WO2000004888A2

WO2000004888A2 - Use of cyclodextrins for the treatment of cerebral ischaemia and central nervous system injury

Info

Publication number: WO2000004888A2
Application number: PCT/GB1999/002311
Authority: WO
Inventors: Alan John Nelson
Original assignee: Vernalis Research Limited
Priority date: 1998-07-20
Filing date: 1999-07-19
Publication date: 2000-02-03
Also published as: GB9815785D0; AU5050899A; WO2000004888A3

Abstract

The use of a substituted or unsubstituted cyclodextrin as the active agent in the manufacture of a medicament for the treatment of cerebral ischaemia or central nervous system injury.

Description

Chemical Compounds

The present invention relates to the use of cyclodextrins and their functionalised derivatives as neuroprotectant agents for the treatment of cerebral ischaemia or central nervous system injury, both in their own right and in admixture with other drugs which are effective in treating the sequelae of cerebral ischaemia or central nervous system injury.

Cyclodextrins are naturally occurring macrocyclic clathrate molecules which were first isolated in 1891. They are typically formed by the action of bacillus macerans amylase on starch to form cyclic α-1,4 linked D-glucopyranose units. The nomenclature of these macrocycles is as follows: α-, β- and γ-cyclodextrins are made up of six, seven and eight hexose units respectively.

β-Cyclodextπn

Cyclodextrins and their functionalised derivatives have only recently been recognised as useful pharmaceutical excipients. The constituent glucose residues are arranged in a circle with a toroidal shape in which all the primary hydroxy groups are on the wider base of the toroid. There are no hydroxy groups on the inside of the circle of glucose residues and consequently the cavity of the toroid has a non-polar character. In a hydrated state the cavity of α-, β- and γ-cyclodextrins is filled with 6, 11 or 17 water molecules, respectively, and these molecules of water may be replaced with an overall gain in energy by molecules of a non-polar compound. The formation of such complexes of cyclodextrins with drugs is a rapidly reversible reaction and these complexes exist both in solution and in crystalline state. The main driving force for such complex formation appears to be the release of the enthalpy-rich water molecules from the cavity which lowers the energy of the system.

Previously, high production costs restricted the application of cyclodextrins, but recent advances in biotechnology have led to the commercial availability of highly purified cyclodextrins. These compounds have found use in pharmaceutical formulation for drug solubilisation and stabilisation (Loftsson, T. and Brewster, M.E. Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization. J. Pharm. Sci., 1996, 85, 1017-1025), and in particular in enhancement of drug solubility in aqueous solutions. Cyclodextrins are therefore utilised for in vivo drug delivery, although regulatory approvals for human cyclodextrin formulations have so far only taken place in Europe and Japan (Rajewski, R.A. and Stella, V.J. Pharmaceutical applications of cyclodextrins. 1. In vivo drug delivery. J. Pharm. Sci., 1996, 85, 1142-1169).

Various derivatised cyclodextrins have been prepared and have been studied as pharmaceutical excipients. These derivatives have different complexing properties and solubilities compared to the unsubstituted cyclodextrins. For example, β-cyclodextrin and hydroxypropyl- β-cyclodextrin (HPCD) have aqueous solubilities of 1.8% and 60% w/v respectively. HPCD, when mixed with water, has a major advantageous effect on the solubility of drugs, by up to three orders of magnitude (Pitha, J. et al, Hydroxypropyl-β-cyclodexrrin: preparation and characterization; effects on solubility of drugs. Int. J. Pharmaceutics., 1986, 29 73-82). Similar effects have been seen by other groups (Loftsson, T. and Bodor, N., Effects of 2-hydroxypropyl-β-cyclodextrin on the aqueous solubility of drugs and transdermal delivery of 17β-estradiol. Acta Pharm. Nord., 1989, 1, 185-194).

Stroke is a severe public health problem and is reputedly the third largest disease in the industrialised world in terms of mortality. Some 500,000 - 600,000 people become victims of this disease each year in North America alone, and it is the leading cause of chronic disability in this region (Kuller, L.H. Incidence rates of stroke in the eighties: the end of the decline in stroke? Stroke, 1989, 20, 841-843). The high proportion of stroke victims requiring long term institutional care increases further the burden of cerebral ischaemia on society. An effective and widely applicable medical treatment for acute stroke would have an enormous public health impact but, as yet, no such treatment has been found (Dorman, P.J. et al, Recently developed neuroprotective therapies for acute stroke. CNS Drugs, 1996, 5, 457-474).

Commercially available cyclodextrins have been used to enhance the aqueous solubility of a limited range of neuroprotectant drugs for use in some animal models of stroke. For example, the antioxidants U-101033E and U-104067F have been examined in a gerbil bilateral carotid artery occlusion ischaemia model where post-ischaemic degeneration of nigrostriatal neurones was observed. Animals treated with the vehicle 40% hydroxypropyl-β-cyclodextrin showed a 42% loss of nigrostriatal neurones after 28 days and the drugs tested were shown to attenuate this loss (Andrus, P.K. et al, Neuroprotective effects of the novel brain-penetrating pyrrolopyrimidine antioxidants U-101033E and U-104067F against post-ischemic degeneration of nigrostriatal neurons. J. Neurosci. Res., 1997, 47, 650-654). Similar effects were seen with the dopaminergic agonist pramipexole when gerbils were treated with the vehicle 40% hydroxypropyl-β- cyclodextrin (Hall, E.D. et al., Neuroprotective effects of the dopamine D₂/D₃ agonist pramipexole against post-ischaemic or methamphetamine-induced degeneration of nigrostriatal neurons. Brain Res. 1996, 742, 80-88). In these scientific articles no mention was made of a pharmacological effect of β-cyclodextrin or its derivatives alone.

Flunarizine in a vehicle of β-cyclodextrin was also compared to this vehicle alone in an ischaemia model (Cohan, S.L. et al, Effect of flunarizine on electroencephalogram recovery and brain temperature in gerbils after brain ischaemia. Stroke, 1992, 23, 229- 33). No mention is made of a neuroprotectant effect of β-cyclodextrin alone. Prostaglandin El complexed with α-cyclodextrin (PGE.CD) was found to have some protective effects against acute cerebral ischaemia (Nonaka, S. et al., Effects of PGE1- alpha-cyclodextrin on experimental cerebral ischaemia. Yakuri to Chiryo, 1992, 20, 4857-72). EP-A-0501552 discloses the anti-stroke properties of lubeluzole (4-[(2- benzothiazolyl)methylamino]-alpha-[(3,4-difluorophenoxy)methyl]-l-piperidine ethanol) as the active ingredient in a composition which may also contain cyclodextrins as a complexant and/or solubilizer. Finally, DMSO and hydroxypropyl-β-cyclodextrin have been used as diluents in the study of the effect of a platelet activating factor antagonist, BN 50739, in the Pulsinelli - Brierley 4 vessel occlusion model of cerebral ischaemia (Sun, D and Gilboe, D.D. Effect of the platelet-activating factor antagonist BN 50739 and its diluents on mitochondrial respiration and membrane lipids during and following cerebral ischaemia. J. Neurochem. 1994, 62, 1929-1938).

It has now been found that the use of cyclodextrins, and in particular substituted β- cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin, combined with water or saline, in the presence or absence of other drugs for treating cerebral ischaemia, results in a reduction of infarct size following cerebral ischaemia. The cerebral ischaemia may be caused by stroke, cardiac arrest or drowning (global ischaemia), central nervous system injury (such as traumatic head injury or cerebral oedema), subarachnoid haemorrhage or other forms of interruption of blood supply to regions of the brain.

Thus, according to the present invention there is provided use of a substituted or unsubstituted cyclodextrin as the active agent in the manufacture of a medicament for the treatment of cerebral ischaemia or central nervous system injury.

The treatment of cerebral ischaemia according to the present invention is applicable to: transient ischaemic attack, stroke (thrombotic stroke, ischaemic stroke, embolic stroke, haemorrhagic stroke, lacunar stroke) subarachnoid haemorrhage, cerebral vasospasm, neuroprotection for stroke, peri-natal asphyxia, drowning, cardiac arrest and subdural haematoma.

The treatment of central nervous system injury according to the present invention is applicable to: traumatic brain injury, neurosurgery (surgical trauma), neuroprotection for head injury, raised intracranial pressure, cerebral oedema, hydrocephalus and spinal cord injury.

According to a further aspect of the present invention there is provided a method of treating cerebral ischaemia or central nervous system injury comprising administration to a subject in need of such treatment an effective dose of a substituted or unsubstitited cyclodextrin as the active agent. As used herein the term "treatment" includes prophylactic treatment.

The present invention may employ α-, β- or γ-cyclodextrins.

The primary hydroxy groups of the cyclodextrin may be substituted and the degree of substitution may vary from 0 to 100%. In a preferred embodiment, 50% to 90% of the hydroxy groups of the cyclodextrin are substituted. Commercially available substituted cyclodextrins typically have about 60% to 80% substitution.

The substituent group(s) may be the same or different, preferably the same, and are selected from groups which do not adversely affect the activity of the cyclodextrin. For example, the substituent group(s) can be alkyl (e.g. methyl, ethyl, butyl, pentyl), carboxyalkyl (e.g. carboxymethyl), hydroxyalkyl (e.g. hydroxyethyl, 2-hydroxypropyl, 2-hydroxybutyl), sulphoalkyl (e.g. sulphobutyl), esters (e.g. acetyl, propionyl, butyryl, succinyl, benzoyl, palmityl), sulphates (toluenesulphonyl), phosphates and sugars (e.g. glucosyl, maltosyl).

As used herein, the term "alkyl" means a branched or unbranched, cyclic or acyclic, saturated or unsaturated, functionalised or unfunctionalised hydrocarbyl radical.

Preferably, the substituent group is hydroxyalkyl and more preferably is 2- hydroxypropyl.

Where the cyclodextrin is substituted, substituted β-cyclodextrins are preferred, particularly 2-hydroxypropyl-β-cyclodextrin.

The cyclodextrins may be combined with a second drug useful in the treatment of cerebral ischaemia or central nervous system injury, the components being in the same formulation or in separate formulations for administration simultaneously or sequentially.

In a preferred embodiment, the second drug is an excitatory amino acid antagonist. Other drugs which may be combined with the cyclodextrins in the present invention are selected from: an AMPA antagonist; an NMDA antagonist; a glycine site antagonist; a glycine site partial agonist; a glycine site agonist; a kainate receptor antagonist; an iron chelator; an antioxidant; a free radical scavenger; an inhibitor of leucocyte adhesion and migration; a calcium antagonist; a modulator of calcium influx; a sodium channel blocker; a glutamate release inhibitor; a potassium channel opener; a naaladase inhibitor; an immunophyllin ligand; a rotamase inhibitor; nerve growth factor; FMRF inhibitor; adenosine kinase inhibitor; adenosine receptor agonist; a calpain inhibitor; a caspase inhibitor; β-adrenoceptor agonist; opioid receptor antagonist; endothelin receptor antagonist; a PLA₂ inhibitor; a PKC inhibitor; a promotor of neuronal repair; serotonin agonist; 5HT_la agonist; insulin-like growth factor; neurotrophic factor; nitric oxide synthetase inhibitor; BDNF enhancer; Bradykinin antagonists; Vitamin E; Coenzyme Q; anti-oedema agent; anticoagulant/thrombolytic; antiinflammatory agent; a metabotropic glutamate receptor ligand; a GABA agonist; a GABA potentiator; an agent-reducing levels of lactate and a membrane stabilising agent.

The present invention also encompasses the cyclodextrins for use as an active agent in therapy generally.

The present invention may be employed in respect of a human or animal subject, more preferably a mammal, more preferably a human subject.

The cyclodextrin may be in saline, water or another pharmaceutically acceptable carrier.

The cyclodextrins may be administered in dosage amounts in the range of 50mg to 250g, preferably 250mg to lg, per day per person and acute or repeated dosage forms are suitable.

Any suitable route may be employed for providing the patient with an effective dosage of the cyclodextrin. For example, oral, rectal, parenteral (intravenous, intramuscular), transdermal, subcutaneous and the like may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules and the like. The most suitable route in any given case will depend on the severity of the condition being treated. The preferred route of administration is the intravenous route. The compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. Such methods all include the step of bringing the active ingredient into association with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the cyclodextrin as the active ingredient with liquid carriers or finely divided solid carriers or both, according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration.

For example, in preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, and the like may be used in the case of oral solid preparations such as, for example, powders, capsules and tablets. If desired, tablets may be coated by standard aqueous or non-aqueous techniques. Pharmaceutical compositions suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, or aerosol sprays each containing a predetermined amount of the active ingredient as a powder or granules, a solution or a suspension in an aqueous liquid, an oil-in- water emulsion, or a water-in-oil liquid emulsion.

Solutions for injection may be prepared by dissolving the cyclodextrin as the active ingredient and possible additives in a part of the solvent for injection, preferably sterile water, adjusting the solution to desired volume, sterilisation of the solution and filling in suitable ampoules or vials. Any suitable additive conventionally used in the art may be added, such as tonicity agents, preservatives, antioxidants, etc. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulating agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g. sterile water, before use.

In addition to the common dosage forms set out above, the compounds of the present invention may also be administered by controlled release means and/or delivery devices such as those described in United States Patent Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 3,630,200; 4,008,719; 4,687,660; and 4,769,027, the disclosures of which are hereby incorporated by reference.

The invention will now be described with reference to the following examples. It will be appreciated that what follows is by way of example only and that modification of detail may be made without departing from the scope of the invention.

EXPERIMENTAL

In vivo cerebral ischaemia: the rat transient middle cerebral artery occlusion (MCAO) model

This model of middle cerebral artery occlusion (MCAO) employs the intraluminal filament technique in the rat (Zhao, Q. et al, Hyperthermia complicates middle cerebral artery occlusion induced by an intraluminal filament. Brain Res., 1994, 649, 253- 259; Zhao, Q. et al, Delayed treatment with the spin trap alpha-phenyl-N-tert-butyl nitrone (PBΝ) reduces infarct size following transient middle cerebral artery occlusion in rats. Acta Physiol. Scand., 1994, 152, 349-350). In this ischaemia model, the animal was anaesthetised and the carotid artery exposed. A chamfered monofilament suture (3/0) was introduced into the ligated carotid artery, past the bifurcations of the external and common carotid, the internal carotid and the pterygopalatine artery, into the intracranial circulation where it lodged in the narrow proximal anterior carotid occluding the middle cerebral artery.

After 90 min., the filament was removed, allowing re-circulation. At the commencement of re-circulation, drug administration started. All drugs were administered via the tail vein, in a volume of 5 ml/kg. Three conditions were employed. "Untreated" rats received no treatment. "Saline" treated rats received 5 ml/kg 0.9% saline as an intravenous bolus at the time of reperfusion. Drug treated rats received 5 ml/kg of a solution of 45% w/v 2-hydroxypropyl-β-cyclodextrin (HPCD; Aldrich Chemical Company; molar substitution = 0.8) in water as an intravenous bolus at the time of reperfusion. Groups sizes were n = 10. Twenty four hours following reperfusion, the animal was assessed neurologically using a modification of the scale developed by Bederson (Bederson, J.B. et al, Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke, 1986, 17, 472-476). After completion of the neurological assessment the animal was deeply anaesthetised and perfused via the transcardiac route with 3% triphenyl tetrazolium chloride at 37°C. Following this the brain was rapidly removed and sliced into 0.5 mm sections. Using this technique, viable tissue was stained dark red and infarcted tissue remained unstained. The area of infarction on each section was measured, and the total volume of infarction in the hemisphere, cortex and striatum computed, using the Kontron image analysis system. Measurement of infarct volume and neurological status was performed blind with regard to allocation of the animal to a treatment group.

The results of the experiments are shown in Table 1 and Figure 1. Table 1 and Figure 1 show infarct volumes in male Lister Hooded rats after 90 min. of transient MCAO followed by 24 hours of reperfusion.

The significantly lower infarct volumes observed in animals treated with 2- hydroxypropyl-β-cyclodextrin, in comparison with untreated and saline treated animals, demonstrates the use of the cyclodextrin in treating cerebral ischaemia.

Table 1. Effects on infarct volume after transient middle cerebral artery occlusion in male Lister Hooded rats

Total infarct volume (mm³)

Mean SEM

Untreated 88.52 17.68

Saline 91.20 20.89

HPCD 45.96 16.28

Cortical infarct volume (mm³)

Mean SEM

Untreated 50.70 14.63

Saline 54.05 12.68

HPCD 25.47 10.33

Striatal infarct volume (mm³)

Mean SEM

Untreated 23.90 4.68

Saline 24.08 6.85

HPCD 11.96 4.34

In vivo cerebral ischaemia: the gerbil transient forebrain ischaemia model

This model is described by Cross, A.J. et al. (Br J Pharmacol 1991, 104, 406-411, Neuroprotective activity of chlormethiazole following transient forebrain ischaemia in the gerbil). Male Mongolian gerbils (B & K Universal), weight 60-80 g, were individually on a 12 hour light: 12 hour dark cycle (lights on at 07:00) with food and water available ad libitum. Gerbils were anaesthetised with Saffan (alphaxalone, 45 mg/kg plus alphadalone, 15 mg/kg i.p.). They were placed on an electrically heated mat and under heating lamps to maintain body temperature at 37 °C as monitored by a rectal probe. Under a dissecting microscope the carotid arteries were exposed via a midline incision, and separated from the vagus nerve. Both common carotids were then occluded with aneurysm clips, the effectiveness of the occlusion being confirmed visually. Following 6 minutes of bilateral occlusion the clips were removed, reperfusion confirmed visually and the neck wound sutured. Temperature control was maintained until recovery from anaesthesia was complete. Four days following induction of ischaemia, gerbils were deeply anaesthetised with Euthesate, and perfused via the heart with 10% formalin/saline containing 5% sucrose. Perfused brains were then immerse fixed in 10% formalin/saline containing 30% sucrose for three days, prior to rapid freezing and sectioning at 25 microns on a cryostat. Staining for Nissl substance was performed using Cresyl Violet. Representative sections for each animal were compared at the level of the dorsal hippocampus and rated according to the schedule:

Score % Protection

0 0-10

1 10 to 40 1.5 40-60

2 60-90

3 90-100

The experiments were performed using HPCD administered as three intraperitoneal doses of an 80 percent solution of 22.5 percent HPCD, in a dose volume of 4 ml/kg, 30 min, 60 min and 120 min following five minutes of transient forebrain ischaemia. The results of the experiment are shown in Figure 2 which illustrates the neuroprotective effect of HPCD in Male Mongolian gerbils.

Claims

1. The use of a substituted or unsubstituted cyclodextrin as the active agent in the manufacture of a medicament for the treatment of cerebral ischaemia or central nervous system injury.

2. A method of treating cerebral ischaemia or central nervous system injury comprising administration to a subject in need of such treatment an effective dose of a substituted or unsubstituted cyclodextrin as the active agent.

3. A use or method according to claim 1 or 2, wherein the subject is human.

4. A use or method according to claim 1, 2 or 3 wherein the cyclodextrin is an ╬▒-, ╬▓- or ╬│-cyclodextrin.

5. A use or method according to claim 1, 2, 3 or 4 wherein the cyclodextrin is unsubstituted.

6. A use or method according to claim 1, 2, 3 or 4 wherein the cyclodextrin is substituted.

7. A use or method according to claim 6 wherein 50% to 90% of the hydroxy groups of the cyclodextrin are substituted.

8. A use according to claim 6 or 7 wherein the substituent group is selected from alkyl, carboxyalkyl, hydroxyalkyl, sulphoalkyl, esters, sulphates, phosphates and sugars.

9. A use according to claim 6 or 7 wherein the substituent group is selected from methyl, ethyl, butyl, pentyl, carboxymethyl, hydroxyethyl, 2-hydroxypropyl, 2- hydroxybutyl, sulphobutyl, acetyl, propionyl, butyryl, succinyl, benzoyl, palmityl, toluenesulphonyl, glucosyl and maltosyl.

10. A use or method according to claim 6 or 7 wherein the substituent group is 2- hydroxypropyl.

11. A use or method according to claim 6 or 7 wherein the cyclodextrin is 2- hydroxypropyl-╬▓-cyclodextrin.

12. A use or method according to any preceding claim wherein the cyclodextrin is in combination with a second drug useful in the treatment of cerebral ischaemia or central nervous system injury, the components being in the same formulation or in separate formulations for administration simultaneously or sequentially.

13. The use or method according to claim 12 wherein the second drug is an excitatory amino acid antagonist.

14. The use or method according to claim 12 wherein the second drug is selected from: an AMPA antagonist; an NMDA antagonist; a glycine site antagonist; a glycine site partial agonist; a glycine site agonist; a kainate receptor antagonist; an iron chelator; an antioxidant; a free radical scavenger; an inhibitor of leucocyte adhesion and migration; a calcium antagonist; a modulator of calcium influx; a sodium channel blocker; a glutamate release inhibitor; a potassium channel opener; a naaladase inhibitor; an immunophyllin ligand; a rotamase inhibitor; nerve growth factor; FMRF inhibitor; adenosine kinase inhibitor; adenosine receptor agomst; a calpain inhibitor; a caspase inhibitor; ╬▓-adrenoceptor agonist; opioid receptor antagonist; endothelin receptor antagonist; a PLA₂ inhibitor; a PKC inhibitor; a promotor of neuronal repair; serotonin agonist; 5HT_la agonist; insulin-like growth factor; neurofrophic factor; nitric oxide synthetase inhibitor; BDNF enhancer; Bradykinin antagonists; Vitamin E; Coenzyme Q; anti-oedema agent; anticoagulant thrombolytic; antiinflammatory agent; a metabotropic glutamate receptor ligand; a GABA agonist; a GABA potentiator; an agent-reducing levels of lactate and a membrane stabilising agent.

15. A use or method according to any preceding claim wherein the cyclodextrin is in saline, water or other pharmaceutically acceptable carrier.

6. For use in therapy, a cyclodextrin as set out in any of claims 1 to 11.