hsp70 gene expression. An inducer of Hsp70 may also induce other heat shock proteins.
In one embodiment the bioactive agent is capable of increasing the intracellular concentration (or levels) of Hsp70 by amplifying Hsp70 gene expression. In one embodiment the bioactive agent is capable of increasing the intracellular concentration (or level) of Hsp70 by amplifying Hsp70 gene expression, wherein said bioactive agent is a hydroxylamine derivative, such as a hydroxylamine derivative small molecule.
Examples of such hydroxylamine derivatives include arimoclomol, iroxanadine, bimoclomol, BGP-15, their stereoisomers and the acid addition salts thereof.
It is an aspect of the present disclosure to provide a small molecule inducer of one or more heat shock proteins, including Hsp70, for use in treating a frontotemporal disorder.
In one embodiment there is provided use of a small molecule inducer of one or more heat shock proteins, including Hsp70, for the manufacture of a medicament for the treatment of a frontotemporal disorder.
In one embodiment there is provided a method of treating a frontotemporal disorder, said method comprising one or more steps of administering a small molecule inducer of one or more heat shock proteins, including Hsp70, to an individual in need thereof.
Arimoclomol
In one embodiment the small molecule inducer of Hsp70 is selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride (arimoclomol), its stereoisomers and the acid addition salts thereof. Arimoclomol is further described in e.g. WO 00/50403.
In one embodiment the small molecule inducer of Hsp70 is selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride (arimoclomol), its optically active (+) or (−) enantiomer, a mixture of the enantiomers of any ratio, and the racemic compound, furthermore, the acid addition salts formed from any of the above compounds with mineral or organic acids constitute objects of the present disclosure. All possible geometrical isomer forms of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride belong to the scope of the disclosure. The term “the stereoisomers of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride” refers to all possible optical and geometrical isomers of the compound.
If desired, the N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride or one of its optically active enantiomers can be transformed into an acid addition salt with a mineral or organic acid, by known methods.
In one embodiment the small molecule inducer of Hsp70 is the racemate of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride.
In one embodiment the small molecule inducer of Hsp70 is an optically active stereoisomer of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride.
In one embodiment the small molecule inducer of Hsp70 is an enantiomer of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride.
In one embodiment the small molecule inducer of Hsp70 is selected from the group consisting of (+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride and (−)-(S)—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride.
In one embodiment the small molecule inducer of Hsp70 is an acid addition salt of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride.
In one embodiment the small molecule inducer of Hsp70 is selected from the group consisting of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride citrate (BRX-345), and N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride maleate (BRX-220).
In one embodiment the small molecule inducer of Hsp70 is selected from the group consisting of (+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride citrate; (−)-S—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride citrate; (+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride maleate; and (−)-S—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride maleate.
BGP-15
In one embodiment the small molecule inducer of Hsp70 is N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamide, dihydrochloride (BGP-15), its stereoisomers and the acid addition salts thereof.
In one embodiment the small molecule inducer of Hsp70 is selected from N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamide, dihydrochloride (BGP-15), its optically active (+) or (−) enantiomer, a mixture of the enantiomers of any ratio, and the racemic compound, furthermore, the acid addition salts formed from any of the above compounds with mineral or organic acids constitute objects of the present disclosure. All possible geometrical isomer forms of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamide, dihydrochloride belong to the scope of the disclosure. The term “the stereoisomers of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamide, dihydrochloride” refers to all possible optical and geometrical isomers of the compound.
Iroxanadine
In one embodiment the small molecule inducer of Hsp70 is selected from 5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine (iroxanadine), its stereoisomers and the acid addition salts thereof. Iroxanadine is further described in e.g. WO 97/16439 and WO 00/35914.
In one embodiment the small molecule inducer of Hsp70 is selected from 5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine (iroxanadine), its optically active (+) or (−) enantiomer, a mixture of the enantiomers of any ratio, and the racemic compound, furthermore, the acid addition salts formed from any of the above compounds with mineral or organic acids constitute objects of the present disclosure. All possible geometrical isomer forms of 5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine belong to the scope of the disclosure. The term “the stereoisomers of 5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine” refers to all possible optical and geometrical isomers of the compound.
If desired, the 5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine or one of its optically active enantiomers can be transformed into an acid addition salt with a mineral or organic acid, by known methods.
In one embodiment the small molecule inducer of Hsp70 is the racemate of 5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine.
In one embodiment the small molecule inducer of Hsp70 is an optically active stereoisomer of 5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine.
In one embodiment the small molecule inducer of Hsp70 is an enantiomer of 5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine.
In one embodiment the small molecule inducer of Hsp70 is selected from the group consisting of (+)-5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine and (−)-5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine.
In one embodiment the small molecule inducer of Hsp70 is an acid addition salt of 5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine.
In one embodiment the small molecule inducer of Hsp70 is selected from the group consisting of 5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine citrate, and 5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine maleate.
In one embodiment the small molecule inducer of Hsp70 is selected from the group consisting of (+)-5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine citrate; (−)-5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine citrate; (+)-5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine maleate; and (−)-5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine maleate.
Bimoclomol
In one embodiment the small molecule inducer of Hsp70 is selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride (bimoclomol) its stereoisomers and the acid addition salts thereof. Bimoclomol is further described in e.g. WO 1997/16439.
In one embodiment the small molecule inducer of Hsp70 is selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride (bimoclomol), its optically active (+) or (−) enantiomer, a mixture of the enantiomers of any ratio, and the racemic compound, furthermore, the acid addition salts formed from any of the above compounds with mineral or organic acids constitute objects of the present disclosure. All possible geometrical isomer forms of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride belong to the scope of the disclosure. The term “the stereoisomers of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride” refers to all possible optical and geometrical isomers of the compound.
If desired, the N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride or one of its optically active enantiomers can be transformed into an acid addition salt with a mineral or organic acid, by known methods.
In one embodiment the small molecule inducer of Hsp70 is the racemate of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride.
In one embodiment the small molecule inducer of Hsp70 is an optically active stereoisomer of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride.
In one embodiment the small molecule inducer of Hsp70 is an enantiomer of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride.
In one embodiment the small molecule inducer of Hsp70 is selected from the group consisting of (+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride and (−)-(S)—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride.
In one embodiment the small molecule inducer of Hsp70 is an acid addition salt of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride.
In one embodiment the small molecule inducer of Hsp70 is selected from the group consisting of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride citrate, and N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride maleate.
In one embodiment the small molecule inducer of Hsp70 is selected from the group consisting of (+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride citrate; (−)-S—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride citrate; (+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride maleate; and (−)-S—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride maleate.
Inducers for Treatment
In one embodiment there is provided a bioactive agent capable of increasing the intracellular concentration of Hsp70 by amplifying Hsp70 gene expression, wherein said bioactive agent is a hydroxylamine derivative,
wherein said bioactive agent is selected from the group consisting of:
In one embodiment said frontotemporal disorder is associated with a mutation in the VCP gene, and/or is associated with one or more of TDP-43 mislocalisation, cytoplasmic ubiquitin aggregation, p-tau lesions, p62 and LC3 expression or aggregation, or stress granule formation.
In one embodiment there is provided a bioactive agent capable of increasing the intracellular concentration of Hsp70 by amplifying Hsp70 gene expression, wherein said bioactive agent is a hydroxylamine derivative,
wherein said bioactive agent is selected from the group consisting of:
In one embodiment there is provided a compound selected from the group consisting of (+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride citrate; (−)-S—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride citrate; (+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride maleate; and (−)-S—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride maleate, for use in the treatment of a frontotemporal disorder, such as a frontotemporal disorder selected from the group consisting of frontotemporal lobar degeneration (FTLD), frontotemporal dementia (FTD), IBM with early-onset PDB and FTD (IBMPFD), inclusion body myopathy (IBM) with FTD, Paget's disease of bone (PDB) with FTD, IBMPFD with amyotrophic lateral sclerosis (ALS) (IBMPFD-ALS) and ALS-FTD.
Other Inducers of Heat Shock Proteins
In one embodiment the bioactive agent is an inducer of Hsp70. Any means for inducing Hsp70 expression is envisioned to be encompassed herewith, some of which are outlined herein below.
In one embodiment the inducer of Hsp70 is sub-lethal heat therapy. Increasing the temperature of an individual is a potent inducer of HSPs including Hsp70, and as such sub-lethal heat therapy is a means for inducing Hsp70. In one embodiment, sub-lethal heat therapy comprises increasing the temperature of an individual to a core temperature of about 38° C., such as about 39° C., for example about 40° C., such as about 41° C., for example about 42° C., such as about 43° C.
Psychological stress such as predatory fear and electric shock can evoke a stress induced eHsp70 release, a process which is suggested to be dependent on cathecholamine signaling. Further, adrenaline and noradrenalin can evoke Hsp70 release.
A number of compounds have been shown to induce (or co-induce) HSPs, including Hsp70. In one embodiment the inducer of Hsp70 is selected from the group consisting of: membrane-interactive compounds such as alkyllysophospholipid edelfosine (ET-18-OCH3 or 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine); anti-inflammatory drugs including cyclooxygenase 1/2 inhibitors such as celecoxib and rofecoxib, as well as NSAIDs such as acetyl-salicylic acid, sodium salicylate and indomethacin; dexamethasone; prostaglandins PGA1, PGj2 and 2-cyclopentene-1-one; peroxidase proliferator-activated receptor-gamma agonists; tubulin-interacting anticancer agents including vincristine and paclitaxel; the insulin sensitizer pioglitazone; anti-neoplastic agents such as carboplatin, doxorubicin, fludarabine, ifosfamide and cytarabine; Hsp90 inhibitors including geldanamycin, 17-AAG, 17-DMAG, radicicol, herbimycin-A and arachidonic acid; proteasome inhibitors such as MG132, lactacystin, Bortezomib, Carfilzomib and Oprozomib; serine protease inhibitors such as DCIC, TLCK and TPCK; Histone Deacetylase Inhibitors (HDACi) including SAHA/vorinostat, Belinostat/PXD101, LB-205, LBH589 (panobinostat), FK-228, CI-994, trichostatin A (TSA) and PCI-34051; anti-ulcer drugs including geranylgeranylacetone (GGA), rebamipide, carbenoxolone and polaprezinc (zinc L-carnosine); heavy metals (zinc and tin); cocaine; nicotine; alcohol; alpha-adrenergic agonists; cyclopentenone prostanoids; L-type Ca++ channel blockers, such as L-type Ca++ channel blockers that also inhibits ryanodine receptors, such as lacidipine; ryanodine receptor antagonists such as DHBP (1,1′-diheptyl-4,4′-bipyridium; as well as herbal medicines including paeoniflorin, glycyrrhizin, celastrol, dihydrocelastrol, dihydrocelastrol diacetate and curcumin.
In one embodiment the inducer of Hsp70 is a proteasome inhibitor. In one embodiment the proteasome inhibitor is selected from the group consisting of Bortezomib, Carfilzomib, Oprozomib, MG132 and lactacystin.
In one embodiment the inducer of Hsp70 is a HDAC inhibitor. In one embodiment the HDACi is selected form the group consisting of SAHA/vorinostat, Belinostat/PXD101, LB-205, LBH589 (panobinostat), FK-228, CI-994, trichostatin A (TSA) and PCI-34051.
Membrane Fluidizers
In one embodiment the inducer of Hsp70 is is a membrane fluidizer. Treatment with a membrane fluidizer may also be termed lipid therapy.
Besides the denaturation of a proportion of cellular proteins during heat (proteotoxicity), a change in the fluidity of membranes is also proposed as being a cellular thermo-sensor that initiates the heat shock response and induces HSPs. Indeed, chemically induced membrane perturbations—analogous with heat induced plasma membrane fluidization—are capable of activating HSP, without causing protein denaturation.
In one embodiment the inducer of Hsp70 is a membrane fluidizer selected from the group consisting of benzyl alcohol, heptanol, AL721, docosahexaenoic acid, aliphatic alcohols, oleyl alcohol, dimethylaminoethanol, A2C, farnesol and anaesthetics such as lidocaine, ropivacaine, bupivacaine and mepivacaine, as well as others known to the skilled person.
Heat Shock Protein 70
It is also an aspect to provide Hsp70, or a functional fragment or variant thereof, for use in treating a frontotemporal disorder.
In one embodiment there is provided use of Hsp70, or a functional fragment or variant thereof, for the manufacture of a medicament for the treatment of frontotemporal disorder.
In one embodiment there is provided a method of treating a frontotemporal disorder, said method comprising one or more steps of administering Hsp70, or a functional fragment or variant thereof, to an individual in need thereof.
It is understood that Hsp70, or a functional fragment or variant thereof, as defined herein can be any natural or synthetic product, and may be produced by any conventional technique known to the person skilled in the art.
In one embodiment, Hsp70, or a functional fragment or variant thereof, is purified from a natural source. Said natural source may be any plant, animal or bacteria which expresses, or may be induced to express, Hsp70 in a form suitable for administering to an individual in need thereof.
In a particular embodiment, Hsp70, or a functional fragment or variant thereof, is made synthetically. It follows that Hsp70, or a functional fragment or variant thereof, in one embodiment is a recombinant protein made by conventional techniques and as such is denoted rHsp70.
The Hsp70 as defined herein, synthetic or natural, may have a sequence which is derived from any suitable species of plant, animal or bacteria. In one embodiment, said rHsp70 is derived from a mammal. Said mammal may be selected form the group consisting of human (Homo sapiens), mouse (Mus musculus), cow, dog, rat, ferret, pig, sheep, and monkey. In another embodiment, said rHsp70 is derived from bacteria.
Hsp70 is characterized in part by having a very high degree of interspecies sequence conservation, thus possibly allowing for Hsp70 derived from one species to be used in another species without eliciting a harmful immune response.
In one particular embodiment, said rHsp70 has a sequence derived from human Hsp70.
In one particular embodiment, said rHsp70 has a sequence derived from more than one species. Said Hsp70, or a functional fragment or variant thereof, may thus in one embodiment be a chimera.
In one embodiment Hsp70 is meant to denote any of the two inducible Hsp70 family members with loci names HSPA1A and HSPA1B.
In one embodiment said Hsp70 is selected from HSPA1A (SEQ ID NOs:1 and 2) and HSPA1B (SEQ ID NOs:4 and 5), or a functional fragment or variant thereof. In SEQ ID NO:2 the initiator methionine (M at position 1) of SEQ ID NO:1 is removed. In SEQ ID NO:5 the initiator methionine (M at position 1) of SEQ ID NO:4 is removed. In vivo this occurs by post-translational processing.
In one embodiment, the Hsp70 is selected from any one of SEQ ID NO:s 1, 2, 4 and 5, or functional fragments or variants thereof, including any naturally occurring variants thereof, such as variants derived from molecule processing and/or amino acid modifications (including any acetylation, phosphorylation and methylation).
In one embodiment, the Hsp70 protein has 100% identity to wild-type Hsp70 protein. In another embodiment, the Hsp70 protein has less than 100% identity to the wild-type Hsp70 protein, such as 99.9 to 95% identity, for example 95 to 90% identity, such as 90 to 85% identity, for example 85 to 80% identity, such as 80 to 75% identity, for example 75 to 60% identity to the wild-type protein. Regardless of the degree of identity, any fragment or variant of Hsp70 that retains its relevant biological effects is encompassed herewith.
In one embodiment said variant of Hsp70 has 99.9 to 99% identity, for example 99 to 98% identity, such as 98 to 97% identity, for example 97 to 96% identity, such as 96 to 95% identity, for example 95 to 94% identity, such as 94 to 93% identity, for example 93 to 92% identity, such as 92 to 91% identity, for example 91 to 90% identity, such as 90 to 85% identity, for example 85 to 80% identity, such as 80 to 75% identity, for example 75 to 70% identity, such as 70 to 65% identity, for example 65 to 60% identity to Hsp70 selected from HSPA1A (SEQ ID NOs:1 and 2) and HSPA1B (SEQ ID NOs: 4 and 5), or a fragment thereof.
In one embodiment, the bioactive agent is Hsp70. In one embodiment, said Hsp70 is full length Hsp70. In one embodiment said Hsp70 is HSPA1A, or a functional fragment or variant thereof. In one embodiment said Hsp70 is SEQ ID NO:1 or 2, or a functional fragment or variant thereof.
It is also an embodiment to provide a functional fragment or variant of Hsp70. As defined herein, a functional fragment or variant is any fragment or variant of Hsp70 which retains the capability of one or more of:
In one embodiment, the bioactive agent is a functional fragment or variant of Hsp70.
In one embodiment, the bioactive agent is a functional fragment or variant of Hsp70, in which Hsp70 is modified by one or more deletion(s), addition(s) or substitution(s) of the wild type Hsp70.
In one embodiment, the bioactive agent is a naturally occurring variant of Hsp70, or a fragment of a naturally occurring variant of Hsp70.
In one embodiment a variant of Hsp70 comprises one or more of D-A at position 10, E-D at position 110, D-A at position 199, K-R at position 561, N-acetylalanine at position 2, N6-acetyllysine at position 108, N6-acetyllysine at position 246, N6-acetyllysine at position 348, N6,N6,N6-trimethyllysine at position 561, phosphoserine at position 631, phosphoserine at position 633 and phosphothreonine at position 636. In one embodiment a naturally occurring variant of Hsp70 is Isoform 1 wherein amino acids of position 96-150 are missing (PODMV8-2).
In one embodiment, a functional fragment or variant of Hsp70 is a variant of Hsp70 in which one or more amino acids has been substituted (or mutated). Said substitution(s) comprises equivalent or conservative substitution(s), or a non-equivalent or non-conservative substitution(s). The term Hsp70 and variants thereof also embraces post-translational modifications introduced by chemical or enzyme-catalyzed reactions, as are known in the art, and chemical modifications such as ubiquitination, labeling, pegylation, glycosylation, amidation, alkylation and esterification. In one embodiment said Hsp70 has been post-translationally modified, including including acetylation, phosphorylation and methylation at any position.
In one embodiment 0.1 to 1% of the amino acid residues of wild type Hsp70 has been substituted, such as 1 to 2%, for example 2 to 3%, such as 3 to 4%, for example 4 to 5%, such as 5 to 10%, for example 10 to 15%, such as 15 to 20%, for example 20 to 30%, such as 30 to 40%, for example 40 to 50%, such as 50 to 60%, for example 60 to 70%, such as 70 to 80%, for example 80 to 90%, such as 90 to 100% amino acid residues.
In one embodiment 1-2, 2-3, 3-4, 4-5 of the amino acid residues of wild type Hsp70 has been substituted, such as 5 to 10, for example 10 to 15, such as 15 to 20, for example 20 to 30, such as 30 to 40, for example 40 to 50, such as 50 to 75, for example 75 to 100, such as 100 to 150, for example 150 to 200, such as 200 to 300, for example 300 to 400, such as 400 to 500 amino acid residues.
In one embodiment, the Hsp70 or functional fragment or variant of Hsp70 is a fusion protein. In one embodiment, said Hsp70 or functional fragment or variant of Hsp70 is fused to a tag.
An “equivalent amino acid residue” refers to an amino acid residue capable of replacing another amino acid residue in a polypeptide without substantially altering the structure and/or functionality of the polypeptide. Equivalent amino acids thus have similar properties such as bulkiness of the side-chain, side chain polarity (polar or non-polar), hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral or basic) and side chain organization of carbon molecules (aromatic/aliphatic). As such, “equivalent amino acid residues” can be regarded as “conservative amino acid substitutions”.
The classification of equivalent amino acids refers in one embodiment to the following classes: 1) HRK, 2) DENQ, 3) C, 4) STPAG, 5) MILV and 6) FYW Within the meaning of the term “equivalent amino acid substitution” as applied herein, one amino acid may be substituted for another, in one embodiment, within the groups of amino acids indicated herein below:
The wild type Hsp70 protein has a total length of 641 amino acids (640 amino acids after removal of initiator methionine at position 1). A fragment of Hsp70 is in one embodiment meant to comprise any fragment with a total length of less than the wild type protein, such as having a total length of is 5 to 25 amino acids, such as 25 to 50 amino acids, for example 50 to 75 amino acids, such as 75 to 100 amino acids, for example 100 to 125 amino acids, such as 125 to 150 amino acids, for example 150 to 175 amino acids, such as 175 to 200 amino acids, for example 200 to 225 amino acids, such as 225 to 250 amino acids, for example 250 to 275 amino acids, such as 275 to 300 amino acids, for example 300 to 325 amino acids, such as 325 to 350 amino acids, for example 350 to 375 amino acids, such as 375 to 400 amino acids, for example 400 to 425 amino acids, such as 425 to 450 amino acids, for example 450 to 475 amino acids, such as 475 to 500 amino acids, for example 500 to 525 amino acids, such as 525 to 550 amino acids, for example 550 to 575 amino acids, such as 575 to 600 amino acids, for example 600 to 625 amino acids, such as 625 to 640 amino acids derived from Hsp70.
A fragment of Hsp70 is in one embodiment a truncated version of the wild type protein. A fragment may be truncated by shortening of the protein from either the amino-terminal or the carboxy-terminal ends of the protein, or it may be truncated by deletion of one or more internal regions of any size of the protein.
In one embodiment the Hsp70 is a variant of a fragment, i.e. a fragment of Hsp70 as defined herein wherein one or more amino acids are substituted as defined herein.
It is appreciated that the exact quantitative effect of the functional fragment or variant may be different from the effect of the full-length molecule. In some instances, the functional fragment or variant may indeed be more effective than the full-length molecule.
The present disclosure also relates to variants of Hsp70, or fragments thereof, wherein the substitutions have been designed by computational analysis that uses sequence homology to predict whether a substitution affects protein function (e.g. Pauline C. Ng and Steven Henikoff, Genome Research, Vol. 11, Issue 5, 863-874, May 2001).
Ectopic Expression of Hsp70
In one embodiment, Hsp70, or a functional fragment or variant thereof, is expressed from a vector. In one embodiment Hsp70, or a functional fragment or variant thereof, is administered to an individual in need thereof in the form of a vector.
The vector used for expressing Hsp70, or a functional fragment or variant thereof, is in one embodiment selected from the group consisting of: viral vectors (retroviral and adenoviral) or non-viral vectors (e.g. plasmid, cosmid, bacteriophage).
In one embodiment, said vector comprises one or more of an origin of replication, a marker for selection and one or more recognition sites for a restriction endonuclease. In another embodiment, said vector is operably linked to regulatory sequences controlling the transcription of said Hsp70, or a functional fragment or variant thereof, in a suitable host cell.
In one embodiment there is provided a method for producing Hsp70, or a functional fragment or variant thereof, as described herein; said method comprising the steps of providing a vector encoding said Hsp70, or a functional fragment or variant thereof, and expressing said vector either in vitro, or in vivo in a suitable host organism, thereby producing said Hsp70, or a functional fragment or variant thereof.
In one embodiment there is provided an isolated recombinant or transgenic host cell comprising a vector encoding Hsp70, or a functional fragment or variant thereof, as defined herein.
In one embodiment there is provided a method for generating a recombinant or transgenic host cell, said method comprising the steps of providing a vector encoding Hsp70, or a functional fragment or variant thereof, introducing said vector into said recombinant or transgenic host cell and optionally also expressing said vector in said recombinant or transgenic host cell, thereby generating a recombinant or transgenic host cell producing said Hsp70, or a functional fragment or variant thereof.
In another embodiment there is provided a transgenic, mammalian organism comprising the host cell producing said Hsp70, or a functional fragment or variant thereof. In a further embodiment, the transgenic, mammalian organism comprising the recombinant or transgenic host cell according to the present disclosure is non-human. The transgenic host cell can be selected from the group consisting of a mammalian, plant, bacterial, yeast or fungal host cell.
To improve the delivery of the DNA into the cell, the DNA must be protected from damage and its entry into the cell must be facilitated. Lipoplexes and polyplexes, have been created that have the ability to protect the DNA from undesirable degradation during the transfection process. Plasmid DNA can be covered with lipids in an organized structure like a micelle or a liposome. When the organized structure is complexed with DNA it is called a lipoplex. There are three types of lipids that may be employed for forming liposomes; anionic (negatively charged), neutral, or cationic (positively charged). Complexes of polymers with DNA are called polyplexes. Most polyplexes consist of cationic polymers and their production is regulated by ionic interactions.
In one embodiment, the vector comprising Hsp70, or a functional fragment or variant thereof, may be used for gene therapy. Gene therapy is the insertion of genes into an individual's cells and tissues to treat a disease, such as a hereditary disease in which a deleterious mutant allele is replaced with a functional one.
In another embodiment, Hsp70, or a functional fragment or variant thereof, may be administered as naked DNA. This is the simplest form of non-viral transfection. Delivery of naked DNA may be performed by use of electroporation, sonoporation, or the use of a “gene gun”, which shoots DNA coated gold particles into a cell using high pressure gas.
Composition
Whilst it is possible for the bioactive agents to be administered as the raw chemical, it is in some embodiments preferred to present them in the form of a pharmaceutical formulation. Accordingly, also provided herewith is a composition, such as a pharmaceutical composition, i.e. a pharmaceutically safe composition, comprising a bioactive agent as defined herein. The composition in one embodiment comprises a pharmaceutically and/or physiologically acceptable carriers or excipients.
Pharmaceutical compositions containing a bioactive agent of the present disclosure may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 2000.
It is thus an aspect to provide a composition, such as a pharmaceutical composition, comprising a bioactive agent that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, for use in the treatment of a frontotemporal disorder.
Administration and Dosage
A bioactive agent or composition comprising the same as defined herein is in one embodiment administered to individuals in need thereof in pharmaceutically effective doses or a therapeutically effective amount.
A therapeutically effective amount of a bioactive agent is in one embodiment an amount sufficient to cure, prevent, reduce the risk of, alleviate or partially arrest the clinical manifestations of a given disease or disorder and its complications. The amount that is effective for a particular therapeutic purpose will depend on the severity and the sort of the disorder as well as on the weight and general state of the subject. An amount adequate to accomplish this is defined as a “therapeutically effective amount”.
In one embodiment, the composition is administered in doses of 1 μg/day to 100 mg/day; such as 1 μg/day to 10 μg/day, such as 10 μg/day to 100 μg/day, such as 100 μg/day to 250 μg/day, such as 250 μg/day to 500 μg/day, such as 500 μg/day to 750 μg/day, such as 750 μg/day to 1 mg/day, such as 1 mg/day to 2 mg/day, such as 2 mg/day to 5 mg/day, or such as 5 mg/day to 10 mg/day, such as 10 mg/day to 20 mg/day, such as 20 mg/day to 30 mg/day, such as 30 mg/day to 40 mg/day, such as 40 mg/day to 50 mg/day, such as 50 mg/day to 75 mg/day, or such as 75 mg/day to 100 mg/day, such as 100 mg/day to 150 mg/day, such as 150 mg/day to 200 mg/day, or such as 200 mg/day to 250 mg/day, such as 250 mg/day to 300 mg/day, such as 300 mg/day to 400 mg/day, such as 400 mg/day to 500 mg/day, such as 500 mg/day to 600 mg/day, such as 600 mg/day to 700 mg/day, such as 700 mg/day to 800 mg/day, such as 800 mg/day to 900 mg/day, such as 900 mg/day to 1000 mg/day.
In one embodiment, the bioactive agent or composition is administered at a dose of 1 μg/kg body weight to 100 mg/kg body weight; such as 1 to 10 μg/kg body weight, such as 10 to 100 μg/day, such as 100 to 250 μg/kg body weight, such as 250 to 500 μg/kg body weight, such as 500 to 750 μg/kg body weight, such as 750 μg/kg body weight to 1 mg/kg body weight, such as 1 mg/kg body weight to 2 mg/kg body weight, such as 2 to 5 mg/kg body weight, such as 5 to 10 mg/kg body weight, such as 10 to 20 mg/kg body weight, such as 20 to 30 mg/kg body weight, such as 30 to 40 mg/kg body weight, such as 40 to 50 mg/kg body weight, such as 50 to 75 mg/kg body weight, or such as 75 to 100 mg/kg body weight.
In one embodiment, a dose is administered one or several times per day, such as from 1 to 6 times per day, such as from 1 to 5 times per day, such as from 1 to 4 times per day, such as from 1 to 3 times per day, such as from 1 to 2 times per day, such as from 2 to 4 times per day, such as from 2 to 3 times per day. In one embodiment, a dose is administered less than once a day, such as once every second day or once a week.
Routes of Administration
It will be appreciated that the preferred route of administration will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated, the location of the tissue to be treated in the body and the active ingredient chosen.
Systemic Treatment
In one embodiment, the route of administration allows for introducing the bioactive agent into the blood stream to ultimately target the sites of desired action.
In one embodiment the routes of administration is any suitable route, such as an enteral route (including the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal and intraperitoneal administration), and/or a parenteral route (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal administration).
Appropriate dosage forms for such administration may be prepared by conventional techniques.
Parenteral Administration
Parenteral administration is any administration route not being the oral/enteral route whereby the bioactive agent avoids first-pass degradation in the liver. Accordingly, parenteral administration includes any injections and infusions, for example bolus injection or continuous infusion, such as intravenous administration, intramuscular administration or subcutaneous administration. Furthermore, parenteral administration includes inhalations and topical administration.
Accordingly, the bioactive agent or composition is in one embodiment administered topically to cross any mucosal membrane of an animal, e.g. in the nose, vagina, eye, mouth, genital tract, lungs, gastrointestinal tract, or rectum, for example the mucosa of the nose, or mouth, and accordingly, parenteral administration may also include buccal, sublingual, nasal, rectal, vaginal and intraperitoneal administration as well as pulmonal and bronchial administration by inhalation or installation. In some embodiments, the bioactive agent is administered topically to cross the skin.
In one embodiment, the intravenous, subcutaneous and intramuscular forms of parenteral administration are employed.
Local Treatment
In one embodiment, the bioactive agent or composition is used as a local treatment, i.e. is introduced directly to the site(s) of action. Accordingly, the bioactive agent may be applied to the skin or mucosa directly, or the bioactive agent may be injected into the site of action, for example into the diseased tissue or to an end artery leading directly to the diseased tissue.
Combination Treatment
It is also an aspect to provide a bioactive agent that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, for use in the treatment of a frontotemporal disorder, in combination with other treatment modalities.
Thus, in one embodiment, the bioactive agent is administered to an individual in need thereof in combination with at least one other treatment modality, such as conventional or known treatment modalities for frontotemporal disorders
Administering more than one treatment modality in combination may occur either simultaneously, or sequentially. Simultaneous administration may be two compounds comprised in the same composition or comprised in separate compositions, or may be one composition and one other treatment modality performed essentially at the same time. Sequential administration means that the more than one treatment modalities are administered at different time points, such as administering one treatment modality first, and administering the second treatment modality subsequently. The time frame for administering more than one treatment modality sequentially may be determined by a skilled person in the art for achieving the optimal effect, and may in one embodiment be between 30 minutes to 72 hours.
The treatment modalities in the form of chemical compounds may be administered together or separately, each at its most effective dosage. Administering more than one compound may have a synergistic effect, thus effectively reducing the required dosage of each drug.
It is also an aspect to provide a composition comprising, separately or together, i) a bioactive agent that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, and ii) other treatment modalities, for use in the treatment of a frontotemporal disorder.
In one embodiment other treatment modalities, or conventional or known treatment modalities for frontotemporal disorders.
In one embodiment the bioactive agent that increases the intracellular concentration and/or activity of one or more heat shock proteins, including Hsp70, is administered in combination with, and/or formulated as a combination product, with one or more further active ingredients.
Treatment of Mutant VCP Mice and VCP Patient iPSC-Derived Motor Neurons with Arimoclomol Ameliorates FTD and ALS Pathology
Fronto-temporal Dementia (FTD) is the most common type of dementia presenting in those under the age of 65, with an incidence of approximately 3.5 per 100,000 in England while, Amyotrophic lateral sclerosis (ALS) has an incidence of 2 per 100,000. Unfortunately, to date, there is no cure for either of these debilitating diseases. While extensive research effort is directed towards identifying the cause of these diseases, there is clear evidence of protein dyshomeostasis in the brain and spinal cord with the presence of misfolded and aggregated proteins (1). Valosin containing protein (VCP) is a central protein in normal protein degradation pathways. Mutations in this protein can give rise to ubiquitin-positive proteinaceous aggregates and mislocalisation of nuclear TDP-43, an RNA modulating protein which become translocated to the cytoplasm. As these are both prominent pathological features of both FTD and ALS targeting protein mishandling may be an effective therapeutic approach for these diseases.
To investigate this possibility, we studied the effects of augmenting the heat shock response (HSR) in neural tissues of a transgenic mouse model of multisystem proteinopathy, also known as Inclusion Body Myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD). Pathology in these mice is caused by over-expression of mutant human VCP (A232E mutation) which causes the severest form of multisystem proteinopathy also in patients. In addition, we also examined the effects of arimoclomol in human iPSC-derived motor neurons from mVCP patients and confirmed
The HSR is an endogenous cytoprotective response to cell stress, which involves an upregulation in the expression of key molecular chaperones called heat shock proteins (HSP), in an attempt to improve protein handling and restore cellular protein homeostasis. We have previously shown that pharmacological up-regulation of the heat shock response (HSR), with a co-inducer of the HSR called Arimoclomol, attenuates disease in mouse models of neurodegenerative diseases including ALS (2) as well as Spinal Bulbar Muscular Atrophy (3).
In addition we have recently shown that treatment with Arimoclomol attenuates muscle pathology in mutant VCP (mVCP) mice, which recapitulates characteristic features of the inflammatory myopathy Inclusion body myopathy (IBM) in skeletal muscle (4,5). These results showed that treatment of mVCP mice with Arimoclomol led to decreased protein aggregation and TDP-43 mislocalisation, as well as reduced myofibre atrophy and degeneration. While we observed no significant reduction in grip strength relative to body weight in transgenic mice with wildtype human VCP (wt-VCP) between 4 to 14 months of age, mVCP mice showed a significant, 44.1% reduction in grip strength during this period. Interestingly, in mVCP mice treated with Arimoclomol, there was no significant decline in grip strength throughout the duration of the study. Furthermore, in vivo assessment of maximal tetanic force of the hindlimb extensor digitorum longus (EDL) muscles of 14 month old mice revealed a significant decrease in force generated by EDL muscles of mVCP mice compared to wt-VCP controls. However, treatment of mVCP mice with Arimoclomol prevented this reduction in muscle force. These results show that there is a significant loss of muscle force in mVCP mice between 4 and 14 months of age, and that this is prevented by chronic treatment with Arimoclomol.
These beneficial effects of Arimoclomol on muscle pathology in mVCP mice are likely to result, at least in part, to an increase in the expression of HSPs, since Western blot analysis of muscle from mVCP mice treated with Arimoclomol showed a two-fold increase in the expression of HSP70 compared to that of untreated mVCP mice.
Methods
Transgenic Mouse Colonies: Colonies of mutant Valosin Containing Protein (VCP) (A232E) and control wild type VCP (wtVCP) transgenic mice were maintained at the UCL Institute of Neurology under license from the UK Home office.
Arimoclomol treatment: Male mutant VCP (mVCP) mice were treated with Arimoclomol (120 mg/kg daily; orally in drinking water), from the start of symptom onset at 4 months, until close to end-stage at 14 months. Transgenic mice over-expressing wild-type human VCP (wt-VCP) were used as controls, and 10 male mice per group were studied; a sample size sufficient to test for statistical significance at P<0.05 in a single sex group.
Motor Unit Counts:
In vivo physiology was carried out acutely at 14 months of age on terminally anaesthetized mice to quantify the number of motor units in the extensor digitorum longus (EDL) muscle in the hindlimb of mice in all experimental groups. Briefly, Isometric contractions were elicited by stimulating the Extensor Digitorum Longus (EDL) motor nerve using pulses of 0.02 ms duration and supramaximal intensity via electrodes. Contractions were elicited by stimulation of the sciatic nerve. The number of motor units in the EDL muscles was determined by stimulating the motor nerve with stimuli of increasing intensity, resulting in stepwise increments in twitch tension because of successive recruitment of motor axons.
Motor Neuron Counts and Area Measurements:
20 μm spinal cord sections from L3-L6 were stained with gallocyanin to visualise neurons for quantification (5 mice per group). Sciatic pool motor neurons were counted from every 3rd section as seen under a Leica light microscope. 20 images of the sciatic pool regions of the spinal cord were taken per animal to measure the size distribution of motor neurons present in this area (at ×20 magnification). A minimum of 3 mice per experimental group were assessed. The soma area of motor neurons was determined by drawing around individual Nissl stained (gallocyanin) motor neuron cell bodies in cross-sectional images of L4-L5 region of the spinal cord. This was recorded using Leica Application Suite V3.8 analysis software and presented as a percentage of the total number of motor neurons per group.
Immunohistochemistry:
Brain and spinal cords were harvested from mice in all experimental groups following transcardial perfusion with saline followed by 4% paraformaldehyde (PFA). Brain and spinal cords were then kept in 4% PFA for 12 hours before being transferred to a 30% sucrose solution. Cross-sections of brain and spinal cords were cut at 20 μm and blocked in 10% Normal Goat serum with 0.1% Triton X100 in PBS before primary antibodies were added for 1 hour at room temperature. Primary antibodies: Rabbit anti-TDP-43 [1:500], Rabbit anti-ubiquitin [1:500], mouse anti-phospho tau (AT8) [1:100], Rabbit/mouse anti-Beta-3 tubulin [1:100], mouse anti-HSP70 [1:100], Mouse anti-p62 [1:200], Rabbit anti-LC3 [1:500]Rabbit anti-Iba1 [1:100], anti neurofilament 2H3 [1:20], Synaptic vesicle protein [1:20], Fluoromyelin Red myelin stain [1:300]. Fluorescently labelled or biotinylated secondary antibodies were used 1:1000 for 2 hours at room temperature. 4′6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) was used to counterstain for nuclei in all fluorescent images. Imaging of tissue was using a standard Leica light/fluorescent microscope or a LSM 780 confocal microscope.
α-Bungarotoxin-tetramethylrhodamine was used to fluorescently label neuromuscular junction endplates for 1 hour at RTP.
Fluorescent images were visualised under a Leica fluorescent microscope and analysed using Leica Application Suite software (Leica Microsystems, Germany).
Human iPSC Derived Motor Neuron Generation:
iPSCs were maintained on Geltrex (Life Technologies) with Essential 8 Medium media (Life Technologies), and passaged using EDTA (Life Technologies, 0.5 mM). All cell cultures were maintained at 37° C. and 5% carbon dioxide. Motor neuron (MN) differentiation was carried out using a previously published protocol (Hall et al., 2017). Briefly, iPSCs were first differentiated to neuroepithelium by plating to 100% confluency in chemically defined medium consisting of DMEM/F12 Glutamax, Neurobasal, LGlutamine, N2 supplement, non-essential amino acids, B27 supplement, 3-mercaptoethanol (all from Life Technologies) and insulin (Sigma). Treatment with small molecules from day 0-7 was as follows: 1 μM Dorsomorphin (Sigma), 2 μM SB431542 (Sigma), and 3 μM CHIR99021 (Sigma). At day 8, the neuroepithelial layer was enzymatically dissociated using dispase (GIBCO, 1 mg/ml), plated onto Geltrex coated plates and next patterned for 7 days with 0.5 μM retinoic acid and 1 μM Purmorphamine. At day 14 spinal cord MN precursors were treated with 0.1 μM Purmorphamine for a further 4 days before being terminally differentiated in 0.1 μM Compound E (Sigma) to promote cell cycle exit. Cells were treated with 10 μM Arimoclomol for 24 hours following terminal differentiation and fixed in PFA for immuno-labelling.
Human Brain Samples:
Frozen human brain samples were obtained from the Queen Square Brain Bank for Neurological Disorders, UCL Institute of Neurology. Cortical sections were received cryosectioned at 12 μm onto glass slides. Immunohistochemistry and immunofluorescent staining was conducting using standard histology protocols. Primary antibodies used are as follows: Rabbit anti-TDP-43 [1:500], mouse anti-HSP70 [1:100], Mouse anti-p62 [1:200], Rabbit anti-LC3 [1:500]. DAPI was used 1:1000 to label nuclei.
Loss of motor units and neurons in mVCP mice is prevented by Arimoclomol treatment Physiological data from our in vivo study has shown that there is a reduced number of motor units innervating the hindlimb muscles of mVCP mice compared to wild-type VCP controls, and that this reduction in motor unit survival is prevented in mVCP mice treated with Arimoclomol (
Quantification of the number of motor neurons in the sciatic pool (L3-6) of the spinal cord reveals a significant reduction in motor neuron survival in mVCP mice compared to controls (
TDP-43 Pathology in mVCP Mice is Attenuated with Arimoclomol Treatment
The C-terminal portion of the nuclear protein TDP-43 becomes mislocalised to the cytoplasm in the brain and spinal cord of mVCP mice, with nuclear clearance of TDP-43 observed in brain tissue (
Intracellular Ubiquitin Protein Aggregation and Extracellular p-Tau Detected in m VCP Mice is not Detected in mVCP Mice Treated with Arimoclomol
mVCP mice develop ubiquitin-positive intracellular aggregates in both brain and spinal cord tissue (
Increased Protein Degradation in Grey and White Matter and Myelin Degeneration in mVCP Spinal Cord is Improved by Arimoclomol Treatment
p62 (sequestosome) shuttles aberrant proteins to the proteasome and for autophagy for degradation, and LC3 is a marker of autophagy. Our results show a substantial increase in p62 expression in spinal cord white and grey matter in mVCP mice compared to controls (
The accumulation of p62, which is normally cleared when associated with proteins undergoing degradation, suggests a possible defect in autophagy in mVCP mice. We therefore looked at the expression of LC3, a protein which is recruited to the autophagosomal membrane before being degraded in the autolysosomal lumen, thereby indicating autophagic activity in a cell (9). In mVCP spinal cord, we detected a substantially increased expression of LC3 in oligodendrocytes associated with abnormal myelin, providing further evidence of defective autophagy in these cells (
Arimoclomol Treatment Enhances HSP70 Expression in mVCP Mouse Brain and Spinal Cord
Heat shock protein 70 (HSP70) expression is a key marker of the heat shock response in cells. This protein is increased in the brain and spinal cord of mVCP mice and further augmented in the brain and spinal cord of mVCP mice treated with Arimoclomol (
Cell Death is Prevented in the Brain of mVCP Mice Treated with Arimoclomol
Mutations in VCP cause <1% of all FTD cases20, and a third of patients diagnosed with multisystem proteinopathy (MSP) caused by mutations in VCP go on to develop FTD8. We therefore examined the brain of mVCP mice for FTD-like pathology.
Apoptosis in the brain was assessed by Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay for apoptotic cells, where nuclei containing double-stranded breaks in the DNA fluoresce green (Fluoroscein-tagged), indicating DNA degradation at the later stage of apoptosis (
Stress Granule Markers Detected in Aggregates in m VCP Cells not Seen in Arimoclomol Treated Animals
Three markers of stress granules, Tia1, FMRP and G3BP, were used to detect the presence of these RNA-containing structures (
Neuromuscular Junction (NMJ) Defects are Prevented in mVCP Mice Treated with Arimoclomol
The NMJ is the chemical synapse connecting a motor neuron to the muscle fibre it innervates and therefore preservation of its morphology and function is crucial for muscle contraction to be elicted. In mVCP mice, there was clear evidence of NMJ disruption and denervation (
Pathological Hallmarks of VCP Pathology are Present in mVCP Patient-Derived iPSC Motor Neurons and are Improved Following Treatment with Arimoclomol.
TDP-43 mislocalisation is a characteristic hallmark for both FTD and ALS pathology. In this study we assessed the expression pattern of TDP-43 in iPSC-derived motor neurons derived from patients with mutations in VCP (
TDP-43 Mislocalisation and Increased HSP70 Levels are Present in Human FTD Patient Brain Tissue
To confirm that the pathology observed in mVCP mouse brain and spinal cord, and mVCP patient iPSC-derived motor neurons, and that the beneficial effects of arimoclomol on these characteristics are clinically relevant, we also assessed the pattern of TDP-43 expression in port-mortem tissue from patients with different forms of fronto-temporal dementia (FTD;
Protein Degradation Markers Seen in mVCP Mice are Also Present in FTD Patient Brain
We also assessed the expression of markers of protein degradation, p62 and LC3, in FTD patient brain, which were both altered in brain of mVCP mice. Both p62 and LC3 were present in cytoplasmic aggregates in all four patient samples (
We have previously shown that muscle pathology in mVCP mice is attenuated following treatment with Arimoclomol (5). In this study we have extended these findings to examine the effects of Arimoclomol on the brain and spinal cord of these mice with a mutation in the key protein-handling protein VCP and corroborated our findings in VCP patient-derived iPSC motor neurons. Furthermore, the key pathological features of disease observed in mVCP mouse spinal cord and brain which is ameliorated following treatment with arimoclomol, are also a feature of pathology in postmortem human brain tissue from patients with a number of forms of FTD.
In mVCP mice, skeletal muscles recapitulate characteristic features of the inflammatory myopathy Inclusion body myositis including formation of ubiquitinated aggregates, TDP-43 mislocalisation, as well as changes in mitochondrial morphology, function and degeneration of muscle fibres. These myopathic changes correlated with a reduction in grip strength in mVCP mice compared to controls. Treatment with Arimoclomol attenuated all of these disease features in mVCP mice (5).
Electrophysiological assessment of the mVCP mice has shown that the decline in muscle strength and muscle force generation corresponds to a reduction in the number of motor units. In these studies, the hind limb muscle EDL was assessed in the mVCP mice for maximal tetanic force generation which revealed a 31.5% reduction in force (5), correlating with a 30% reduction in the number of EDL motor units (
Two motor neuron sub-types are present on the spinal cord motor pool—large alpha neurons, which innervate extrafusal muscle fibers of skeletal muscle and are directly responsible for initiating their contraction, and smaller gamma neurons which innervate the intrafusal muscle fibres of muscle spindles, specialized sensory organs. Alpha motor neurons are selectively vulnerable in ALS. Examination of the size distribution of sciatic motor neurons show a clear shift in the size distribution of surviving motor neurons on mVCP mice, towards smaller neurons compared to WT and wtVCP controls (
In mVCP mice treated with Arimoclomol from the start of symptom onset at 4 months of age to 14 months of age, motor neuron survival is improved and motor unit number is maintained. There is also no significant change in the size distribution of motor neurons in mVCP mice treated with Arimoclomol compared to controls, with little or no shift in size distribution compared to controls. As a result, the muscle force generated by the EDL muscle in Arimoclomol treated mVCP mice is significantly greater than in untreated mice (
To investigate the pathological changes that may have played a role in the death of motor neurons in mVCP mice and reduction of muscle function, key pathological changes which are hallmarks of neurodegenerative diseases were investigated in these cohorts of mice.
TDP-43 (transactive response DNA binding protein 43 kDa) is a protein involved in RNA metabolism and is ubiquitously expressed in most tissues, normally within the nucleus of cells (6). This RNA-binding protein is cleaved by activated Caspase 3/7 following cell stress cytoplasm (7). The translocated C-terminus of TDP-43 is detected in the brain and spinal cord of ALS and FTD patients. In mVCP mice, we observed an increase in mislocalised TDP-43 in the brain and spinal cord compared to control mice. However, in mice treated with Arimoclomol there was a clear reduction in cytoplasmic staining for TDP-43 (
Protein dyshomeostasis has been proposed to play a key role in the pathogenesis of neurodegenerative diseases in which protein aggregation is commonly observed (1). Analysis of protein aggregation in the mVCP mice showed cytoplasmic ubiquitin-positive aggregates in the spinal cord and brain with aggregates seen in the cortex and midbrain (
FTD is commonly referred to as a tauopathy due to the presence of hyper-phosphorylated tau (p-tau) lesions in the brains of FTD patients (8). Interestingly, in the brain of mVCP mice, immunostaining for phosphorylated tau (antibody AT8) revealed large extracellular lesions in the cortex, which were not present in the control animals (
Interestingly, cell death was noted in the brain of mVCP mice following a TUNEL apoptosis assay which revealed dying cells in layer 1 of the cortex (
To determine whether the improvements in brain and spinal cord pathology observed in Arimoclomol treated mVCP mice were a result of the co-induction of the HSR, these tissues were immuno-stained for HSP70. HSP70 expression was upregulated in the brain and spinal cord of mVCP mice compared to control animals (
Our results show clear signs of neuronal death in the brain and spinal cord of mVCP mice, reminiscent of human FTD and ALS, and link this degeneration to our previously published data which also shows pathology in the muscle of mVCP mice. To determine whether pathology seen in spinal cord motor neurons affects the interface with muscle fibres at the neuromuscular junction we examined the neuromuscular junction. Our results show evidence for disrupted endplate structure and denervation in muscle sections of mVCP mice (
To test whether the data from our in vivo mVCP mouse studies was corroborated in human cells, we examined mVCP-patient derived iPSC motor neurons. We focused on TDP-43 cytoplasmic mislocalisation as a key pathological outcome measure in ALS and FTD, and a pathological feature of all three tissues assessed in vivo in mVCP mice (ie in muscle, in spinal cord and in the cortex). Under basal conditions mVCP patient iPSC-motor neurons showed cytoplasmic mislocalisation of TDP-43, which was not observed in cells from healthy controls or importantly, in cells treated with Arimoclomol. Moreover, HSP70 levels in mVCP MNs was increased under basal conditions compared to healthy controls, and was augmented in mVCP patient iPSC-motor neurons treated with Arimoclomol, demonstrating successful co-induction of the HSR by arimoclomol in human neurons.
In order to confirm that the key pathological features observed in tissues of mVCP mice and in mVCP patient iPSC-derived motor neurons which are ameliorated by treatment with arimoclomol are a good readout of the human disease, we also examined the expression of TDP-43 and HSP70 in postmortem samples of brain from patients with a range of FTD subtypes, including a patient with FTD-MND. In all patient brains we identified cells containing mislocalised TDP-43 and an upregulation of HSP70 levels. In addition we also examined signs of disrupted autophagy and protein mishandling in the FTD patient brain samples and found evidence of cytoplasmic LC3 and p62 aggregates in neurons and glia, with p62 also associating with neurofibrillary tangles in the brain of an FTD-MAPT patient. These findings indicate that common pathomechanisms may be the cause of disease in all these patients.
In conclusion, the results of this study show that expression of mutant VCP in transgenic mice results in brain and spinal cord pathology reminiscent of FTD and ALS respectively, replicating key pathological hallmarks of these neurodegenerative diseases, including TDP-43 mislocalisation, ubiquitin-positive and p62 positive protein aggregation as well as lesions of phosphorylated tau in the brain and cell death in both the spinal cord and brain. Denervation at the NMJ and abnormal endplate structure links the neuronal findings to the myopathy seen previously and demonstrates that multiple tissues can be affected in mVCP mice, as observed in patients with multisystem proteinopathy (MSP) where FTD, ALS and IBM can all coexist in individual patients. Importantly, in this study we have shown that in both the mouse model and the patient-derived MNs, treatment with Arimoclomol led to an amelioration of all the pathological changes observed. Since the same pathological characterizes are also observed in FTD patient postmortem brain tissue, these results suggest that induction of Hsp70 exemplified by treatment with Arimoclomol in FTD patients may be a beneficial therapeutic strategy.
| Number | Date | Country | Kind |
|---|---|---|---|
| EP17172669.8 | May 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/063662 | 5/24/2018 | WO | 00 |
