Field of the invention

This invention relates to new uses of mebendazole.

Background of the invention

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent form of polycystic kidney disease, and the most common inherited progressive kidney disorder, with an estimated prevalence in Europe of 1 in 2525. Characterised by fluid-filled renal cysts which appear in utero and expand throughout life, ADPKD causes enlarged kidneys and leads eventually to end stage renal disease in 50% of patients by the age of 60. In addition, extrarenal manifestations, including hepatic cysts and subarachnoid hemorrhage, vary in frequency and severity between patients.

Mutations in the PKD1 gene are responsible for approximately 80% of ADPKD cases, while mutations in the PKD2 gene account for approximately 15% of cases, with the remaining cases genetically unresolved or due to rare mutations in other genes. PKD1 and PKD2 encode the polycystin proteins, PCI and PC2, respectively, which - form a core complex in the primary cilium of renal epithelial cells and regulate intracellular signalling pathways. However, these become dysregulated upon ADPKD-associated mutations.

At present, the available treatments for ADPKD are mainly limited to managing the symptoms and the conditions caused by ADPKD (such as high blood pressure, pain, or surgery to remove kidney stones, etc). An existing medication that is indicated to slow kidney function decline in adults at risk of rapidly progressing ADPKD is tolvaptan, however this drug is known to have common and occasionally severe side effects, such as liver injury.

Autosomal recessive polycystic kidney disease (ARPKD) is a much rarer form of polycystic kidney disease, with an estimated prevalence in the USA of 1.2 in 100,000. As with ADPKD, ARPKD is also characterized by multiple fluid-filled renal cysts which appear in utero and expand to cause enlarged kidneys. In the case of ARPKD, however, the disease course is much more severe, with many patients who survive the neonate period progressing to end stage renal disease by late childhood or early adulthood. Extrarenal manifestations are common, and can be severe, with pulmonary hypoplasia claiming approximately 30% of ARPKD patients in the neonatal period, and hepatic fibrosis occurring in many patients with varying severity.

Unlike ADPKD, there are no disease-modifying treatments approved for ARPKD, and the only treatment available is symptomatic (mechanical ventilation to treat pulmonary hypoplasia, and antihypertensives to treat high blood pressure etc). Mutations in the PKHD1 gene are responsible for almost all cases of ARPKD. PKHD1 encodes the protein fibrocystin, which localises to the primary cilium of renal epithelial cells. While the function of fibrocystin has not been completely elucidated, it has been shown to interact with the ciliary PC1/PC2 complex implicated in ADPKD, and a number of the same intracellular pathways driving cyst growth are shared between ARPKD and ADPKD. This suggests that therapies targeting disease progression through the inhibition of cyst growth may be effective in both conditions.

Despite various efforts to identify a more effective therapy for treating ADPKD, and ARPKD, no such successful therapies have been reported to date. Especially given the prevalence of these diseases, there is clearly a need for new and effective therapies to treat or prevent ADPKD and ARPKD.

Mebendazole is a broad spectrum benzimidazole anthelmintic that is used in humans and animals for the treatment of various parasitic infections, including those caused by Whipworm, Hookworm, Roundworm, Tapeworm and Threadworm. Its primary mode of action in parasites is via binding to the colchicine-sensitive site of tubulin, thus blocking polymerisation and subsequent microtubule formation, leading to impaired uptake of glucose, a reduced production of energy, and eventually paralysis and death. The selective toxicity of mebendazole is thought to be due to a higher affinity for parasitic over mammalian tubulin targets. Mebendazole has the systematic name (5-benzoyl-lH- benzimidazol-2-yl)carbamic acid methyl ester.

Summary of the invention

The present invention is a composition comprising mebendazole, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of ADPKD and ARPKD. As will be evident from the in vitro and in vivo data presented below, mebendazole is effective in treating and preventing ADPKD and ARPKD.

A first aspect of the invention is a composition comprising mebendazole, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of ADPKD or ARPKD.

A second aspect of the invention is use of mebendazole, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment or prevention of ADPKD or ARPKD.

A third aspect of the invention provides a method of treating or preventing ADPKD or ARPKD comprising administering the patient with a composition comprising mebendazole or a pharmaceutically acceptable salt thereof. Description of the figures

Figure 1 shows the results of mebendazole on cyst growth and cell death in the absence of a ddAVP stimulus in in vitro ADPKD assays.

Figure 2 shows the results of mebendazole on cyst growth and cell death in the presence of a ddAVP stimulus in in vitro ADPKD assays.

Figure 3 shows the results of mebendazole on cyst growth and cell number in the absence and presence of an AVP stimulus in in vitro ARPKD assays

Figure 4 shows the results of mebendazole on kidney cyst burden in an animal model of polycystic kidney disease.

Figure 5 shows the results of mebendazole on increased kidney weight in an animal model of polycystic kidney disease.

Figure 6 shows the results of mebendazole on elevated blood urea levels in an animal model of polycystic kidney disease.

Detailed description

In the present invention, and as demonstrated by the below in vitro and in vivo data, mebendazole inhibits the growth of cysts, without causing an increase in cell death, and is therefore effective at treating and preventing ADPKD and ARPKD.

The term "mebendazole" as used herein refers to any one of the solid state forms: polymorph A, polymorph B, polymorph C, or a mixture of polymorphs A, and/or B and/or C.

In an embodiment, mebendazole refers to mebendazole polymorph A. In another embodiment, mebendazole refers to mebendazole polymorph B. In another embodiment, mebendazole refers to polymorph C.

In an alternative embodiment, mebendazole refers to a mixture of polymorphs A, and/or B and/or C. Suitably the mixture is a mixture of polymorphs A and B, or polymorphs A and C, or polymorphs B and C or polymorphs A, B and C. By the term "treatment" or "treating" as used herein, we refer to therapeutic (curative) treatment. Treatment also includes stopping the disease from developing or slowing further progression of the disease. For example, treatment may include preventing a cyst from growing bigger or slowing a cyst's growth rate.

By the term "prevention" or "preventing" as used herein, we refer to "prophylactic" treatment, which includes administering mebendazole to a patient that has mutations in the PKD1 and/or PKD2 and/or PKHD1 gene.

"Patient" and "subject" are used interchangeably and refer to the subject that is to be administered the mebendazole. Preferably the subject is a human. ADPKD and ARPKD are highly allelically heterogeneous diseases. The Mayo Clinic PKD database currently lists 1273 pathogenic PKD1 mutations and 202 pathogenic PKD2 mutations, many of which are unique to individual families. As such there are no prototypical common mutations that represent the diseases. In one embodiment the patient has a mutation in the PKD1 gene and/or the PKD2 gene, preferably the mutation is pathogenic. In one embodiment, the patient has a mutation in the PKHD1 gene, preferably the mutation is pathogenic.

In one embodiment, mebendazole is used for the treatment or prevention of ADPKD or ARPKD, wherein the patient has had or is going to have surgery to remove some or all of cysts caused by the ADPKD or ARPKD. This may be particularly advantageous if the cysts are large and/or expands across tissue boundaries, and/or large in number, so it is difficult to remove them all by surgery and/or a quick removal of at least some of it is desired/beneficial.

The term "surgery" has its normal meaning in the art. Surgery is an invasive technique with the fundamental principle of physical intervention on organs/organ systems/tissues for diagnostic or therapeutic reasons.

As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base.

The present invention is directed to a composition comprising mebendazole, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of ADPKD or ARPKD. In one embodiment the composition comprising mebendazole, or a pharmaceutically acceptable salt thereof, is for use in the treatment or prevention of

ADPKD. In another embodiment the composition comprising mebendazole, or a pharmaceutically acceptable salt thereof, is for use in the treatment or prevention of ARPKD.

In an alternative embodiment, the present invention is directed to a composition comprising mebendazole, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of ADPKD or ARPKD, wherein mebendazole is the only active agent in the composition. By only active agent it is meant that the composition does not contain other components which may be used in the treatment or prevention of ADPKD or ARPKD.

The compositions of the invention may contain a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant any diluent or excipient, such as fillers or binders, that is compatible with the other ingredients of the composition, and which is not deleterious to the recipient. The pharmaceutically acceptable carrier can be selected on the basis of the desired route of administration, in accordance with standard pharmaceutical practices.

In the present invention, the composition may be administered in a variety of dosage forms. In one embodiment, the composition may be formulated in a format suitable for oral, rectal, parenteral, intranasal or transdermal administration or administration by inhalation or by suppository. The composition may be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. Preferably, the composition is formulated such that it is suitable for oral administration, for example tablets and capsules. Tablets and capsules may be prepared with binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, celluloses or polyvinylpyrrolidone; fillers, such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine; lubricants, such as magnesium stearate, talc, polyethylene glycol, or silica; and surfactants, such as sodium lauryl sulfate. Liquid compositions may contain conventional additives such as suspending agents, for example sorbitol syrup, methyl cellulose, sugar syrup, gelatin, carboxymethyl-cellulose, or edible fats; emulsifying agents and surfactants such as lecithin, or acacia; vegetable oils such as almond oil, coconut oil, cod liver oil, or peanut oil; preservatives such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Liquid compositions may be encapsulated in, for example, gelatin to provide a unit dosage form.

The composition may also be administered parenterally, whether intraperitoneally, subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques.

The composition may also be administered by inhalation. An advantage of inhaled medications is their direct delivery to the area of rich blood supply in comparison to many medications taken by oral route. Thus, the absorption is very rapid as the alveoli have an enormous surface area and rich blood supply and first pass metabolism is bypassed.

The present invention also provides an inhalation device containing the composition of the present invention. Typically said device is a metered dose inhaler (MDI), which contains a pharmaceutically acceptable chemical propellant to push the medication out of the inhaler.

The composition may also be administered by intranasal administration. The nasal cavity's highly permeable tissue is very receptive to medication and absorbs it quickly and efficiently. Nasal drug delivery is less painful and invasive than injections, generating less anxiety among patients. By this method absorption is very rapid and first pass metabolism is usually bypassed, thus reducing inter-patient variability. Further, the present invention also provides an intranasal device containing the composition according to the present invention.

The composition may also be administered by transdermal administration. For topical delivery, transdermal and transmucosal patches, creams, ointments, jellies, solutions or suspensions may be employed. The present invention therefore also provides a transdermal patch containing the composition.

The composition may also be administered by sublingual administration. The present invention therefore also provides a sub-lingual tablet comprising the composition. The composition may also be formulated with an agent which reduces degradation of the substance by processes other than the normal metabolism of the patient, such as anti-bacterial agents, or inhibitors of protease enzymes which might be the present in the patient or in commensural or parasite organisms living on or within the patient, and which are capable of degrading the compound.

Liquid dispersions for oral administration may be syrups, emulsions and suspensions.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspension or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for injection or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

In an embodiment of the invention, the composition is administered in an effective amount to treat or prevent ADPKD or ARPKD. An effective dose will be apparent to one skilled in the art, and is dependent on a number of factors including age, sex, weigh, which the medical practitioner will be capable of determining.

In a preferred embodiment, the composition comprises 25 to 3500 mg, more preferably 500 to 3000 mg, yet more preferably 1000 to 2600 mg of mebendazole.

In one embodiment, a single dose of the composition of the invention comprises between 10 mg/kg to 100 mg/kg, preferably 20 mg/kg to 70 mg/kg, more preferably 30 mg/kg to 60 mg/kg, yet more preferably 40 mg/kg to 50 mg/kg of mebendazole based on the subject's weight (kg). In an embodiment of the invention, the composition is administered at least once a day. Preferably it is administered as a single daily dose.

Alternatively, the composition may be administered once a day, twice a day, three times a day or four times a day.

Preferably the single daily dose is 25 to 3500 mg, more preferably 500 to 3000 mg, yet more preferably 1000 to 2600 mg of mebendazole.

In an embodiment of the invention, the composition comprises 12.5 to 1750 mg and is administered twice daily. Preferably each dose is 12.5 to 1750 mg, more preferably 250 mg to 1500 mg, yet more preferably 500 mg to 1300 mg of mebendazole.

In another embodiment, each dose of the composition of the invention administered twice daily comprises between 5 mg/kg to 50 mg/kg, preferably 10 mg/kg to 35 mg/kg, more preferably 15 mg/kg to 30 mg/kg, yet more preferably 20 mg/kg to 25 mg/kg of mebendazole based on the subject's weight (kg). In an embodiment of the invention, the composition comprises 8 mg to 1170 mg and is administered three times daily. Preferably each dose is 8 mg to 1170 mg, more preferably 170 mg to 1000 mg, yet more preferably 330 mg to 870 mg of mebendazole.

In another embodiment, each dose of the composition of the invention administered thrice daily comprises between 1 mg/kg to 35 mg/kg, preferably 5 mg/kg to 25 mg/kg, more preferably 10 mg/kg to 20 mg/kg of mebendazole based on the subject's weight (kg).

In an embodiment of the invention, the composition comprises 6 mg to 875 mg and is administered four times daily. Preferably, each dose is 6 mg to 875 mg, more preferably 125 mg to 750 mg, yet more preferably 250 mg to 650 mg of mebendazole.

In another embodiment, each dose of the composition of the invention administered four times daily comprises between 1 mg/kg to 20 mg/kg, preferably 5 mg/kg to 15 mg/kg, more preferably 8 mg/kg to 13 mg/kg of mebendazole based on the subject's weight (kg).

Preferably, the dosage regime is such that the total daily dosage of mebendazole does not exceed 3500 mg.

In order to treat or prevent ADPKD or ARPKD, the composition comprising mebendazole may be used in a chronic dosage regime i.e. chronic, long-term treatment. Suitably the regime lasts for at least one month, suitably at least two months, such as at least three months.

The present invention also relates to use of mebendazole, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment or prevention of ADPKD or ARPKD. This embodiment of the invention may have any of the preferred features described above.

The present invention also relates to a method of treating or preventing ADPKD or ARPKD comprising administering the patient with a composition comprising mebendazole or a pharmaceutically acceptable salt thereof. This embodiment of the invention may have any of the preferred features described above. The method of administration may be according to any of the routes described above.

For the avoidance of doubt, the present invention also embraces prodrugs which react in vivo to give a compound of the present invention.

Experimental Section

Example 1 - In vitro drug testing utilizing primary ADPKD patient kidney cells

In this study the efficacy of mebendazole was investigated. Primary ADPKD patient kidney cells derived from resected patient kidneys were cultured in Kidney Culture Medium (OcellO). Patient genotypes were as follows: PKD04 (PKD1: 10594C<T Gln3532*), PKD05 (PKD1: c.5622G>A p.Trp1874*), PKD06 (PKD1: c.5861dupA p.(Asn1954Lysfs*36). Mutations were sequenced in cystic patient tissue that was used to derive these cellular models.

Cells were mixed with PrimCyst-Gel (OcellO BV). 15pL of cell-gel mix was pipetted to 384- well plates using a CyBio Felix 96/250 robotic liquid dispenser. Gel-cell mix was plated at a final cell density of 450 objects per well. After gel polymerization at 37°C for 30 minutes, 33pL culture medium was added to each well. Cells were grown in gel for 24 hours, after which the cells were co-exposed or not with the cystogenic stimulus l-deamino-8-D- arginine vasopressin (ddAVP) and one the following molecules: toxic control compound Staurosporine, or mebendazole.

After 48 hours, cultures were fixed with 4% Formaldehyde and simultaneously permeabilized with 0.2% Triton-XlOO and stained with 0.25pM rhodamine-phalloidin and 0.1% Hoechst 33258 in lx PBS for 2 days at 4°C, protected from light. After fixation and staining, plates were washed with lx PBS, sealed with a Greiner SilverSeal and stored at 4°C prior to imaging.

Imaging was done using Molecular Devices ImageXpress Micro XLS with a 4x NIKON objective. For each well around 35 images in the Z-direction were made for both channels, capturing the whole z-plane in each image. Image analysis was performed using Ominer™ software (OcellO BV). Cysts were segmented using detection of Hoechst-stained nuclei and Rhodamine-phalloidin-stained cellular f-actin. Cyst area was determined by calculating for the area in px of each object in every in-focus plain. This was averaged per well. Fraction of dead cells as an indicator of toxicity was calculated as the amount of nuclei without actin signal relative to the total amount of nuclei, both as count-measurements. Cyst area was normalised to DMSO-treated controls, and presented as a Z-score normalised value.

Mebendazole had a purity of >95%. After solubilisation in DMSO, mebendazole was tested in an eight point concentration curve in quadruplicate across all three ADPKD patient- derived cell models.

Plots were created with GraphPad Prism 8 (GraphPad Software, La Jolla, CA). Results

Mebendazole in the absence of ddAVP stimulus

Results for mebendazole in the absence of ddAVP stimulus can be seen in Figure 1. Mebendazole reduces cyst growth in a concentration-dependent manner without causing an increase in cell death. After 48 hours of culture, mebendazole inhibits the growth of cysts in three cell models of ADPKD: PKD04 (IC50 = 367nM), PKD05 (IC50 = 744nM), PKD06 (IC50 = 689nM). Error bars represent standard deviation.

Mebendazole in the presence of ddAVP stimulus

Results for mebendazole in the presence of ddAVP stimulus can be seen in Figure 2. Mebendazole reduces cyst growth to below the level seen in the absence of a cystogenic stimulus (ddAVP) in a concentration-dependent manner without causing an increase in cell death. After 48 hours of culture, mebendazole inhibits the growth of cysts in three cell models of ADPKD: PKD04 (IC50 = 672nM), PKD05 (IC50 = 465nM), PKD06 (IC50 = 639nM). Error bars represent standard deviation.

Conclusions

Mebendazole inhibits the growth of cysts in vitro in three cell models of ADPKD. It is thus expected that mebendazole will reduce, treat and prevent ADPKD.

Example 2 -.In vitro drug testing utilizing primary patient-derived ARPKD kidney cells

Primary ARPKD cells were derived from patient kidneys. Patient genotype was determined as PKHD1: p.Leu4037Pro.

Cells were cultured in biogels containing RenalCyte media (DiscoveryBiomed) in 384-well plates at a density of 5500 cells/well. The day after plating, cells were co-exposed or not with the cystogenic stimulus arginine vasopressin (AVP), alongside varying concentrations of mebendazole. Additional mebendazole and AVP stimulus was added on culture days 4, 8 and 11.

After 14 days of culture, automated endpoint imaging of cells was conducted with a Biotek Cytation 5 to determine Total Cyst Area per well in pixels ² . Following endpoint imaging, the CellTitre-Glo cell viability assay was performed, and luminescence in raw luminescent units (RLU) determined and used as a proxy for cell number per well. Mebendazole had a purity of >95%. After solubilisation in DMSO, mebendazole was tested in a four-point concentration curve with n = 10 wells/condition in this patient-derived ARPKD cell model.

Results were analysed and plotted with GraphPad Prism 9 (GraphPad Software, La Jolla, CA).

Results

Results for mebendazole in the ARPKD cell model can be seen in Figure 3. In the presence and absence of the cystogenic stimulus arginine vasopressin (AVP), mebendazole inhibits the growth of cysts and the proliferation of cells in a concentration-dependent manner in an ARPKD cell model across 14 days of treatment. * = p<0.05 and **** = p<0.0001 versus untreated control, Dunnett's multiple comparisons test. Error bars represent standard deviation.

Conclusions

Mebendazole inhibits the growth of cysts and proliferation of cells in a cell model of ARPKD.

It is thus expected that mebendazole will reduce, treat and prevent ARPKD.

Example 3 - In vivo drug testing using an animal model of polycystic kidney disease

Materials and Methods

Animals

Ksp-TamCre x PkdlLox mice on a C57/BL6 background were bred, then housed in their litters in IVC cages from Innovive with sterilized corncob bedding (irradiated, dust-free supplied by Innovive, USA) and a shelter-like device (if feasible; PLEXX, The Netherlands) to stimulate natural patterns of behavior. Animals were maintained at 21°C ± 2°C and a relative humidity between 40 to 70%, on a 12/12 light/dark cycle with air renewed 8-20 times per hour. During the in-life period, the animals had free access to standard pelleted food (Diet No. V1534-703 valid until 05/2020, supplied by SNIFF, Germany) and water. To induce a kidney-specific knockout of Pkdl, mice were dosed orally on both postnatal days 10 and 11 (PND10 and 11) with 25mg Tamoxifen/kg bodyweight (Pkdl KO). Non- tamoxifen treated mice were used as Pkdl wildtype controls (Pkd1 WT).

Compounds

Mebendazole was formulated in 10% DMSO / 90% HP-β-CD (20% w/v in water) and dosed by daily intraperitoneal injection (IP) from PND14 to PND27 at 15mg/kg. As a positive control, everolimus was formulated in 5% EtOH / 7% PEG400 / 5% Tween80 / 83% water and dosed by daily oral gavage (PO) from PND14 to PND27 at 4mg/kg. Compound treatment groups were compared with matched vehicle control groups (control for IP: 10% DMSO/ 90% HP-β-CD / no mebendazole; control for PO: 5% EtOH / 7% PEG400 / 5% TweenSO / 83% water / no mebendazole).

Sample processing

On PND27, animals were deeply anaesthetized with isoflurane, and sacrificed by exsanguination through cardiac needle puncture. Whole blood was collected for urea measurements. Kidneys were weighed and fixed in 10% formalin overnight, before paraffin embedding, sectioning, and staining with H8iE for assessment of cystic area.

Statistical analysis

All data were analysed with GraphPad Prism 9.2,0 using One-way ANOVA, with planned comparisons between vehicle control and compound-treated groups. Non-tamoxifen treated mice were not included in statistical analysis.

Results

Mebendazole treatment reduces kidney cvst burden in polvcvstic kidney disease

Daily treatment with IP mebendazole at 15mg/kg or the positive control, PO everolimus at 4mg/kg, reduces the kidney cyst burden induced by kidney-specific knockout of Pkdl compared with respective vehicle controls. The results are shown in Figure 4. Each symbol represents one animal, and error bars represent standard deviation. **** = p < 0.0001 Holm-Sidak multiple comparison; n = 8-13 per group.

Mebendazole treatment ameliorates increased kidney weight in polvcvstic kidney disease Daily treatment with IP mebendazole at 15mg/kg or the positive control, PO everolimus at 4mg/kg, ameliorates the increased kidney weight induced by kidney-specific knockout of Pkdl compared with respective vehicle controls. The results are shown in Figure 5. Kidney weight (KW) is normalised to body weight (BW). Each symbol represents one animal, and error bars represent standard deviation. **** = p < 0.0001 Holm-Sidak multiple comparison; n = 8-13 per group.

Mebendazole treatment ameliorates elevated blood urea levels in polycystic kidney disease Daily treatment with IP mebendazole at 15mg/kg or the positive control, PO everolimus at 4mg/kg, ameliorates the increase in blood urea levels induced by kidney-specific knockout of Pkdl compared with respective vehicle controls. The results are shown in Figure 6. Each symbol represents one animal, and error bars represent standard deviation. **** = p < 0.0001 Holm-Sidak multiple comparison; n = 8-13 per group.

Conclusion

Mebendazole reduces kidney cyst burden, ameliorates increased kidney weight, and ameliorates elevated blood urea in an animal model of polycystic kidney disease. It is thus expected that mebendazole will reduce, treat and prevent ADPKD and ARPKD.