Patent Application
20240390356
The present invention belongs to the pharmaceutical sector and refers to haloperidol or a pharmaceutically acceptable salt or solvate thereof, as well as pharmaceutical compositions comprising the same, for use in methods for preventing and/or treating spinal muscular atrophy.
Spinal muscular atrophy (SMA) is a rare genetic neuromuscular disorder caused by loss of alpha-motor neurons in spinal cord and brain nuclei. The muscle atrophy in SMA was believed to be solely a consequence of muscle fiber denervation; however, there is more and more evidence that muscle defects take place independently and even prior to motor neuron degeneration. The experiments in SMA murine model and human cells revealed defects in the myoblast's proliferation, maturation and fusion processes. Every 1 in 6000 to 10 000 children is affected by the disease and every 1 in 40 people is a carrier of the responsible mutation, which shows a recessive autosomal inheritance pattern.
SMA is primarily caused by mutations in the SMN1 gene. However, as a result of duplication in the genome, the SMN locus (chromosome location 5q13) contains two inverted copies of SMN, called SMN1 (telomeric) and SMN2 (centromeric). The SMN1 and SMN2 genes are almost identical with only 5 nucleotide changes: one in intron 6 (G/A), exon 7 (C/T), exon 8 (G/A) and two in intron 7 (AA/GG). The difference in the coding regions of both genes is a C-to-T transition at position +6 of exon 7 in SMN2. This change is critical, as it results in a different splicing pattern. In SMN1, exon 7 (Ex7) is included, resulting in a full-length and functional SMN protein. However, the C-to-T transition in SMN2 causes Ex7 exclusion in most cases, resulting in a C-terminally truncated SMN protein (Δ7-SMN). Nevertheless, a fraction of the SMN2 mRNAs include Ex7 and results in a functional, full-length SMN protein (FL-SMN), which is, however, insufficient to compensate for the SMN1 loss. Additionally, the truncated SMN protein comprising 282 instead of 294 amino acids has an impaired oligomerization capability and is highly unstable.
All the patients suffering SMA carry one or more copies of the SMN2 gene, which is mostly translated into the unfunctional truncated SMN isoform and produces only a small part of functional protein.
Despite even the most severe cases of SMA normally manifest at postnatal stages, there are studies that report increased motor neuron loss, decreased myotube size and abnormal synaptic contacts at neuromuscular junctions (NMJ), at the prenatal stages. In fact, even though motor neurons are the main cell type affected in SMA physiopathology, there is increasing evidence of the multisystemic character of SMA, directly affecting muscles, heart, brain and pancreas.
The discovery of the mutated SMN1 gene and its unaffected SMN2 copy, allowed linking the SMN2 copy number with the disease severity (see table 1). In fact, the number of SMN2 copies is inversely correlated with the disease severity because the amount of functional protein translated from the SMN2 transcripts partially compensates for SMN deficiencies caused by the mutations in SMN1.
On the other hand, despite the fact that the presence of the SMN2 copy does not fully compensate for the SMN1 loss of function, it constitutes an important therapeutic target in many aspects. In fact, the specific enhancement of SMN2 Ex7 inclusion is a promising therapeutic approach, which increases the full-length transcript levels and, as a consequence, the functional SMN protein levels. The full-length to truncated protein ratio in patients is approximately 1:4, and accordingly, a therapy increasing the Ex7 inclusion will be an efficient therapeutic approach.
Two approaches are currently being explored: one involves the use of Ex7 inclusion-promoting antisense oligonucleotides (ASOs), and the other one is based on small molecules with similar splicing switching properties.
ASOs are single-stranded oligonucleotides designed to be complementary to a targeted RNA sequence. The ASOs can be designed for modulation of splicing or RNA processing. This strategy was proposed for designing therapeutic oligonucleotides that modify the SMN2 pre-mRNA alternative splicing, favoring the Ex7 inclusion in the final transcript. For instance, recently, a 2′-O-methoxyethyl-ASO-based therapy called nusinersen (Spinraza™) has become the first FDA-approved therapy for severe SMA, although additional clinical trials are still being carried out. ASOs do not cross the hematoencephalic barrier when administered in a systemic route and, accordingly, nusinersen requires intrathecal administration. The nusinersen administration protocol includes two phases: one initial charge doses, with a frequency of four injections during two months and, subsequently, a maintenance phase wherein injections are administered every four months.
Another SMA therapy recently approved (2020) for adults and children older than 2 months, is risdiplam (Evrysdi®). Risdiplam interacts directly with the spliceosome to promote exon 7 inclusion in the pre-mRNA of SMN2 producing an increase in the level of the full length functional SMN protein. It is a liquid drug administered orally, since it is capable of crossing the hematoencephalic barrier. This allows treatment without hospitalization and a systemic distribution of the drug, not only through the central nervous system but also in other organs and peripheric tissues.
Using the same therapeutic target, i.e. promoting the expression of the SMN protein, Zolgensma™ (Onasemnogene abeparvovec) was approved in 2019 for pediatric SMA patients under the age of 2 years. Onasemnogene abeparvovec is a gene therapy for the replacement of SMN1, independently of gene SMN2, i.e., it is directed directly to the dysfunctional gene causing SMA. This replacement is achieved using a non-replicating adeno-associated virus capsid (scAAV9). The administration of this therapy consists of a single intravenous systemic application, since it crosses the hematoencephalic barrier and also enters motor neurons, muscle and other tissues where SMN is highly expressed, resulting in a systemic and durable increase of SMN1 expression as well as a longer survival of the patient, since the protein is ubiquitously expressed.
Despite the existence of several approved therapies, there is still the need for therapeutic alternatives to overcome the disadvantages and limitations of the current approved drugs. In fact, the currently marketed therapies include several limitations or disadvantages: invasive administration routes (nusinersen), efficacy dependent on patient age and type (more effective when patients are treated at earlier stages), adverse effects and high cost.
For instance, nusinersen administration route is highly invasive, since it is administered intrathecally, several times per year, during the whole life of the patient's life and, accordingly, requires hospitalization periods. Moreover, this administration route may include several difficulties for patients with serious scoliosis or having suffered a spinal fusion. Analogously, the presence of low levels of the SMN protein in peripheric tissues, due to the lack of systemic availability of the drug due to the administration route and the consequence thereof is also a concern when using nusinersen.
The development of drugs that are systemically administered, such as onasemnogene abeparvovec, or with oral administration, such as risdiplam, has overcome some of the issues raised by nusinersen administration route. However, onasemnogene abeparvovec has been only approved for patients of age 2 or younger, and the presence of preexistent anti-AAV9 antibodies in the SMA patient population, or the presence of antibodies developed after the treatment may also be an issue, since it may reduce the therapeutic benefits of this therapy and increase the immune response risks.
Additionally, the occurrence of important adverse effects may appear in patients being administered those treatments. Among those adverse effects are headaches and back pain due to nusinersen administration, acute hepatic damage, bleeding and cardiac damage due to onasemnogene abeparvovec. Additionally, since risdiplam has not been used for long periods, it is still unknown the possible adverse effects that may cause long term, as well as the risks that may pose in other areas. Moreover, risdiplam features a low capability to cross the hematoencephalic barrier.
Lastly, one of the most important limitations of all the approved therapies is their high cost, which prevents access thereto to many patients. For instance, the first treatment year with nusinersen currently represents a cost of about 750,000 USD, and 375,000 USD for the following years of chronic treatment. On the other hand, the price of risdiplam represents about 340,000 USD per year and the single dose onasemnogene abeparvovec is the world's most expensive drug costing the required single dose about 2.1 million USD.
Accordingly, there is still a need to develop new treatments for SMA, with a more convenient, more affordable and less invasive administration regime, which are able to provide good levels of the full-length, functional SMN protein and have a good capability to cross the hematoencephalic barrier. In particular, a drug capable of increasing the amount of full-length functional SMN protein in different body tissues, in particular, in motor neurons, with a simple administration route and accessible costs, is still needed.
The present invention relates to haloperidol, or a pharmaceutically acceptable salt, ester or solvate thereof, for use in a method for preventing and/or treating spinal muscular atrophy. This method may result in an increase of full length functional SMN protein levels.
Another aspect of present invention also refers to haloperidol, or a pharmaceutically acceptable salt, ester or solvate thereof, for use in a method for preventing and/or treating spinal muscular atrophy, wherein said method increases the SMN2 transcript expression and/or increases SMN2 exon 7 inclusion and, accordingly, results in an increase of full length functional SMN protein levels.
Additionally, present invention refers to a pharmaceutical composition comprising haloperidol, or a pharmaceutically acceptable salt, ester or solvate thereof, and at least a pharmaceutically acceptable excipient; for use in a method for preventing and/or treating spinal muscular atrophy. This method may result in an increase of functional SMN protein levels.
Yet another aspect of present invention refers to a pharmaceutical composition comprising haloperidol, or a pharmaceutically acceptable salt, ester or solvate thereof, and at least a pharmaceutically acceptable excipient; for use in a method for preventing and/or treating spinal muscular atrophy, wherein said method increases the SMN2 transcript expression and/or increases of SMN2 exon 7 inclusion and, accordingly, results in an increase of full length functional SMN protein levels.
Finally, another aspect of present invention refers to a pharmaceutical composition comprising haloperidol and moxifloxacin, or a pharmaceutically acceptable salt, ester or solvate thereof, and at least a pharmaceutically acceptable excipient, wherein said pharmaceutical composition comprises a molar ratio of haloperidol: moxifloxacin from 1:5 to 1:10.
The present invention refers to haloperidol, or a pharmaceutically acceptable salt, ester or solvate thereof, for use in a method for preventing and/or treating spinal muscular atrophy. This method can result in an increase of full length functional SMN protein levels.
Haloperidol is an antipsychotic drug, known to cross the hematoencephalic barrier, and is a strong antagonist of the dopamine receptor 2, which is indicated for the treatment of the manifestations of several psychotic disorders including schizophrenia, acute psychosis, Tourette syndrome, and other severe behavioral disorders. Additionally, haloperidol has also been used for the management of chorea associated with Huntington's disease or in palliative care, probably, as an antiemetic agent.
To that respect, present invention shows that, surprisingly, in addition to the metabolic effects known in the art, haloperidol administration can result in an increase of full length functional SMN protein levels, an effect that makes said compound useful in the treatment of spinal muscular atrophy.
In fact, Example 1 shows that haloperidol produced a significant increase in SMN protein levels and, in particular, produced a significant increase in the SMN2 functional full-length isoform, as shown in Example 2.
Accordingly, present invention refers to haloperidol, or a pharmaceutically acceptable salt, ester or solvate thereof, for use in a method for preventing and/or treating spinal muscular atrophy, wherein said method results in an increase of full length functional SMN protein levels, in particular, wherein said method increases the SMN2 transcript expression and/or increases of SMN2 exon 7 inclusion.
As used herein a full length functional SMN or SMN2 protein can be any full length functional SMN or SMN2 protein. This also implies that any isoforms of this protein are envisioned. The present invention also contemplates that the functional full length SMN protein or SMN2 protein has a sequence that has a percent identity of at least 85%, 90%, 95%, 96%, 97%, 98% or 99% or 100% to a sequence as set forth in SEQ ID No. 5. The present invention contemplates that the functional full length SMN protein has a sequence as set forth in any one of SEQ ID No. 5 to SEQ ID No. 8.
In accordance with the present invention, the term “identity” or “percent identity” in the context of the SMN or SMN2 protein of sequence of SEQ ID No. 5 refers to two or more polypeptide sequences that are the same or that have a specified percentage of amino acids that are the same (e.g. at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity) when compared to SEQ ID No. 5 and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison as known in the art or by manual alignment and visual inspection. Sequences having, for example 85% or greater identity are considered to be substantial identical. Those having skill in the art know how to determine percent identity between/among sequences by using, for example, algorithms such as those based on CLUSTALW computer program (Thompsom Nucl. (1994)) or FASTDB (Brutlag et al. (1990)) as known in the art. Also available to those having skill in this art are BLAST and PSI-BLAST algorithms (Altschul et al. (1977)). The BLASTP program for amino acid sequences uses as default a word size (W) of 6, an expect threshold of 0.05, and a comparison of both strands. Furthermore, the BLOSUM 62 scoring matrix (Henikoff and Henikoff (1992)) can be used. For example, BLAST2.6. which stands for Basic Local Alignment Search Tool (Altschul. Nucl. Acids Res. 25 (1997), 3389-3402; Altschul. (1993), 290-300; Altschul et al. (1990), 403-410, can be used to search for local sequence alignments.
It is further envisioned that the method of the present invention increases the number of synaptic contacts of neuromuscular junctions compared to the number of synaptic contacts of neuromuscular junctions before administering haloperidol. It is also contemplated that the method of the present invention reduces the number of denervated endplates compared to the number of denervated endplates before administering haloperidol. How the synaptic contacts of neuromuscular junctions or the denervated endplates can be measured is outlined in example 9 and
Further, it is envisioned that the method of the present invention increases the fiber area, fiber perimeter and Ferret's diameter compared to the fiber area, fiber perimeter and Ferret's diameter before administering haloperidol. How the fiber area, fiber perimeter and Ferret's diameter can be measured is outlined in example 8 and
The term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt, which upon administration to the patient is capable of providing (directly or indirectly) a compound as described herein. Such salts preferably are acid addition salts with physiologically acceptable organic or inorganic acids, or alkali addition salts with acceptable organic or inorganic bases. Examples of the acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate, phosphate, and organic acid addition salts such as, for example, lactate, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulphonate and p-toluenesulphonate. Examples of the alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, N,N-dialkylenethanolamine, triethanolamine and basic aminoacids salts. However, it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the invention since those may be useful in the preparation of pharmaceutically acceptable salts. Procedures for salt formation are conventional in the art. Preferably, said pharmaceutically acceptable haloperidol salt is haloperidol lactate.
The term “pharmaceutically acceptable ester” refers to any form of the active compound in accordance with the invention in which the hydroxyl group of haloperidol forms an ester with a carboxylic acid. Preferably, said pharmaceutically acceptable haloperidol ester is haloperidol decanoate.
The term “pharmaceutically acceptable solvate” in accordance with this invention should be understood as meaning any form of the active compound in accordance with the invention in which said compound is bonded by a non-covalent bond to another molecule (normally a polar solvent), including especially hydrates and alcoholates.
Present invention shows, therefore, that haloperidol, a pharmaceutically acceptable salt, ester or solvate thereof, is useful in the prevention and/or treatment of SMA. In this sense, the examples of present application show that, in addition to the effect of haloperidol in the SMN protein levels, treatment with haloperidol had, as a consequence effects on myoblasts and motor neurons.
In fact, SMN-deficient myoblasts, as those from SMA patients, have been reported to have impaired fusion capabilities. To that respect, myoblasts treated with haloperidol significantly increased the fusion index, recovering this capacity up to 66.7% (compared to a fusion rate of about 68% for healthy myoblasts) when patient myoblasts have reduced it to 50% (
Finally, in vivo assays carried out in a murine SMA model showed that haloperidol administration resulted in a significant weight increase in treated SMA mice, and, in addition, the behavioral assessment showed that haloperidol administration improved motor performances (
Accordingly, present invention also refers to a pharmaceutical composition comprising haloperidol, or a pharmaceutically acceptable salt, ester or solvate thereof, and at least a pharmaceutically acceptable excipient; for use in a method for preventing and/or treating spinal muscular atrophy. The method can result in an increase of full length functional SMN protein levels, in particular, wherein said method can increase the SMN2 transcript expression and/or increases of SMN2 exon 7 inclusion and, accordingly, results in an increase of full length functional SMN protein levels.
It is further envisioned that the pharmaceutical composition of the present invention increases the number of synaptic contacts of neuromuscular junctions compared to the number of synaptic contacts of neuromuscular junctions before administering or in the absence of said pharmaceutical composition or compared to a control. It is also contemplated that the pharmaceutical composition of the present invention reduces the number of denervated endplates compared to the number of denervated endplates before administering said pharmaceutical composition or compared to a control. How the synaptic contacts of neuromuscular junctions or the denervated endplates can be measured is outlined in example 9 and
Further, it is envisioned that the pharmaceutical composition of the present invention increases the fiber area, fiber perimeter and Ferret's diameter compared to the fiber area, fiber perimeter and Ferret's diameter the absence of said pharmaceutical composition. How the fiber area, fiber perimeter and Ferret's diameter can be measured is outlined in example 8 and
The excipient included in the pharmaceutical compositions for use according to present invention, refers, for the purpose of present invention, to an inert ingredient such as, but not limited to, cosolvents, surfactants, oils, humectants, emollients, preservatives, stabilizers and antioxidants.
Another aspect of present invention refers to the use of haloperidol, or a pharmaceutically acceptable salt, ester or solvate thereof, or to the use of a pharmaceutical composition comprising the same, for the manufacturing of a medicament for the prevention and/or treatment of spinal muscular atrophy. Said medicament can result in an increase of full length functional SMN protein levels and, in particular wherein administering said medicament can increase the SMN2 transcript expression and/or increases of SMN2 exon 7 inclusion.
Yet another aspect of present invention refers to a method of preventing and/or treating spinal muscular atrophy, wherein said method comprises administering an effective amount of haloperidol, or a pharmaceutically acceptable salt, ester or solvate thereof, or of a pharmaceutical composition comprising the same, to a subject in need thereof. Preferably, said method results in an increase of full length functional SMN protein levels and, in particular, said method increases the SMN2 transcript expression and/or increases of SMN2 exon 7 inclusion.
In a preferred embodiment of the invention, the subject is a pediatric subject. In another preferred embodiment of the invention, the subject is an adult.
A preferred aspect of the invention refers to haloperidol, or a pharmaceutically acceptable salt, ester or solvate thereof, or to a pharmaceutical composition comprising the same, for use in a method for preventing and/or treating spinal muscular atrophy, wherein said haloperidol, or a pharmaceutically acceptable salt, ester or solvate thereof, or pharmaceutical composition comprising the same is administered to a pediatric subject or to an adult.
For the purposes of present invention, an effective amount of the compounds disclosed herein, when said effective amount is referred to a therapeutic use of said compounds, is that which provides a therapeutic effect in the subject or an objectively identifiable improvement in the health condition of said subject as noted by the clinician or other qualified observer.
In a preferred embodiment, said pharmaceutically acceptable salt, ester or solvate thereof, is haloperidol lactate or haloperidol decanoate. Preferably, the spinal muscular atrophy is type I, type II, type Ill or type IV. In this regard, type 1 develops in 6-months-old or younger babies; type 2 appears in babies, who are 7 to 18 months old; type 3 develops at 19 months of age or older and type 4 affects adults.
More preferably, the spinal muscular atrophy is type IA, type IB, type IC, type IIA, type IIB, type IIIA, type IIIB or type IV.
In one embodiment of the present invention, the pharmaceutical compositions disclosed herein may optionally comprise at least a second therapeutic agent having additive or synergistic biological activities. For the purposes of present invention, the term “therapeutic agent” should be taken as synonyms and mean a chemical entity that exerts therapeutic effects when administered to human or animal beings.
Accordingly, in a preferred embodiment of present invention haloperidol, or said pharmaceutically acceptable salt, ester or solvate thereof, or the pharmaceutical composition comprising the same, is administered prior to, together with, or after administering an additional therapeutic agent for use in the treatment of spinal muscular atrophy.
In an embodiment of present invention said additional therapeutic agent is an SMN gene replacement therapy, an SMN2 splicing modifier, a muscle enhancing agent, a p38 MPAK inhibitor, an antimyostatin, a calcium channel modifier or troponin activation therapy.
Preferably, said additional therapeutic agent is selected from the group consisting of: moxifloxacin, nusinersen, onasemnogene abeparvovec (AVXS-101), risdiplam (RO703406 or RG7916), BIIB110, Columbia/NU-p38aMAPK inhibitor, branaplam (LMI070), monoclonal antibody SRK-015, albuterol, splicing modifier RG7800, recombinant protein RO7239361, reldesemtiv, azithromycin, bortezomib and trochostatin A or a combination thereof.
Moxifloxacin (IUPAC name 1-cyclopropyl-7-[(1S,6S)-2,8-diazabicyclo[4.3.0]nonan-8-yl]-6-fluoro-8-methoxy-4-oxoquinoline-3-carboxylic acid is an antibiotic with a quinolone structural core that is able to increase the full length SMN protein levels by promoting exon 7 inclusion in SMN2 in a concentration-dependent manner.
Nusinersen is a 2′-O-methoxyethyl phosphorothioate-modified antisense oligonucleotide that promotes exon 7 inclusion in SMN2 transcripts by blocking through the steric hindrance of a splicing silencer.
Onasemnogene abeparvovec, known previously as AVXS-101 (Zolgensma®) is a gene replacement therapy that uses a non-replicating adeno-associated virus capsid (scAAV9) to deliver a functional copy of the SMN gene to motor neuron cells.
Risdiplam, also known as RO703406 or RG7916 (Evrysdi®), is a small molecule that promotes exon 7 inclusion in pre-mRNA of SMN2, providing a higher expression of the full-length functional SMN protein.
In a preferred embodiment, said additional therapeutic agent is moxifloxacin, nusinersen or risdiplam, or a combination thereof. More preferably, said additional therapeutic agent is nusinersen or risdiplam, or a combination thereof. Even more preferably, said additional therapeutic agent is moxifloxacin or a pharmaceutically acceptable salt, ester or solvate thereof.
In fact, the combined administration of haloperidol and moxifloxacin provides a surprisingly improved activity, providing a synergic effect, as shown in example 7. In fact, example 7, as well as
Thus, even more preferably, haloperidol, or said pharmaceutically acceptable salt, ester or solvate thereof, or the pharmaceutical composition comprising the same, is administered prior to, together with, or after administering moxifloxacin, providing said combined administration a synergic (higher than additive) activity, in the prevention and/or treatment of spinal muscular atrophy, when compared to the activity that said compounds provide when administered separately. In a preferred embodiment, the haloperidol for use, or a pharmaceutically acceptable salt, ester or solvate thereof is administered together with moxifloxacin, or a pharmaceutically acceptable salt, ester or solvate thereof, preferably in a pharmaceutical composition comprising haloperidol and moxifloxacin, or a pharmaceutically acceptable salt, ester or solvate thereof, and at least one pharmaceutically acceptable excipient.
More preferably said haloperidol for use is administered prior to, together with, or after administering moxifloxacin at a molar ratio of haloperidol: moxifloxacin from 1:5 to 1:10; or said haloperidol for use is administered in a pharmaceutical composition comprising haloperidol and moxifloxacin, or a pharmaceutically acceptable salt, ester or solvate thereof, at a molar ratio of haloperidol: moxifloxacin from 1:5 to 1:10.
Accordingly, another aspect of present invention refers to a pharmaceutical composition comprising haloperidol and moxifloxacin, or a pharmaceutically acceptable salt, ester or solvate thereof, and at least a pharmaceutically acceptable excipient, wherein said pharmaceutical composition comprises a molar ratio of haloperidol: moxifloxacin from 1:5 to 1:10.
One embodiment refers to said pharmaceutical composition comprising haloperidol and moxifloxacin, or a pharmaceutically acceptable salt, ester or solvate thereof, and at least a pharmaceutically acceptable excipient, wherein said pharmaceutical composition comprises a molar ratio of haloperidol: moxifloxacin from 1:5 to 1:10; for use as a medicament, and in particular, for use in the prevention and/or treatment of spinal muscular atrophy.
Also disclosed is the use of said pharmaceutical composition comprising haloperidol and moxifloxacin, or a pharmaceutically acceptable salt, ester or solvate thereof, and at least a pharmaceutically acceptable excipient, in the manufacturing of a medicament for the prevention and/or treatment of spinal muscular atrophy, and wherein said medicament comprises a molar ratio of haloperidol: moxifloxacin from 1:5 to 1:10.
Further disclosed is a method of preventing and/or treating spinal muscular atrophy wherein said method comprises administering an effective amount of a pharmaceutical composition comprising haloperidol and moxifloxacin, or a pharmaceutically acceptable salt, ester or solvate thereof, and at least a pharmaceutically acceptable excipient, to a subject in need thereof, wherein said pharmaceutical composition comprises a molar ratio of haloperidol: moxifloxacin from 1:5 to 1:10.
Additionally, the combined administration of haloperidol with risdiplam and/or nusinersen provides also a surprisingly improved activity (data not shown). Accordingly, more preferably, haloperidol, or said pharmaceutically acceptable salt, ester or solvate thereof, or the pharmaceutical composition comprising the same, is administered prior to, together with, or after administering risdiplam and/or nusinersen.
Additionally, the dosage of the compounds and compositions disclosed in accordance with the invention varies depending on the compound and the condition being treated for example the age, weight, and clinical condition of the recipient patient, as well as their use in combination with other therapeutic agents. Other factors include: the route of administration, the patient, the patient's medical history, the severity of the disease process, and the potency of the particular compound. The dose, or effective amount, should be sufficient to ameliorate symptoms or signs of the disease treated without producing unacceptable toxicity to the patient.
One preferred embodiment disclosed herein refers to the route of administration, which may be any route that effectively transports the compounds and compositions disclosed in present disclosure, to the appropriate or desired sites of action, such as oral, nasal, topical, pulmonary, transdermal, intrathecal or parenteral, e. g., rectal, subcutaneous, intravenous, intraurethral, intramuscular, intranasal, ophthalmic solution or an ointment.
Preferably, haloperidol, or said pharmaceutically acceptable salt, ester or solvate thereof, or the pharmaceutical compositions comprising haloperidol alone or in combination with other therapeutic agents, and in particular comprising haloperidol and moxifloxacin, is administered orally, intrathecally or parenterally, more preferably orally, intrathecally or via transdermal administration route.
For oral administration, either solid or fluid unit dosage forms can be prepared. For preparing solid compositions such as tablets, the compounds for use according to present invention, are mixed into formulations with conventional ingredients such as talc, magnesium stearate, di-calcium phosphate, magnesium aluminum silicate, calcium sulphate, starch, lactose, acacia, methylcellulose, and functionally similar materials as pharmaceutical diluents or carriers.
Capsules are prepared by mixing the compounds for use according to present invention with an inert pharmaceutical diluent and filling the mixture into a hard gelatin capsule of appropriate size. Soft gelatin capsules are prepared by machine encapsulation of slurry of the compound with an acceptable vegetable oil, light liquid petrolatum or other inert oil. Fluid unit dosage forms for oral administration such as syrups, elixirs and suspensions can be prepared. The water-soluble forms can be dissolved in an aqueous vehicle together with sugar, aromatic flavoring agents and preservatives to form syrup. An elixir is prepared by using a hydroalcoholic (e. g., ethanol) vehicle with suitable sweeteners such as sugar and saccharin, together with an aromatic flavoring agent. Suspensions can be prepared with an aqueous vehicle with the aid of a suspending agent such as acacia, tragacanthin, methylcellulose and the like.
The pharmaceutical compositions include the compounds described in present application associated with pharmaceutically acceptable excipients, which may be a carrier or a diluent, as a way of example. Such compositions can be included in a capsule, sachet, paper or other container. In making the compositions, conventional techniques for the preparation of pharmaceutical compositions may be used. For example, the compounds for use according to present invention may be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier that may be in the form of an ampoule, capsule, sachet, paper, or other containers. When the carrier serves as a diluent, it may be solid, semi-solid, or liquid material that acts as a vehicle, excipient, or medium for the active compound. The compounds for use according to present invention can be adsorbed on a granular solid container for example in a sachet. Some examples of suitable carriers are water, salt solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, lactose, terra alba, sucrose, cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hydroxymethylcellulose, and polyvinylpyrrolidone. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. Said compositions may also include wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. The compositions for use according to the invention may be formulated so as to provide quick, sustained, or delayed release of the compounds for use according to present invention after administration to the patient by employing procedures well-known in the art.
The pharmaceutical compositions can be sterilized and mixed, if desired, with auxiliary agents, emulsifiers, salt for influencing osmotic pressure, buffers and/or coloring substances and the like, which do not deleteriously react with the compounds for use according to present invention.
In addition to the compounds for use according to present invention, the compositions for use according to the invention may include, depending on the composition and mode of delivery desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which include vehicles commonly used to form pharmaceutical compositions for animal or human administration. The diluents are selected so as not to unduly affect the biological activity of the combination.
Examples of such diluents that are especially useful for injectable formulations are water, the various saline, organic or inorganic salt solutions, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may include additives such as other carriers; adjuvants; or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like.
Furthermore, excipients can be included in the compositions disclosed. Examples include, but are not limited to, cosolvents, surfactants, oils, humectants, emollients, preservatives, stabilizers and antioxidants. Any pharmacologically acceptable buffer may be used, such as, tris or phosphate buffers. Effective amounts of diluents, additives, and excipients are those amounts that are effective in obtaining a pharmaceutically acceptable formulation in terms of solubility, biological activity, etc.
The pharmaceutical compositions comprising the compounds for use according to present invention may be incorporated into a microsphere. The compounds for use according to present invention can be loaded into albumin microspheres, from which it is possible to recover such microspheres in a dry powder for nasal administration. Other materials suitable for the preparation of microspheres include agar, alginate, chitosan, starch, hydroxyethyl starch, albumin, agarose, dextran, hyaluronic acid, gelatin, collagen, and casein. The microspheres can be produced by various processes known to the person skilled in the art, such as a spray drying process or an emulsification process.
Another preferred embodiment of the invention is the dosage scheme of the compounds for use according to present invention. The term “unit dosage form” refers to physically discrete units suitable as unitary dosages for subjects, e.g., mammalian subjects, e.g. humans, dogs, cats, and rodents, each unit containing a predetermined quantity of active material calculated to produce the desired pharmaceutical effect in association with the required pharmaceutical diluent, carrier or vehicle. The specifications for the unit dosage forms of this invention are dictated by and dependent on (a) the unique characteristics of the compounds and extracts disclosed above herein and the particular effect to be achieved and (b) the limitations inherent in the art wherein said compounds or extracts are used in humans and animals. Examples of unit dosage forms are tablets, capsules, pills, powder packets, wafers, suppositories, granules, cachets, teaspoonfuls, tablespoonfuls, dropperfuls, ampoules, vials, aerosols with metered discharges, segregated multiples of any of the foregoing, and other forms as herein described. The compositions disclosed herein can be included in kits, which can contain one or more-unit dosage forms of the composition and instructions for use.
Slow or extended-release delivery systems, including any of a number of biopolymers (biological-based systems), systems employing liposomes, colloids, resins, and other polymeric delivery systems or compartmentalized reservoirs, can be utilized with the compositions described herein to provide a continuous or long-term source of the therapeutic compound.
In an embodiment of present invention, the haloperidol, or said pharmaceutically acceptable salt, ester or solvate thereof, and more preferably haloperidol decanoate, or a pharmaceutical composition comprising the same, is administered by transdermal administration route, preferably using a skin patch or by intrathecal administration route, preferably using a pump with intrathecal port catheter. Accordingly, one embodiment of the invention refers to a skin patch comprising haloperidol, or a pharmaceutically acceptable salt, ester or solvate thereof, or a pharmaceutical composition comprising the same, and more preferably comprising a pharmaceutical composition comprising haloperidol and moxifloxacin, or a pharmaceutically acceptable salt, ester or solvate thereof. In particular, one embodiment refers to said skin patch as a medicament and, in particular, for use according to present invention, in the prevention and treatment of spinal muscular atrophy. Said skin patch will slowly release the haloperidol, or the pharmaceutical composition comprising the same, dissolved in an oil with intramuscular delivery (haloperidol decanoate), or even through an intrathecal administration route using a pump with intrathecal port catheter.
Sequences referred to herein:
The examples of the present invention described below aim to illustrate its preferred embodiments without limiting its scope of protection.
Haloperidol was evaluated in fibroblasts derived from patients with type II spinal muscular atrophy.
Fibroblasts derived from SMA patients (GM03813; Coriell Cell Repositories) were grown at 37° C. in cell culture bottles with Dulbecco's Modified Eagle Medium (DMEM; Gibco) and 4.5 g/ml glucose. The medium was supplemented with penicillin (100 U/ml) and streptomycin (100mg/ml) [1% P/S; Gibco] and 10% inactivated fetal bovine serum (FBS; Gibco).
For RNA extraction 400,000 GM03813 fibroblasts were seeded in 60 mm culture dishes in 4 ml of supplemented DMEM medium. The following day the medium was changed, and the candidate compound was added at its maximum non-toxic concentration. The cells were incubated for 48 hours and then washed with 1× PBS and collected in sterile 1.5 ml tubes. The same was done for the evaluation of the effect of haloperidol on the level of SMN protein (example 2) with the difference that 1,000,000 fibroblasts were used in a 100 mm cell culture plate and that the cells were collected after 2 days with a RIPA protein extraction buffer supplemented with a protease inhibitor cocktail (cOmplete; Roche).
For immunostaining 40,000 fibroblasts cells were seeded in a 400 μl 24-well plate with supplemented DMEM medium followed by medium change with the treatment of the corresponding candidate compound. Cells were incubated for 72 h with candidate compounds and fixed in 4% paraformaldehyde for 10 min. All cell cultures were incubated at 37° C. with 5% CO2.
Fibroblasts derived from SMA type Il patients were incubated with haloperidol at three concentrations for 72 h. 10 μg of protein extracted from cells treated with haloperidol were separated by electrophoresis with acrylamide/bis-acrylamide gels SDS-PAGE with the Mini-protean Electrophoresis System (Bio-Rad). After electrophoresis was carried out, the proteins were transferred to a nitrocellulose membrane (GE Healthcare) using a semi-dry electro-transfer system (Bio-Rad) for one hour with a constant voltage of 15 V. The membranes were incubated with a blocking solution (5% skim milk in PBST, 1× PBS, 0.1% Tween 20) for at least 1 h at room temperature. They were then incubated at 4° C. overnight, with the SMN primary antibody blocking solution, MANSMA1 (11F3; Developmental Studies Hybridoma Bank), and subsequently incubated with a peroxidase-conjugated anti-mouse IgG secondary antibody (Sigma) for 1 hour at room temperature. β-actin was used as a loading control, using an antibody against β-actin and a peroxidase-conjugated anti-rabbit IgG.
Chemiluminescence was detected using SuperSignal West Pico Sensitivity Substrate (Pierce) and acquired with ImageQuant LAS 4000 (GE Healthcare). The SMN levels were measured by Western blotting and the results obtained were compared to those of a sample treated with solvent (0.5% DMSO). Statistical significance was calculated using the Student's t-test (* p<0.05, ** p<0.01, *** p<0.001).
The results shown in table 2, below, indicate that haloperidol produced a significant increase in SMN protein levels compared to the control with DMSO.
Haloperidol's effect on the SMN2 transcript was also evaluated.
Extraction of RNA from cells was carried out with ReliaPrep™ RNA Cell Miniprep System (Promega), following the manufacturer's instructions. This extraction system includes DNase I treatment and, therefore, the reverse transcription of RNA from cells does not include this step. RNA concentration and salt contamination were measured with the NanoDrop spectrophotometer (ThermoFisher). The samples were stored at −80° C. 1 ρg of total RNA was retrotranscribed using SuperScript III retrotranscriptase (Invitrogen) following the manufacturer's instructions. Genomic DNA contamination was monitored in the RNA samples without the addition of retrotranscriptase.
For the quantification of the SMN2 full length (FL) and SMN2 without exon 7 (Δ7) transcripts, qPCR Probe Mix (Solis Biodyne) was used. The reaction was carried out in a multiplex manner, in which the signal from the SMN2 and GAPDH probe was read in a single reaction. 10 ng of cDNA was used as a standard and the concentration of both probes was 250 nM.
Primer sets for detection of SMN2 FL or Δ7 isoforms were used at a final concentration of 500 nM while the concentration of the primer set used for GAPDH was 150 nM. All reactions were carried out using a Step-One Real-Time PCR thermocycler (Applied Biosystems). In all cases, the experiment included 3 biological samples and three technical replicates for each sample. Expression levels were normalized to the reference gene using a 2-ΔΔCt method. Primers were obtained from Naryshkin, N. A. et al. Science (2014) 345, 688-693.
The selected SMA fibroblast line has 3 copies of the SMN2 gene and homozygous deletion of Ex7-Ex8 in SMN1 and therefore only SMN2 gene transcripts were measured. The conditions for cell cultures and extraction protocols are described in Example 1.
Haloperidol increases SMN protein levels and significantly increases the SMN2 FL isoform as shown in table 4 below:
As seen in table 4, the FL isoform, which includes exon 7, increases together with haloperidol concentration, while the Δ7 isoform that excludes exon7 does not change in a dose-dependent manner. The quantitative analysis of the isoforms showed that at the highest concentration haloperidol doubled the amount of FL SMN2 transcript, which is consistent with what has been observed on the SMN protein level.
SMN-deficient myoblasts have been reported to have impaired fusion capabilities. We tested haloperidol for its ability to restore myoblasts' ability to form multinuclear myotubes.
The myoblasts derived from SMA type II patients (KM1150SMAII7PV; Institut de Myologie, Paris) and the control myoblasts (AB1190; Institut de Myologie, Paris) were grown at 37° C. with 5% CO2 in cell culture bottles with a KMEM medium that consisted of 4 vol of DMEM Glutamax 4.5 g (Gibco)+1 vol of 199 medium (Gibco) supplemented with 20% inactivated fetal bovine serum (FBS; Gibco), fetuin (25 μg/ml; Life Technologies), hEGF (5 ng/ml; Life Technologies), bFGF (0.5 ng/ml; Life Technologies), insulin (5 g/ml; Sigma), dexamethasone (0.2 μg/ml; Sigma), penicillin (100 U/ml) and streptomycin (100 mg/ml) [1% P/S; Gibco].
For myoblast immunostaining, 40,000 cells were seeded in a 24-well plate (previously covered with collagen) with 400 μl of KMEM medium and incubated overnight. The following day the medium was changed for the DMEM Glutamax 4.5 g (Gibco) differentiation medium supplemented with 2% horse serum (Sigma); penicillin (100 U/ml) and streptomycin (100 mg/ml) [1% P/S; Gibco], gentamicin (50 μg/ml; Sigma) and insulin (10 μg/ml; Sigma). After 72 h the medium was changed, and the corresponding compound treatment was administered (2.5 μM haloperidol; 1 μM risdiplam). The cells were incubated for 72 h with the compounds and fixed in 4% paraformaldehyde for 10 minutes. All cell cultures were incubated at 37° C. with 5% CO2.
SMN-deficient myoblasts have been reported to have impaired fusion capabilities. We tested haloperidol for its ability to restore myoblasts' ability to form multinuclear myotubes. For this, we used two immortalized cell lines: SMA type II patient-derived (SMA; ref. KM1150SMAII7PV) and healthy control (CON; ref. AB1190). To do this, myoblasts were treated with 2.5 M haloperidol for 72 h, and the cells were stained for Desmin, a specific protein of muscle cells. Nuclei were stained with DAPI. Myoblasts were seeded in 24-well plates (previously collagen-treated), in 400 μL KMEM, with 40,000 cells per well, and were incubated overnight at 37° C. The following day the cells were changed to differentiation medium in which they were incubated for 3 days. The following day, the cells were changed in medium and treated with DMSO (0.5%), haloperidol at 2.5 μM and risdiplam at 1 μM (positive control) diluted with 0.5% DMSO. The cells were incubated for 72 h and fixed with 4% paraformaldehyde for 10 minutes. The cellular-membrane was permeabilized with 1× PBST (1× PBS, 0.3% Triton X-100) for 15 min with subsequent incubation in blocking buffer (1× PBST, 1% normal goat serum) for 1 h at room temperature. Primary monoclonal anti-Desmin antibody (mouse, Millipore) was diluted with blocking buffer (1:50) and incubated overnight at 4° C. The cells were washed 3× with PBS-T (5 min) and biotin-conjugated anti-mouse secondary antibody was diluted with blocking buffer (1:200) and incubated for 1 h at room temperature. After 3 washes with PBS-T the cells were incubated with ABC solution (ABC kit, VECTASTAIN) for 45 minutes at room temperature, followed by washes with PBS-T and incubation with streptavidin-FITC (1:200) for 2 h. Finally, the cells were washed with 1× PBS, and the nuclei were stained with DAPI in mounting medium (Vectashield).
It was observed that myoblasts treated with haloperidol significantly increased the fusion index (number of cells with more than 2 nuclei divided by the total number of cells). The fusion index was calculated for healthy cells treated with DMSO 0.5% (CON 0.5%), cells from patients treated with DMSO 0.5%, cells from patients treated with the risdiplam positive control (1 μM) and cells from patients treated with haloperidol (2.5 μM). Healthy myoblasts were observed to have a fusion index of about 68% when patient myoblasts reduced it to 50%. Both, risdiplam and haloperidol treatments were able to almost fully restore the fusion capabilities of SMA myoblasts, recovering this capacity up to 66.7% (
Human induced pluripotent stem cells (hiPSCs) were derived at the Institute for Stem cell Therapy and Exploration of Monogenic diseases (ISTEM) from human primary fibroblasts obtained from Coriell (for the control: GM03814; for SMA: GM00232) and reprogrammed using sendai vectors expressing OCT4, KLF4, SOX2 and c-Myc as described previously [Fusaki et al., 2009].
For spinal motor neurons (MNs) differentiation from hiPSCs, we used a previously reported protocol [Maury et al., 2015]. HiPSC were incubated in suspension in a neural induction medium containing DMEM/F12 and Neurobasal medium supplemented with B27 (Life Technologies), N2 Supplement (Life Technologies), Pen-Strep 0.1%, β-Mercaptoethanol (β-ME) 0.1% (Life Technologies), Ascorbic Acid (0.5 μM, Sigma-Aldrich), Chir99021 (3 μM, Stemgent), SB431542 (20 μM, Tocris), LDN 193189 (0.2 μM, Stemgent) and Y-27632 (10 μM, Stemcell) at a density of 0.2×106 cells/ml in T25 flask or 0.25×106 cells/ml in spinner flask. After 2 days, caudalization of the neural progenitors was obtained by addition of retinoic acid (RA 0.1 μM) and smoothened agonist (SAG, 0.5 μM). After 1 week in suspension, brain-derived neurotrophic factor (BDNF, 10 ng/ml), glial-derived neurotrophic factor (GDNF, 10 ng/ml), and N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT, 10 nM) were added in the medium without SB431542 and LDN193189. Between day 10 and 14, MN progenitors are converted into MNs. At this step (day 10) embryoid bodies (EBs) were dissociated into single cells with Trypsin-EDTA and plated at 3.000 cells per well in 384-well plates (Corning) coated with poly-ornithin (20 μg/ml, Sigma-Aldrich) and 5 g/ml laminin (Invitrogen) in 35 μl of the same medium with Y-27632 (10 μM, Stemcell). Four days later, 35 μl of N2B27 medium containing different concentrations of Haloperidol was added to each well. A solution of 0.1% DMSO (Sigma) was used as a negative control. Cells were treated every 3 days for the next ten days. For each medium change, 32 μl of medium was replaced by 38 μl of N2B27 medium containing the different concentrations of Haloperidol.
4.2. Quantification of the Effect of Haloperidol on hiPSC-Derived MNs Survival
Cells plated were fixed using 4% PFA (Electron Microscopy Sciences) for 5 min at room temperature. Fixed cells were incubated with a blocking buffer composed of 1X PBS, 1% BSA (Sigma) and 0.1% Triton-X-100 (Sigma) for 30 min at room temperature. Cells were next incubated overnight at 4° C. with primary antibodies (for ISL1: Ref: GT15051 Neuromics, 1:1000; TuJI: Ref: 801201 Biolegend, 1:1000; SMN: Ref: 610647 BDTransduction, 1:200). Each antibody was diluted in a solution of 1X PBS containing 1% BSA. The cells were washed 3 times in PBS and incubated for 1 h at room temperature with secondary Alexa Fluor-488, 568, 647-conjugated secondary antibodies diluted to 1:1000 in a solution of 1X PBS containing 1% BSA (ISL1: 568-donkey anti-goat IgG Ref: A11057, ThermoFisher Scientific; TuJI: 647-donkey anti-rabbit IgG Ref: A31573, ThermoFisher Scientific; SMN: 488-donkey anti-mouse IgG Ref: A21202 ThermoFisher Scientific). In addition, Hoechst 33228 (1:3000, Sigma) nuclear staining was performed during the secondary labeling. Finally, the cells were washed 3 times in PBS (
The survival of MNs was determined by quantifying the number of ISL1-positive cells in each condition. ISL1 positive cells were automatically detected using the high-content analysis system Cellomics Array Scan VTI high content imaging system (Thermofisher-Cellomics). Images were acquired with the 20× objective in High Resolution camera mode on two channels: nuclei (channel 1, HOECHST XF93) and ISL1 (channel 2, Texas-red BGRFR_549_15). Acquisition was done for each condition on 4 different wells with 16 fields per well. Based on the Hoechst channel, nuclei were identified using intensity thresholding, background correction and segmentation parameters. Each nuclear region was then reported on the second channel (Texas-red BGRFR_549_15) to limit the ISL1 detection to the nuclear area. ISL1 staining is identified using a fixed intensity threshold, background correction and segmentation parameters.
In this experiment, the MNs were treated for 10 days starting on day 14 from neural induction. A fresh amount of medium with dissolved drugs was delivered every 3 days until day 24 when the MNs were fixed and immunostained. Healthy MNs were used as a cell survival control as well as the neuroprotective drug kenpaullone, which previously proved to decrease the death rate of SMA MNs in such a designed experimental system.
MN survival was presented as a percentage of ISL1 positive cells counted at day 24 normalized to day 14 of differentiation (
As shown in
SMN protein was also quantified using Homogeneous Time Resolved Fluorescence (HTRF, Cisbio Bioassays) according to the manufacturer's protocol. This technology allows the detection and quantification of SMN protein in 384 well plate format. For this, cells were lysed in 15 μL of lysate buffer and 10 μL of cell lysate were transferred to a 384-well small volume white plate containing 5 μL of the antibody solution (1:100 dilution of anti-SMN d2 and anti-SMN cryptate) (Cisbio). The plate was incubated overnight at room temperature. Fluorescence was measured at 665 nm and 620 nm on a CLARIOstar microplate reader (BMG Labtech). Ratio values of (665 nm emission/620 nm emission)×10 000 were used to calculate ΔF (%) according to the following equation:
The effect of haloperidol in a co-culture study of SMA type I motoneurons and SMA type I myoblasts was also tested according to the method disclosed in Young et al., 2018 (ISERM, Paris).
Skeletal muscle cells were obtained from a quadriceps muscle biopsy of a 5 day-old unaffected infant (CHQ or Ctl) (Edom et al. 1994), a paravertebral muscle biopsy of a 11-year-old infant patient suffering from SMA (KM432-7PV or SMA type I) and a paravertebral muscle biopsy of a 7-year-old infant patient suffering from SMA (KM1150-11PV or SMA type II). Primary cells were isolated from skeletal muscle as described previously (Chaouch et al. 2009). These cells were expanded and differentiated as previously described. Briefly, primary and immortalized myoblasts were expanded in a mix of 199 medium/Dulbecco modified Eagles minimal essential medium (1:4 ratio) supplemented with 20% fetal bovine serum (FBS), 25 μg/mL fetuin, 0.5 ng/ml basic fibroblast growth factor, 5 ng/ml epidermal growth factor, 5 μg/mL insulin. Cells were then cultivated in a growth medium consisting of DMEM-F12 glutamax (Life Technologies) supplemented with 20% FBS (Sigma-Aldrich) and 0.1% penicillin/steptomycin (Life Technologies). Myogenic differentiation was induced by switching growth medium to DMEM supplemented with 2% horse serum (Life Technologies).
For co-culture, spinal MNs must be differentiated from hiPSC as described previously, but the embryoid bodies are dissociated at day 14 of differentiation and frozen. 384 micropatterned-well plates were designed and provided by CYTOO SA (Young et al. 2018). The myoblasts were plated at a density of 5 500 cells per well in myoblast growth medium (DMEM/F12-glutamax supplemented with 20% fetal bovine serum (Sigma-Aldrich) and 0.1% penicillin-streptomycin (ThermoFisher) with Y-27632 (10 μM, Stemcell). After 2 days, the growth medium was replaced by the differentiation medium (DMEM/F12-glutamax supplemented with 2% hors serum (ThermoFisher)). On day 4, the hiPSC-dervied MNs were thawed and plated at a density of 8 000 cells per well in N2B27 medium supplemented by brain derived neurotrophic factor (BDNF, 10 ng/mL, Peprotech), glial-derived neurotrophic factor (GDNF, 10 ng/ml, Peprotech), N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT, 10 μM, Tocris) and Y-27632 (10 μM, Stemcell). Cultures have been maintained up to 11 days which correspond to 4 days of myogenic differentiation and 7 days of co-culture with hiPSC-derived MNs. After fixation with 4% PFA (Electron Microscopy Sciences) for 7 min at room temperature, cells were incubated overnight at 4° C. with primary antibodies (listed in Table 5). The cells were washed 3 times in PBS buffer and incubated for 1 h at room temperature with appropriated Alexa fluorescent labeled secondary antibodies (Invitrogen, 1:1 000) and Hoechst (1 μg/mL).
Co-culture images were acquired using Spinning Disk microscopy (Zeiss) with 20× objective. Each image was taken with z-stack and an orthogonal view was performed on Fiji Software. Islet1 quantification was determined by automatically counting the number of Islet1-positive cells by micropattern. Myotube area was quantified by using Fiji Software to quantify total area of cells stained by MF20 antibody per micropattern. A neuroprotective drug kenpaulone was used as a positive control.
All quantitative data were analyzed with Prism 8. Values are represented as mean value±SD. Student's t-test was used to compare data between two groups (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). One-way analysis of variance (ANOVA) was performed to compare data among three or more groups, followed by a post-hoc Tukey's multiple comparison test. Values of p<0.05 were considered as statistically significant (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001).
Results show that the haloperidol not only increases the survival rate of SMA MNs (
The treatment was administered every 3 days for 7 days from seeding both cell types. It was observed a clear increase in myotubes area (
The efficacy of Haloperidol was then tested (University of Turin, Boido et al.) in SMA Δ7 mice (purchased from Jackson Laboratory, Maine, USA; stock number 005025). This triple mutant mouse harbors two transgenic alleles and a single targeted mutant. The Tg(SMN2*delta7)4299Ahmb allele consists of a SMN cDNA lacking exon 7 whereas the Tg(SMN2)89Ahmb allele consists of the entire human SMN2 gene. Mice that are homozygous for the targeted mutant Smn allele and homozygous for the two transgenic alleles exhibit symptoms and neuropathology similar to patients afflicted with SMA type II. At birth, triple mutants are noticeably smaller than normal littermates; by day 5, signs of muscle weakness appear and become progressively more pronounced over the following week as the mice display an abnormal gait, shakiness in the hind limbs and a tendency to fall over. Mean survival is approximately 13 days.
To discriminate their genotype, at birth pups were tail snipped and genotyped by PCR.
Treated SMA mice subcutaneously received 0.5 mg/Kg Haloperidol, dissolved in 2% DMSO in saline solution (HALO group), whereas control mice received an equal amount of saline solution with 2% DMSO (VHL group). Haloperidol or saline solution were daily administered, from P2 (postnatal day 2) to P12 (day of sacrifice).
During the whole period, the animals underwent a number of behavioral motor tests: tail suspension test, righting reflex, hindlimb suspension test, negative geotaxis (El-Khodor et al. 2008, Boido et al. 2018). Mice were evaluated daily starting from P2 for body weight and behavioral performances:
At P12, Halo and VHL animals were sacrificed, and fresh spinal cords and some skeletal muscles (diaphragm, quadriceps, and gastrocnemius) were rapidly dissected. We then verified the effect of Haloperidol on SMN expression on these samples. Tissues were homogenized on ice using a pestle homogenizer in radioimmunoprecipitation assay buffer (RIPA lysis buffer) (Merck Life Sciences) supplemented with 1 mM PMSF, 1 mM DTT, 2 mM sodium orthovanadate (ThermoFisher Scientific) and 1× cOmplete™ Protease Inhibitor Cocktail (Merck Life Sciences). Sample homogenates were incubated on ice for 20 min before being centrifuged for the insoluble material separation at 14,000×g for 20 min at 4° C. Total protein concentration was assayed on supernatants using Bradford reagent (Bio-Rad). Protein denaturation was performed with NuPAGER LDS Sample Buffer supplemented with NuPAGE® Sample Reducing Agent (ThermoFisher Scientific), by heating at 95° C. for 5 min. SDS-PAGE and transfer were performed on 4-20% Mini-PROTEAN® TGX™ Precast Protein Gels and Trans-Blot® Turbo™ mini nitrocellulose membranes using a Trans-Blot® Turbo™ transfer System (Bio-Rad), respectively. Nonspecific binding sites were blocked using 5% nonfat dried milk in PBS-0.2% Tween-20 (Merck Life Sciences) (PBS-T) for 1 h at RT under shaking. All membranes were incubated overnight at 4° C. under shaking with SMN-antibody solution (mouse-anti-SMN, BD Bioscience 610646) diluted 1:2,000 in 2% nonfat dried milk in PBS-T, and with Vinculin-antibody solution (Monoclonal mouse Anti-Vinculin, Clone Hvin-1, Merck Life Sciences) diluted 1:2,000 in 2% nonfat dried milk in PBS-T as loading control. HRP-conjugated secondary antibody (Goat Anti-Mouse IGG HRP, Bio-Rad) was diluted 1:10,000 in 2% nonfat dried milk in PBS-T and incubated for 1 h at RT under shaking. Immunolabeling was detected with Clarity™ Western ECL Blotting Substrates (Bio-Rad) using the ChemiDoc™ imaging system (Bio-Rad). Densitometric quantitation of bands was performed with Fiji (Image J, NIH).
Haloperidol administration supported a significant weight increase in treated SMA mice, starting from P5 (postnatal day 5) and progressively increasing during days (
Moreover, the behavioral assessment showed that Haloperidol administration in SMA mice improved motor performances in the tail suspension test (
The behavioral results positively correlated with SMN expression (
The motoneurons differentiated from the SMA type 1 induced pluripotent stem cells, obtained as disclosed in example 4, were used to evaluate their survival rate after treatment with haloperidol or moxifloxacin. With that purpose, the SMA type 1 MNs were treated with 7 concentrations of each drug separately and the survival rate was measured after 10 days of treatment. We established the most efficient dose (optimal dose) to increase the motoneurons survival when each drug is administered separately (
Next, the MNs were treated with 49 combinations of different concentrations of haloperidol and moxifloxacin and the survival rate was measured after 10 days of treatment (
The results shown in
To evaluate the presence of a synergistic effect between haloperidol and moxifloxacin we applied the Bliss Independence model which adopts a probabilistic perspective by assuming that under additivity, the drugs' effects do not interfere with one another. This allows to determine the expected result at a dose combination (d1, d2) as the sum of the independent drug responses minus their joint effect under additivity (in analogy with stochastic independence) (Thas et al., 2021; Van der Borght et al., 2017). The analysis was performed using the R software, specifically:
The synergistic and antagonistic effects of drug combinations could be also plotted on a two-dimensional contour plot where the x-axis and y-axis represent doses of haloperidol and moxifloxacin, respectively, as shown in
Based on the results shown in
We verified the efficacy of haloperidol on muscle trophism. We first performed morphological analyses of the H/E-stained quadriceps muscles from mice at postnatal day 12 (P12) after 10 days of treatment with haloperidol at 0.5 mg/kg and compared it to mice treated with sham. Specifically, for the morphological analysis of quadriceps skeletal muscle sections were stained with H/E, as previously described (Valsecchi et al. (2015); Rindt et al. (2015)). To morphologically evaluate mean fiber area, perimeter, minimum and maximum Feret's diameter, more than 100 fibers for each animal were drawn with Neurolucida software (MicroBrightField Inc.) and a computer-assisted microscope (Nikon Eclipse E600 microscope). Data were obtained by the associated data analysis software NeuroExplorer (MicroBrightField): we then averaged the means obtained from single animals. Images of skeletal muscles fibers were acquired with Zeiss Axioscan Z.1 digital slide scanner microscope (Carl Zeiss AG).
We further evaluated the mean fiber area, perimeter, and the Feret's diameters of 100 muscle fibers per animal (
After rapid rehydration in PBS, skeletal muscle sections on 4% gelatin-coated slices were stained for NMJ detection. First, the sections underwent antigen retrieval by sequential incubations with 1% SDS (5 min, RT) and sodium citrate solution heated to 95° C. (pH 6, for 7-10 min). Subsequently, non-specific binding sites were blocked using 4% BSA solution, 4% foetal bovine serum and PBS-0.1% Triton-X for 1:30 h, RT. The sections were incubated overnight with the primary antibody (Neurofilament 145 kDa Antibody, CT, clone 3H11, 1:100, Sigma-Aldrich) diluted in the blocking solution for 24 h at 4° C. The sections were subsequently incubated with appropriate fluorochrome-conjugated secondary antibody (1:30 h, RT) and with α-bungarotoxin (BTX) conjugated with Alexafluor-555 (1:500; 11554187, Invitrogen) diluted in 4% BSA and PBS-0.1% Triton-X (1:30 h, RT). Finally, slices were incubated with DAPI in PBS 1× for 3 min and then cover slipped with anti-fade mounting medium. Images were acquired with a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems).
For NMJ innervation analysis (at least 50 NMJs for animal), the presence and number of NF-positive fibers reaching each NMJ was evaluated and counted with computer-assisted microscope (Nikon Eclipse E600 microscope) to classify the NMJs as mono-innervated, multi-innervated or denervated.
Percentage of multi-innervated, mono-innervated and denervated neuromuscular junctions (NMJs) in quadriceps of sham-and haloperidol-treated delta 7 SMA mice was determined. NMJ innervation was analyzed for the number of neurofilament (NF) contacting the endplate, results show a significant reduction in denervated endplates after treatment with haloperidol (sham: 33.75±1.31%; haloperidol: 9.50±1.50%),
Administration of haloperidol to delta 7 SMA mice improves the skeletal muscle trophism and leads to better muscular innervation. The formation of the NMJs proceeds normally in SMA mouse models, but their maturation stalls (Januel et al. (2022). The lack of SMN protein in those models was suggested to have a central role in the formation of the NMJs Boido and Vercelli (2016); therefore, enhancing the SMN levels can improve reciprocal signaling between motoneurons and muscles, important for the formation and maintenance of NMJs (Fralish et al. (2021). Haloperidol successfully contributed to sustaining the NMJ phenotype and muscular innervation in treated mice and delays the skeletal muscle atrophy.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21383162.1 | Dec 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/087022 | 12/20/2022 | WO |
