WO2017059486A1

WO2017059486A1 - Compositions and methods for the treatment of epilepsy

Info

Publication number: WO2017059486A1
Application number: PCT/AU2016/050933
Authority: WO
Inventors: Gillian Alison Webster
Original assignee: Innate Immunotherapeutics Limited
Priority date: 2015-10-06
Filing date: 2016-10-05
Publication date: 2017-04-13

Abstract

The present invention relates to compositions and methods for the treatment of epilepsy. In particular, the present invention relates to the use of muramyl dipeptide crosslinked to form a microparticle for reducing the frequency and/or severity of epileptic seizures.

Description

Compositions and methods for the treatment of epilepsy Field of the Invention

[0001 ] The present application claims priority from Australian Provisional Patent

Application No. 2015904058 (filed on 6 October 2015), the contents of which are herein incorporated in their entirety by reference.

[0002] The present invention relates to compositions and methods for the treatment of epilepsy. In particular, the present invention relates to the use of muramyl dipeptide crosslinked to form a microparticle for reducing the frequency and/or severity of epileptic seizures.

Background of the Invention

[0003] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[0004] Epilepsy is a neurological disorder in which nerve cell activity in the brain becomes disrupted, causing seizures or periods of unusual behaviour, sensations and sometimes loss of consciousness. Epilepsy refers to a clinical phenomenon rather than a single disease entity, since there are many forms and causes of epilepsy.

[0005] The most common type (60%) of epileptic seizures are convulsive. Of these, one- third begin as generalized seizures, affecting both hemispheres of the brain. Two-thirds begin as partial seizures (which affect one hemisphere of the brain) which may then progress to generalized seizures.

[0006] The remaining 40% of epileptic seizures are non-convulsive. An example of this type is the absence seizure, which presents as a decreased level of consciousness and usually lasts about 10 seconds.

[0007] Known causes of epilepsy include head injuries, brain tumors, lead poisoning, maldevelopment of the brain, genetic and infectious illnesses (e.g., CNS inflammation). However, in half of cases, no definitive cause can be identified. [0008] Nearly 10 percent of the population may have a single unprovoked seizure, with at least two unprovoked seizures generally required for an epilepsy diagnosis. In Australia, the US and Europe, 3-4% of the population will develop epilepsy.

[0009] Treatment with medications can control seizures for about 70 percent of people with epilepsy. Older established antiepileptic drugs (AEDs), such as phenytoin,

carbamazepine, clonazepam, ethosuximide, valproic acid and barbiturates are widely prescribed but may induce a range of side effects, including dizziness, nausea, headache, vomiting, fatigue, vertigo, ataxia, blurred vision, and tremor. While more recent AEDs, including felbamate, gabapentin, lamotrigine, oxcarbazepine, tiagabine, topiramate, vigabartrin, zonisamide and levetiracetam, have improved efficacies and side-effect profiles, about 30% of patients have epilepsy that is not controlled by medication, known as refractory epilepsy.

[0010] Accordingly, there is a need for new agents that are effective in the treatment prevention of epilepsy, in particular new agents for reducing the frequency and/or severity of epileptic seizures that have reduced side effects.

[001 1 ] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

Summary of the Invention

[0012] It has been surprisingly found that muramyl dipeptide crosslinked to form a microparticle (MDP microparticle) increases the seizure threshold in animal epilepsy models.

[0013] According to one aspect, the present invention provides a method for the therapeutic or prophylactic treatment of epileptic seizures in a subject, comprising

administering to the subject a composition comprising muramyl dipeptide crosslinked to form a microparticle.

[0014] According to another aspect, the present invention provides use of muramyl dipeptide crosslinked to form a microparticle for the manufacture of a medicament for therapeutic or prophylactic treatment of epileptic seizures in a subject.

[0015] According to another aspect, the present invention provides a composition comprising muramyl dipeptide crosslinked to form a microparticle for use in therapeutic or prophylactic treatment of epileptic seizures in a subject [0016] In one embodiment, the microparticle comprises DNA fragments.

[0017] In another embodiment, the MDP microparticle is isolated from bacteria.

[0018] In one embodiment, the epileptic seizures are convulsive seizures.

[0019] In one embodiment, the epileptic seizures are non-convulsive seizures.

[0020] In one embodiment, the epileptic seizures are prevented.

[0021 ] In one embodiment, frequency of the epileptic seizures is reduced.

[0022] In one embodiment, severity of the epileptic seizures is reduced.

[0023] In one embodiment, duration of the epileptic seizures is reduced.

[0024] In one embodiment, the composition or medicament comprises one or more pharmaceutically-acceptable excipients, carriers, vehicles or diluents.

[0025] In one embodiment, the composition or medicament is administered to the subject parenterally.

[0026] In one embodiment, the composition or medicament is administered to the subject intravenously.

[0027] In one embodiment, the composition or medicament is administered to the subject orally.

[0028] In one embodiment, the composition or medicament is administered to the subject at a dosage of about 1 μg to about 100μg.

[0029] In one embodiment, the composition or medicament is administered to the subject at a dosage of about 100μg to about 1000μg.

[0030] In one embodiment, the composition or medicament is administered to the subject at a dosage of about 100μg to about 700μg.

[0031 ] In one embodiment, the composition or medicament is administered to the subject at a dosage of about 300μg to about 700μg.

[0032] In one embodiment, the composition or medicament is administered to the subject at a dosage of about 500μg to about 700μg. [0033] In one embodiment, the composition is administered to the subject at a dosage of about 1 , 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975 or 1000 μg.

[0034] In one embodiment, the composition or medicament is administered to the subject once a day.

[0035] In one embodiment, the composition or medicament is administered to the subject once a week.

[0036] In one embodiment, the composition or medicament is administered to the subject once a fortnight.

[0037] In one embodiment, the composition or medicament is administered to the subject once a month.

[0038] The appropriate dosage of MDP microparticles, total amount administered and duration of administration can be easily determined by a medical practitioner based on guidance provided herein, the nature and severity of the epilepsy, and the response by the subject to the treatment. As an example, useful individual dosages may be selected from the range 1 μ9 to 1000μg of MDP microparticles, and may be administered once a day, once a week, once a fortnight or once month depending on the subject's condition, symptoms, tolerance and response to treatment. Doses in a higher range can also be used depending on the requirements, for examples doses in the range of 1000μg to 1500μg of MDP microparticles. Dosages at other frequencies may also be employed. An example of a suitable dosage regimen could be to start with an initial dose of 100 μg followed by escalated doses until appropriate beneficial therapeutic effects are observed in the subject, without significant side-effects. The dosage may be given as single bolus dose or infused over time, or given in divided doses. The total amount of MDP microparticles administered will depend on subject response and tolerance to treatment. The composition may be administered once a day, once a week, once a fortnight or once a month for a total period that depends on the subject's response.

[0039] MDP microparticle-containing compositions or medicaments may be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of MDP microparticle-containing composition which may be combined with a carrier material to produce a single dose may vary depending upon the subject being treated, and the particular mode of administration.

[0040] The MDP microparticle-containing compositions or medicaments may be administered alone or in combination with pharmaceutically acceptable excipients, carriers, vehicles or diluents, in either single or multiple doses. Suitable pharmaceutical acceptable excipients, carriers, vehicles and diluents include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. The compositions formed by combining the MDP microparticle-containing compositions and the pharmaceutically acceptable excipients, carriers, vehicles or diluents are then readily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, injectable solutions and the like. These

pharmaceutical compositions can, if desired, contain additional ingredients such as flavourings, binders, excipients and the like. Thus, for purposes of oral administration, tablets containing various excipients such as L-arginine, sodium citrate, calcium carbonate and calcium phosphate may be employed along with various disintegrates such as starch, alginic acid and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulphate and talc are often useful for tabletting purposes. Solid composition of a similar type may also be employed as fillers in soft and hard filled gelatin capsules.

Appropriate materials for this include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral

administration, the essential active ingredient therein may be combined with various sweetening or flavouring agents, colouring matter or dyes and, if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and combinations thereof. The MDP microparticle-containing compositions may also comprise enterically coated dosage forms.

[0041 ] Suitable formulation protocols and suitable excipients, carriers, vehicles and diluents can be found in standard texts such as Remington: The Science and Practice of Pharmacy, 19^th Ed, 1995 (Mack Publishing Co. Pennsylvania, USA), British Pharmacopoeia, 2000, and the like.

[0042] It will be clear to those skilled in the art that MDP microparticle treatment may form a component of co-therapy in the treatment of epilepsy. Thus, MDP microparticles may be administered in conjunction with other known therapies for epilepsy. Such known therapies include, but are not limited to, phenytoin, carbamazepine, clonazepam, ethosuximide, valproic acid, barbiturates, felbamate, gabapentin, lamotrigine, oxcarbazepine, tiagabine, topiramate, vigabartrin, zonisamide and levetiracetam. In co-therapy treatment, MDP microparticles may be administered simultaneously or sequentially with other treatments.

Definitions

[0043] In the context of the present invention, the terms "muramyl dipeptide crosslinked to form a microparticle" and "MDP microparticle" refer to a microparticle formed by crosslinked repeats of muramyl dipeptide (MDP), wherein the MDP repeats are crosslinked to each other. The MDP microparticle may also contain DNA fragments and/or other agents that stimulate and/or regulate the immune system.

[0044] In the context of the present invention, the words "comprise", "comprising" and the like are to be construed in their inclusive, as opposed to their exclusive, sense, that is in the sense of "including, but not limited to".

[0045] In the context of the present invention, the terms "treatment" or "treating" include preventing a disease, disorder or condition from occurring in a subject which may be predisposed to the disease, disorder or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition, reducing the severity of the disease, disorder and/or condition, or reducing the frequency of the disease, disorder and/or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected.

[0046] In the context of the present invention, the term "prophylactic treatment" includes administration of MDP microparticle-containing compositions prior to clinical manifestation of the disease, disorder or condition (i.e., it protects the host against developing the disease, disorder or condition).

[0047] In the context of the present invention, the term "therapeutic treatment" includes administration of MDP microparticle-containing compositions after manifestation of the disease, disorder or condition (i.e., it is intended to diminish, ameliorate, or stabilize the existing disease, disorder or condition, or side effects thereof).

[0048] In the context of the present invention, the phrase "therapeutically effective amount" refers to an amount of MDP microparticle that produces some desired effect at a reasonable benefit/risk ratio applicable to any medical treatment. In certain embodiments, the term refers to that amount necessary or sufficient to eliminate or reduce medical symptoms for a period of time. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular composition without necessitating undue experimentation. In certain embodiments, compositions are formulated in a manner such that they will be delivered to a subject in a therapeutically effective amount, as part of a prophylactic or therapeutic treatment. The desired amount of the composition to be administered to a subject will depend on absorption, inactivation and excretion rates of the MDP microparticles, as well as the delivery rate of the MDP microparticle.

[0049] In the context of the present invention, the phrase "parenteral administration" refer to modes of administration other than enteral and topical administration, such as injections, and include without limitation intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0050] In the context of the present invention, the phrase "pharmaceutically acceptable" is art-recognized. In certain embodiments, the term includes compositions, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals, human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication,

commensurate with a reasonable benefit/risk ratio.

Brief Description of the Figures

[0051 ] Figure 1 : Seizure threshold as tested by a 6Hz seizure test is increased in mice treated with 100μg or 200μg MIS416 on day -8 and day-1 .

[0052] Figure 2: Seizure threshold as tested by a 6Hz seizure test is increased in mice treated with 100μg MIS416 on day-1 .

[0053] Figure 3: Seizure threshold as tested by a fluorothyl seizure test is increased in mice treated with 100μg MIS416 on day-1 . Preferred Embodiment of the Invention

[0054] Although the invention has been described with reference to certain embodiments detailed herein, other embodiments can achieve the same or similar results. Variations and modifications of the invention will be obvious to those skilled in the art and the invention is intended to cover all such modifications and equivalents.

[0055] The present invention is further described by the following non-limiting examples. Examples

Example 1 : Preparation of MIS416

[0056] Propionibacterium acnes was grown to a mid-stationary growth phase and washed to remove contaminants of bacterial culture origin by employing techniques well known to those in the art. Hydrophobic components contained in the cell walls and cytoplasm were sequentially extracted by successive washes with increasing concentrations of

ethanol/isopropanol/water (10%:10%:80%, 25%:25%:50% and 40%:40%:20%) at elevated temperatures. The isopropanol was then removed with successive washes with decreasing concentrations (80%, 50%, 40% and 20%) of ethanol at elevated temperatures. The resultant microparticles (MIS416) were then suspended in 6M guanidine-HCI and then washed in water for irrigation and its concentration measured by relating its absorbance at 565 nm to the absorbance of turbidity standards.

[0057] MIS416 contains extensively crosslinked MDP, amino-linked L-alanine-D- isoglutamine dipeptides and bacterial DNA fragments. The MIS416 generated by the present methods can have a broad range of sizes (for example, 0.01 to 30 microns) but the most common size range is from 1 to 7 microns. The preferred size is in the range of 0.5 to 3 microns.

[0058] MIS416 can be isolated from natural sources, as described above, or synthesized using well-known synthetic procedures (see, e.g., Liu et al., Bioorganic and Medicinal Chemistry Letters, 10 (12), 2000, pp. 1361 -1363(3); Schwartzman & Ribi, Prep Biochem. 1980; 10(3): 255-67; Ohya et al. Journal of Bioactive and Compatible Polymers, 1993; 8: 351 -364).

[0059] The concentration of MIS416 was adjusted to 0.2 mg/mL in sodium chloride for intravenous administration. Example 2: Effect of MIS416 in animal seizure models

[0060] Mice. 8-10 week male Swiss CD1 mice (Animal Resources Centre, Western Australia) were singly housed with a 12h light/dark cycle and access to food and water ad libitum. All experiments were performed with experimenters blinded to treatment.

[0061 ] Electrical (6 Hz) and chemical (fluorothyl) acute seizure models were used as acute seizure models for testing of seizure thresholds.

[0062] 6Hz test - 0.5% Tetracaine (Sigma Aldrich, Australia) in 0.9% saline was applied to both corneas of the mice. Corneal electroshocks from a rodent electroshock unit (Ugo Basile,Varese, Italy; 6 Hz, 3 second, 0.2 millisecond rectangular pulse width) were given starting at 14 mA. The current was increased step-wise (stimulation intensity) until the mouse exhibited seizures. Seizures were characterized by a stunned or fixed posture, which were also often accompanied by rearing, forelimb clonus and twitching.

[0063] Flurothyl test - Mice were placed individually into a clear plastic chamber (15 cm ^χ 20 cm x 28 cm) and 20 μΙ/min fluorothyl was dripped onto a filter paper placed near the top of the container. The durations between the start of the fluorothyl drip until the first myoclonic seizure and final tonic extension of the hind limbs or death were measured

[0064] Experiment 1 - Mice (n=15/group) were injected intravenously with 100μg/mouse of MIS416 on days -8 and -1 or 200 μg/mouse of MIS416 on days -8 and -1 . On day 0, mice were tested by the 6Hz seizure test.

[0065] Experiment 2 - Mice (n=15/group) were injected intravenously with 100 μg/mouse of MIS416 on day -1 . On day 0, mice were tested by the 6Hz seizure test.

[0066] Experiment 3 - Mice (n=15/group) were injected intravenously with 100 μg/mouse of MIS416 on day -1 . On day 0, mice were tested by the fluorothyl seizure test

[0067] The results from Experiment 1 show that pre-treatment with MIS416 at 100 μg and 200 μg increased the seizure threshold compared to untreated mice (Figure 1 ). This is illustrated by the increase in current that was required to achieve a seizure in MIS416 treated mice compared to that required for untreated mice (vehicle). Both 100 μg and 200 μg treated groups showed a similar increase in seizure threshold. [0068] The results from Experiment 2 confirm that pre-treatment with MIS416 increases the seizure threshold in the 6 Hz model. (Figure 2, ^* p<0.05; unpaired t-test; control versus 100 μg treated group).

[0069] The results from Experiment 3 show that mice treated with 100 μg MIS416 showed an increase in seizure threshold as indicated by the increased time before exhibiting the first generalised seizure compared to untreated controls (Figure 3a). The time until the second seizure (tonic extension) was also enhanced in the MIS416 treated group compared to untreated group (Figure 3b, p<0.05; unpaired t test; control versus treated group).

[0070] The results from Experiment 3 demonstrate that MIS416 treatment induces antiseizure activity as demonstrated by increasing the seizure threshold in a chemical acute seizure model.

Example 3: MIS416 toxicology studies

[0071 ] It has been well established that free/soluble MDP has significant toxicity in vivo. Attempts to reduce MDP toxicity have employed procedures to delay release, such as MDP incorporation into liposomes or other related compounds, or modification of terminal groups. Chemical modification has resulted in marked reduction in activity, and designs which change delivery rate have been difficult to control.

[0072] In vivo toxicology studies for MIS416 were performed as summarized in Table 1 .

Study ID. Study Title Method Quantity of Outcome

MIS416

G6121 MIS416: MIS41 6 was administered 10,000, 15,000 Maximum tolerated

Acute as single escalating doses. 30,000 and dose (MTD) Toxicology Animals were monitored for 45,000 μg/kg established as Study by IV toxic signs and mortality up body weight 15,000 μg/kg body route in to day 15 and subjected to weight

Swiss Albino detailed necropsy at

Mice terminal sacrifice on day 15

G6122 MIS416: MIS41 6 was administered 50, 200, 800 MTD established as

Acute as single escalating doses. and 3200 3,200 μg/kg body Toxicology Animals were monitored for μg kg body weight

Study by IV toxic signs and mortality up weight

route in New to day 15 and subjected to (rabbits)

Zealand detailed necropsy at

White terminal sacrifice on day 15

Rabbits

G6123 MIS416: MIS416 doses selected 1 ,000 3,000 The no-observed- Four Week from acute toxicology study and 10,000 adverse-effect-level Repeated were administered twice μg/kg body (NOAEL) was Dose weekly for 4 weeks. weight (mice) considered to be Study ID. Study Title Method Quantity of Outcome

MIS416

Toxicology Animals were subjected to 1 ,000 μg/kg (or a Study by IV detailed necropsy at total weekly dose of Route in terminal sacrifice. 2,000 μg/kg/week) Swiss Albino

Mice

G6124 MIS416: MIS416 doses selected 50, 500 and The NOAEL was

Four Week from acute toxicology study 5,000 μg/kg considered to be Repeated were administered twice body weight 50 μg/kg injected Dose weekly for 4 weeks. twice weekly

Toxicology Animals were subjected to (100 μg/kg/week) Study by IV detailed necropsy at

Route in New terminal sacrifice.

Zealand

White

Rabbits

1370-002 MIS416: A MIS41 6 was administered 20, 200, 1000 NOAEL in this

26-Week IV once weekly for 26 weeks. μg/kg body study could be Toxicity Animals were subjected to weight considered as Study In detailed necropsy at being close to Rabbits terminal sacrifice. A one 20 μg/kg for the month recovery arm purpose of comprised 2 estimation of animals/sex/group. human safety

margins

Table 1 Summary of toxicology studies

[0073] The toxicity studies, conducted in mice and rabbits for up to 26 weeks duration, provide adequate safety margins to support long term clinical studies at dosage levels in patients up to 20 μg/kg/week.

[0074] The toxicity studies show that MIS416 has significantly lower toxicity than free/soluble MDP.

Claims

1 . A method for therapeutic or prophylactic treatment of epileptic seizures in a subject, comprising administering to the subject a composition comprising muramyl dipeptide crosslinked to form a microparticle.

2. The method according to claim 1 , wherein the microparticle comprises DNA fragments.

3. The method according to claim 1 , wherein the epileptic seizures are convulsive seizures.

4. The method according to claim 1 , wherein the epileptic seizures are non-convulsive seizures.

5. The method according to claim 1 , wherein the epileptic seizures are prevented.

6. The method according to claim 1 , wherein frequency of the epileptic seizures is reduced.

7. The method according to claim 1 , wherein severity of the epileptic seizures is reduced.

8. The method according to claim 1 , wherein duration of the epileptic seizures is reduced.

9. The method according to claim 1 , wherein the composition is administered to the subject parenterally.

10. The method according to claim 1 , wherein the composition is administered to the subject intravenously.

1 1 . The method according to claim 1 , wherein the composition is administered to the subject orally.

12. The method according to claim 1 , wherein the composition is administered to the subject at a dosage of about 1 μg to about 10C^g.

13. The method according to claim 1 , wherein the composition is administered to the subject at a dosage of about 10C^g to about 10OC^g.

14. The method according to claim 1 , wherein the composition comprises one or more pharmaceutically-acceptable excipients, carriers, vehicles or diluents.

15. The method according to claim 1 , wherein the composition is administered to the subject once a day.

16. The method according to claim 1 , wherein the muramyl composition is administered to the subject once a week.

17. The method according to claim 1 , wherein the muramyl composition is administered to the subject once a fortnight.

18. The method according to claim 1 , wherein the muramyl composition is administered to the subject once a month.

19. Use of muramyl dipeptide crosslinked to form a microparticle for the manufacture of a medicament for the therapeutic or prophylactic treatment of epileptic seizures in a subject.