Patent Application
20230133251
The present invention relates to lipid vesicles containing at least one mTOR inhibitor for use in the treatment or prevention of a disease of the central nervous system.
The mammalian target of rapamycin (mTOR) is a serine/threonine kinase with the function of promoting cell growth, proliferation, protein synthesis and survival in response to the environment and cell requirements. This enzymatic complex is formed by two molecular subunits called mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2), both formed by the association of several proteins. Ample research in the last twenty years has demonstrated that mTOR influences growth, metabolism and cell aging in response to environmental and endocrine stimuli.
Rapamycin and the analogues thereof are known as mTOR inhibitors capable of blocking the activity mainly of mTORC1. Rapamycin derivatives (rapalogs) are molecules developed with the objective of improving the pharmacokinetic and toxicological profile of the parent. They share with the latter the same molecular scaffold but have substitutions in the hydroxyl group at the C40 position of the methoxycyclohexanol residue, adapted to modulate the physicochemical properties, biodistribution and metabolism thereof.
The effectiveness of mTOR inhibitors in the treatment of numerous pathologies affecting the central nervous system (CNS) has been amply demonstrated. Various clinical studies have been conducted to assess the benefit of mTOR inhibitors in the treatment of the tuberous sclerosis (TS), a genetic syndrome characterised by the occurrence of tumours in various organs and tissues: mTOR inhibitors are effective in inducing regression of the cerebellar astrocytomas typical of this condition. However, the pathology is often also accompanied by drug-resistant epilepsy and autism spectrum behavioural disorders. Recently, publications have appeared which attest to the effectiveness of mTOR inhibitors, when the administration thereof is brought forward to under the age of 2, in improving the clinical expression of the latter two aspects as well. But no data have been published to suggest a benefit in cases of cognitive disability.
It should thus also be assessed whether other epilepsies as well, neuropsychiatric disorders and cognitive deficits, which are today either incurable or not treatable in a satisfactory manner, may derive a benefit from therapy with mTOR inhibitors at an appropriate dosage.
In 2011 the effect of the oral administration of mTOR inhibitors on a model of mice affected by Alzheimer's disease was published for the first time; an improvement was observed both in the neurofibrillary tangles composed of Tau protein and extra- and intraneuronal accumulations of beta-amyloid protein in the brain (classic anatomopathological characteristics of Alzheimer's disease), with a recovery of learning and memory. The effect seemed due to the induction of autophagy mediated by the inhibition of mTOR. Autophagy is the metabolic process whereby cells degrade substrates produced by them. The increase in this process induces the degradation of pathological protein accumulations in Alzheimer's. The activity of mTOR inhibits it, whereas the inhibition thereof stimulates it. It is not yet completely understood whether the symptomatologic benefit observed is entirely attributable to this phenomenon.
Despite the benefits clinically observed after the systemic administration of mTOR inhibitors in neuro-oncological diseases (e.g. tuberous sclerosis), and in mouse models of neuroinflammatory diseases (e.g. models of multiple sclerosis), neurodegenerative diseases (e.g. models of Alzheimer's disease) and models of many other neurological diseases, there is a difficulty in reaching effective drug concentrations in the central nervous system. Furthermore, the systemic administration of mTOR inhibitors poses serious problems of toxicity tied to the high biodistribution thereof and thus a considerable immunosuppressant effect, in the case of use in therapies such as the ones mentioned above, represents a major undesirable effect.
Cerebrospinal fluid (CSF), the fluid produced by the choroid plexus of the brain ventricles, which fills them and from which it flows out to surround the brain, can be considered an ultrafiltrate of plasma: under physiological conditions, it contains ions, a few proteins and very few cells. If injected here, in the brain ventricles, the drug will be homogeneously distributed in the brain parenchyma.
Its volume varies between 135 and 150 ml, but it is continuously produced at a rate of 500 ml/day. It is absorbed by Pacchioni granulations, protruding formations in the venous sinuses of the dura mater and reintroduced into the bloodstream through venous blood. Therefore, it is entirely replaced about 3.5 times a day.
Consequently, if it is desired to maintain the concentration of a drug in the central nervous system (which has a volume of approx. 1400 ml), it will be necessary to administer it continuously into the cerebrospinal fluid. The continuous turnover with blood circulation produces a systemic absorption of the drug, whose blood concentration will depend on its half-life and distribution volume.
On the basis of these considerations, a therapeutic strategy has been proposed which provides for intrathecal, preferably intraventricular, administration of one or more mTOR inhibitors using intracerebroventricular administration devices. This new therapy has been experimented with on normal and 3xTg-AD mouse models and described in patent EP2846770B1.
However, although they possess a long half-life in bodily fluids, mTOR inhibitors, at body temperature, are unstable molecules that rapidly degrade inside infusion devices. This instability precludes ensuring the sustained effect that should be observed with a gradual administration, above all over a number of weeks. This therefore implies the need for repeated daily administration of mTOR inhibitors, also intrathecally. It is thus necessary to develop a method capable of drastically increasing the stability of these drugs at body temperature inside infusion devices.
A first aspect of the present invention relates to the use of lipid vesicles containing at least one mTOR inhibitor for use in the treatment or prevention of a disease of the central nervous system, preferably a neurodegenerative disease or a tumoral disease with neurological involvement or a neuroinflammatory disease. The vesicles are preferably a single lipid layer; more preferably they are micelles. The vesicles are preferably administered via the intrathecal route, preferably via the intraventricular route.
A second aspect of the present invention relates to a pharmaceutical composition comprising the above-described lipid vesicles.
The composition is preferably formulated in liquid form, preferably as a suspension.
A third aspect of the present invention relates to a method for the treatment or prevention of a neurodegenerative disease or tumoral disease with neurological involvement or a neuroinflammatory disease or a disorder characterised by chronic pain of central origin, which comprises the step of locally administering, into a patient's brain, an effective dose of the lipid vesicles containing at least one mTOR inhibitor or of the composition comprising said vesicles.
The present invention is described below in detail and illustrated by way of example with reference to the appended figures, in which:
In the context of the present invention, “minimum effective concentration” means the minimum concentration at which the desired biological effect is obtained.
In the context of the present invention, “compliance” means the patient's adherence, after careful physician counselling, to a therapy, generally a pharmacological one.
A first aspect of the present invention relates to lipid vesicles containing at least one mTOR inhibitor for use in the treatment or prevention of a disease of the central nervous system, preferably a neurodegenerative disease or a tumoral disease with neurological involvement or a neuroinflammatory disease or drug-resistant epilepsy, preferably West syndrome, or a neurobehavioural autism spectrum disorder, or a disorder characterised by chronic pain of central origin.
Preferably, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Huntington's chorea, frontotemporal dementia, vascular dementia, preferably of the genetically determined CADASIL type (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), prion encephalopathy and amyotrophic lateral sclerosis. In a preferred embodiment, the neurodegenerative disease is Alzheimer's disease.
The tumoral disease with neurological involvement is preferably tuberous sclerosis (TSC).
The neuroinflammatory disease is preferably selected from: multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), Devic's neuromyelitis optica, Rasmussen's syndrome, primary CNS vasculitis and primary progressive aphasia.
In one embodiment, the at least one mTOR inhibitor is selected from: everolimus, rapamycin (sirolimus), PF-04691502, ridaforolimus, temsirolimus and combinations thereof.
In a preferred embodiment of the invention, the at least one mTOR inhibitor is selected from: everolimus, rapamycin and combinations thereof.
The lipid vesicles are preferably in a single layer.
The single-layer lipid vesicles most widely available and used in the biomedical field, above all as carriers for drugs or other molecules of biological or therapeutic importance, are “micelles”. In one embodiment of the invention, therefore, the single-layer lipid vesicles containing at least one mTOR inhibitor can preferably be micelles. As is well known in the sector, micelles are formed from at least one layer of one or more phospholipids.
The lipid layer of the vesicles described here, preferably of the micelles, can consist essentially of one or more saturated phospholipids; in another embodiment, the layer can consist essentially of one or more unsaturated phospholipids. More typically, however, the layer can consist essentially of a mixture of saturated phospholipids and unsaturated phospholipids.
In a preferred embodiment, one or more phospholipids forming the layer of the lipid vesicles can be selected from the group consisting of: dipalm itoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolam ine (DSPE), distearoylphosphatidylcholine (DSPC), dipalm itoylphosphatidylethanolam ine (DPP E), dipalm itoylphosphorylglycerol (DPPG), distearoylphosphorylglycerol (DSPG), dipalmitoylphosphate (DPPA), distearoylphosphate (DSPA), dipalm itoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), palm itoyl-stearoylphosphatidylcoline (PSPC), dioleoylphosphatidylethanolam ine (DOPE), dioleoylphosphatidylcoline (DOPC), palm itoyl-oleoylphosphatidylcoline (POPC), stearoyl-oleoylphosphatidylcoline (SOPC), and combinations thereof.
In a preferred embodiment, the layer of single-layer lipid vesicles, preferably micelles, comprises distearoylphosphatidylethanolamine (DSPE).
Furthermore, another preferred embodiment of the invention envisages that the lipids forming the lipid layer can be modified by binding one or more hydrophilic polymers to them. Typically, one or more hydrophilic polymers can be bound to the polar heads of the phospholipids present in the layer of vesicles, preferably micelles. In other words, the vesicles can carry one or more hydrophilic polymers on their outer surface, typically bound to the polar heads of the phospholipids.
The presence of hydrophilic polymers on the outer surface of the vesicles imparts a longer half-life following parenteral administration. In particular, one or more hydrophilic polymers prevent (or at least render more difficult) the uptake of the vesicles by the reticuloendothelial system, since they form an external barrier that prevents (or at least renders more difficult) the adsorption of plasma proteins by the vesicles themselves. The presence of the one or more hydrophilic polymers on the outer surface thus increases the stability of the vesicles, ensuring that they remain longer in the physiological fluids after administration.
Preferably, the one or more hydrophilic polymers can be selected from the group consisting of: polyethylene glycol (PEG), polyoxazolines, polyvinyl alcohol (PVA), polyglycerol, polyvinylpyrrolidone (PVP), poly-N-hydroxypropyl methacrylamide (polyHPMA), polyaminoacids and combinations thereof.
In a preferred embodiment of the invention, the hydrophilic polymer bound to the phospholipids of the lipid vesicles is polyethylene glycol (PEG). In one embodiment, the PEG is selected from: PEG1000, PEG2000, PEG3400 and PEG5000, preferably PEG2000.
In a preferred embodiment of the invention, the lipid vesicles, preferably the micelles, comprise the phospholipid DSPE bound to the hydrophilic polymer PEG, preferably to the hydrophilic polymer PEG2000.
In one embodiment, the molar ratio between the mTOR inhibitor and vesicle is comprised between 1:5 and 1:10, preferably 1:7.
The vesicles of the present invention are preferably prepared by mixing the at least one mTOR inhibitor with at least one phospholipid forming the layer of lipid vesicles. In one embodiment, at least one mTOR inhibitor and at least one phospholipid are weighed and dissolved in a solvent. The solvent is later removed, preferably by evaporation and/or drying.
The vesicles are then resuspended in an aqueous medium in order to obtain a clear micellar dispersion.
In one embodiment, after their preparation the vesicles are prepared under aseptic conditions using any suitable method, for example after sterilisation of the receptacles in an autoclave and of the solutions by filtration.
In a preferred embodiment of the invention, the vesicles are administered via the intrathecal route, more preferably via the intraventricular route.
Intrathecal administration can be performed in a pulsed manner, preferably at regular intervals, or in a continuous manner.
Intrathecal administration is preferably performed by means of a mechanical or electromechanical device, more preferably by means of an electromechanical infusion pump. The mechanical or electromechanical device can be, for example, an implanted device, i.e. surgically implanted under the scalp and provided with a subcutaneous reservoir, leading off from which there is a catheter extending from the cranial vault until reaching the intraventricular space (similar to an “Ommaya Reservoir”). Alternatively, a pump can be inserted beneath the skin (so-called “ports”) in the periclavicular region, or implanted in the abdomen (infusion pumps). Connected to the pump there is a catheter, which is inserted into the intrathecal space, or reaches the ventricular space, first via the subcutaneous route, then the transcranial route.
The use of micelles for the diffusion of drugs into the central nervous system (CNS) is usually not considered a suitable means, as the ability of micelles to pass through the ependymal barrier (very similar to the blood-brain barrier) is entrusted to the small portion thereof involved in transcytotis.
Surprisingly, the Applicant has demonstrated that incorporating the mTOR inhibitor in a vector, preferably in lipid vesicles, makes it possible to reach a minimum effective concentration of mTOR inhibitors in CSF. In fact, without wishing to be bound to any theory, at body temperature, in a low presence of solutes (under normal conditions and generally in the pathological conditions we intend to treat, CFS is a plasma ultrafiltrate with very low protein and cell concentrations) and below their critical micellar concentration, lipid vesicles, in particular micelles, invariably disintegrate, releasing the drug in the medium. In CSF, the micelles are at a critically low concentration and will disintegrate. Being lipophilic molecules, mTOR inhibitors in CSF can thus freely disseminate in the brain parenchyma. Once in CSF, they are homogeneously diffused in the subarachnoid space, penetrate into the parenchyma, and reach and enter effectively into the target cells.
Furthermore, intrathecal administration makes it possible to avoid the side effects due to systemic administration of mTOR inhibitors. In fact, besides increasing the concentration of mTOR inhibitors in CSF, intrathecal administration prevents the mTOR inhibitors from reaching high concentrations in other regions of the body, reducing the risk of immunosuppressant effects.
Intrathecal administration of mTOR inhibitors, together with the 0 incorporation thereof into lipid vesicles, is capable of increasing both the concentration of mTOR inhibitors in CSF and the stability thereof. In fact, as demonstrated in the experimental section of the present application, micelles increase the stability of mTOR inhibitors at different temperatures; in particular, at 37° C. the everolimus loaded in the micelles demonstrated a much higher stability than everolimus in solution in DMSO.
Intrathecal administration of mTOR inhibitors encapsulated in micelles could thus demonstrate to be a very effective therapy for very severe neurological pathologies, such as, for example, neurodegenerative diseases, tumoral diseases with neurological involvement and neuroinflammatory diseases, while avoiding systemic side effects and enabling a greater stability and concentration of the drug in CSF. In fact, the greater stability of mTOR inhibitors encapsulated in lipid vesicles, preferably in micelles, would enable more extended administrations of the mTOR inhibitors and fewer systemic side effects, thus increasing patient compliance with the therapy.
For the purposes of the above-described uses, the vesicles can preferably be administered at least once or twice a month, the infusion phase lasting 2-3 weeks, thus allowing for 1-2 week break between 2 administrations.
In a preferred embodiment, the vesicles are administered in a single administration or in a number of administrations on a monthly basis or at different time internals according to need.
A second aspect of the present invention relates to a composition comprising the vesicles as described above in detail.
The pharmaceutical composition of the invention can be formulated with suitable excipients known to the person skilled in the art.
According to one embodiment of the invention, the composition is formulated in liquid form, preferably as a suspension.
Alternatively, the composition is prepared extemporaneously by mixing the lyophilised and/or dry composition with a saline solution, water or, preferably, aCSF.
In a preferred embodiment of the invention, the composition is administered via the intrathecal route, more preferably via the intraventricular route.
Intrathecal administration can be performed in a pulsed manner, preferably at regular intervals, or in a continuous manner.
Intrathecal administration is preferably performed by means of a mechanical or electromechanical device, more preferably by means of an electromechanical infusion pump. The mechanical or electromechanical device can be, for example, an implanted device, i.e. surgically implanted under the scalp and provided with a subcutaneous reservoir, leading off from which there is a catheter extending from the cranial vault until reaching the intraventricular space (similar to an “Ommaya Reservoir”).
Alternatively, a pump can be inserted beneath the skin (so-called “ports”) in the periclavicular region, or implanted in the abdomen (infusion pumps). Connected to the pump there is a catheter, which is inserted into the intrathecal space, or reaches the ventricular space, first via the subcutaneous route, then the transcranial route.
A further aspect of the present invention relates to a method for the treatment and/or the prevention of a pathology of the central nervous system, preferably a disease of the central nervous system, preferably a neurodegenerative disease or a tumoral disease with neurological involvement or a neuroinflammatory disease or a drug-resistant epilepsy or a neurobehavioural autism spectrum disorder of unknown origin or a disorder characterised by chronic pain of central origin, which comprises a step of administering to the patient an effective dose of the vesicles of the present invention as described above in detail or of the composition comprising the vesicles.
Preferably, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Huntington's chorea, frontotemporal dementia, vascular dementia, preferably of the genetically determined CADASIL type (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), prion encephalopathy and amyotrophic lateral sclerosis. In a preferred embodiment, the neurodegenerative disease is Alzheimer's disease.
The tumoral disease with neurological involvement is preferably tuberous sclerosis (TSC).
The neuroinflammatory disease is preferably selected from: multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), Devic's neuromyelitis optica, Rasmussen's syndrome, primary CNS vasculitis and primary progressive aphasia.
In a preferred embodiment of the invention, the vesicles are administered via the intrathecal route, more preferably via the intraventricular route. Intrathecal administration can be performed in a pulsed manner, preferably at regular intervals, or in a continuous manner.
Intrathecal administration is preferably performed by means of a mechanical or electromechanical device, more preferably by means of an electromechanical infusion pump.
The mechanical or electromechanical device can be, for example, an implanted device, i.e. surgically implanted under the scalp and provided with a subcutaneous reservoir, leading off from which there is a catheter extending from the cranial vault until reaching the intraventricular space (of the “Ommaya Reservoir” type). Alternatively, a pump can be inserted beneath the skin (so-called “ports”) in the periclavicular region, or implanted in the abdomen (infusion pumps). Connected to the pump there is a catheter, which is inserted into the intrathecal space, or reaches the ventricular space, first via the subcutaneous route, then the transcranial route.
In a preferred embodiment, the vesicles are administered in a single administration or in a number of administrations lasting 15 days, with a 15 day break in between, until remission of the symptoms and normalisation of the disease marker.
Everolimus (Selleck Chemicals, Aurogene, Rome, Italy) was dissolved in DMSO (VWR, Milan, Italy) at a concentration of 1 mg/ml. The everolimus solution was subsequently diluted in 10% v/v DMSO/saline solution until obtaining a final concentration of 10 μg/ml. The resulting solution was then incubated at 4, 25 and 37° C.
Preparation Micelles Loaded with Everolimus
Carefully weighed amounts of everolimus and distearoylphosphoethanolam ine-polyethylene glycol 2000 (DSPE-PEG2000, Lipoid, Switzerland) were dissolved in 3 ml of chloroform (Sigma-Aldrich, Milan, Italy) in a 50 ml flask. The solvent was removed by light evaporation at room temperature under a flow of nitrogen and vacuum dried for 1 hour to eliminate residual traces. The thin film obtained was reconstituted in an exact volume (3 mL) of aqueous medium. After complete reconstitution a clear micellar dispersion was obtained. The dispersion was then incubated at 4, 25 and 37° C.
The drug was quantified using the HPLC method. Briefly, the instrument used is a Portlab STAYER HPLC system equipped with a UV detector, a parallel pump and Triathlon autosampler (Portlab, Rome, Italy). The conditions used are the following: isocratic mode with acetonitrile:water (60:40, v/v) eluted at 0.8 ml/min and a Zorbax C8 column, 4.6 mm ID×250 mm, 5 μm (Agilent, Milan, Italy) equilibrated at 55° C. UV detection was performed at 278 nm. A calibration curve was constructed in the concentration interval of 0.62-10 μg/mL (r2=0.9992).
The samples of solution were injected directly (injection volume 15 μL), whereas the micellar suspension was diluted 200 times in DMSO before being subjected to analysis. In order to reduce the carryover effects and an excessive accumulation of lipids in the column, samples of solution and micelles were injected alternately. The column was periodically washed with chloroform and methanol to avoid the accumulation of lipids and impairment of performance. All measurements were performed in triplicate and the results were expressed as the mean±D.S.
The role of pH and the effect of the medium on the stability of the micelles loaded with everolimus were analysed by incubating the samples in different media at 37° C. Saline solution, 10 mM phosphate-buffered saline, pH 7.4 (PBS, Sigma-Aldrich, Milan, Italy) and artificial cerebrospinal fluid (aCSF) were used as the incubation solution.
Measurements of size distribution were performed using a Nicomp 380 ZLS autocorrelator (PSS Inc., Santa Barbara, Calif., USA) equipped with a Coherent Innova 70-3 He—Ne laser (Laser Innovation, Moonpark, Calif., USA), an avalanche photodiode detector (APD) and a PALS phase analyser. The polydispersity index (PI) and the mean hydrodynamic diameter (MHD) of the micelles loaded with everolimus in saline solution and in PBS were measured in triplicate.
Both HeLa cells (cervical cancer cell line) and SH-SY5Y cells (neuroblastoma cell line) were obtained from the American Type Culture Collection (Manassas, Va., USA) and cultured in Dulbecco's Modified Eagle's medium (DMEM with L-glutamine) supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS), 100 U of penicillin, 100 U of streptomycin in a 5% CO2 humidified incubator at 37° C. Cell viability was verified with the standard Trypan Blue method.
Analysis with MTT Colorimetric Assay
The activity of everolimus in organic solution and in the micelles was determined by evaluating cell proliferation after the treatment. Briefly, the HeLa and SH-SY5Y cells were seeded in a 96-well plate at a concentration of 3000 cells/well and 6000 cells/well, respectively, and were maintained in the culture medium (100 μl) for 24 hours. After incubation at 37, 25 and 4° C. for 7, 14, 35, 50 and 77 days, the samples were suspended in the cell culture medium at a final concentration of 5 nM. Untreated cells, cells treated with a 10% v/v DMSO/saline solution (Veh) and empty micelles in saline solution, both diluted in a manner equivalent to the drug, were analysed as controls. A freshly prepared everolimus solution was used as the reference standard. Cell proliferation was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) assay (Sigma-Aldrich) according to the manufacturer's manual. The cells were mixed with 10 μl of MTT (0.5 mg/ml) and kept in a humidified incubator at 37° C. for 4 hours. Subsequently, 100 μl of a solubilisation solution (10% SDS with 0.01 N HCl) were added to each well. Formazan crystal solubilisation was carried out overnight in an incubator a 37° C. The next day, cell proliferation was evaluated by means of spectrophotometric readings taken with a Beckman Coulter DTX 880 multimode detector (Beckman Coulter Inc., Brea, Calif., USA) using a line at 589 nm and reference at 650 nm.
The toxicity of the micelles was evaluated in HeLa cells by means of the MTT assay. The HeLa cells were seeded in a 96-well plate at a concentration of 3000 cells/well and maintained in 100 μL of medium. The test was carried out after 24 and 48 hours of treatments. The empty micelles, suspended in PBS or in saline solution, were used at a final concentration of 0, 2, 1, 2, 4, 8, 16 and 32×, in reference to the concentration of the loaded micelles, 5 nM, considered as 1×.
At 37° C., the micelles loaded with everolimus showed much higher stability than the solution, as shown in
In order to correlate the stability of everolimus with the preservation of activity, samples incubated under the same conditions as in the stability study were dosed in HeLa and SH-SY5Y cells at established points in time. For this test the cell lines were seeded and maintained for 24 hours at 37° C. prior to treatment. The solution and the micelles loaded with everolimus were tested at a final concentration of 5 nM. As shown in
The solutions clearly showed a drastic decrease in activity at 14, 35 and 50 days, at 37, 25 and 4° C., respectively, data that correlate perfectly with the instability previously observed. All of the micelles, by contrast, were highly stable, remaining completely active at 25 and 4° C., whereas the ones incubated at 37° C. decreased to about 20% at 77 days. These results demonstrate a perfect correspondence between the stability measured and the activity obtained without any difference between the two cell lines. In order to determine the biocompatibility in vitro of the DSPE-PEG2000 micelles, the toxicity of the empty micelles was evaluated in the HeLa and SH-SY5Y cell lines by MTT analysis. The empty micelles, suspended in PBS or in saline solution, were used at a final concentration of 0, 0,2, 1, 2, 4, 8, 16 and 32×, where 1×=equivalent to 5 nM of everolimus. As shown in
Overall, the DSPE-PEG2000 micelles seem to be highly tolerated and safe, at least in vitro, supporting their clinical application.
The evaluation of the stability of DPSE-PEG2000 micelles loaded with everolimus in different media at 37° C. revealed some important differences that are useful for establishing clinical conditions optimal for use of the formulation.
In
The percentage of Everolimus loaded in micelles in PBS decreased until arriving at zero at day 42, whereas the stability in aCSF was nearly double up to day 80. The behaviour in saline solution was nearly completely identical to that observed during the first stability study, which confirmed the already observed stabilisation capacity of the micelles. The high stability in aCSF may suggest a high compatibility with bodily fluids during prolonged administration of the formulation. Naturally, a further destabilisation is to be expected in bodily fluids due to the enzymatic component, which was not taken into consideration in this simulation, but which we predict to be negligible in CSF.
Furthermore, the variations in the size distribution of the micelles over time in PBS and saline solution were studied.
This behaviour is explained by an initial period of organisation of the micelles, which led to a size adjustment in both media. A similar trend was observed for the value of the polydispersity index (PI) in PBS, whereas PI remained more constant in saline solution. In general, the micellar dispersion is finer and more homogeneous in PBS with a PI around 0.4 compared to a PI of about 1.3 in saline solution (
These results, together with the size distribution profiles, suggest that the high stabilisation observed is probably due to an aggregate state of the micellar dispersion in the saline solution. In PBS, by contrast, this aggregate state is disrupted by the presence of phosphate with a consequent reduction in the protective ability of the micelles over time.
The micellar solution of everolimus is more stable than the solution of everolimus in organic solvent, as it is more slowly degraded. In fact, the results obtained from the HPLC assays and MTT assays show that the micellar solution of everolimus is capable of maintaining the molecule of everolimus stable for a number of days, thus improving the effectiveness thereof (
| Number | Date | Country | Kind |
|---|---|---|---|
| 102020000007228 | Apr 2020 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2021/052740 | 4/1/2021 | WO |
