Patent Application
20210220270
The present invention relates to novel formulations of peroxisome proliferator activated receptor (PPAR) modulators and uses of the same in therapy. It also relates to the use of acylhomoserine lactones (PPAR modulators) for enhancing drug delivery via nonparenteral or topical administration routes.
The present invention is concerned, in one aspect, with the treatment or prevention of neurodegenerative conditions, retinal disorders, and brain disorders, as well as pulmonary arterial hypertension, cancer and antifibrotic disorders. Neurodegenerative conditions affect various parts of the central and peripheral nervous systems, and include Parkinson's Disease, Alzheimer's Disease and Huntington's Disease. Retinal disorders may include retinal degenerative conditions such as glaucoma, age-related macular degeneration (AMD), diabetic retinopathy and optic neuritis. Brain disorders may include traumatic brain injury, stroke, cerebral palsy, e.g. as caused by neonatal hypoxia, and cancer, including brain tumours.
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the superfamily of nuclear hormonal receptors. These receptors were identified in the hepatocytes of rodents in 1990, and the name comes from their ability to induce peroxisome proliferation. PPARs interact directly with PPAR gamma coactivators 1-alpha (PGC-1a) and 1-beta (PGC-1β) in the regulation of mitochondrial biogenesis, through the detection and control of lipid homeostasis. These receptors also regulate the expression of genes coding for uncoupling proteins (UCPs). UCPs are transporters in the inner mitochondrial membrane involved in the control of thermogenesis, ROS production, and oxidative function. By binding to a specific sequence in the promoter region of target genes, PPARs are able to regulate gene transcription. Activated PPARs can also directly inhibit transcription factors. These functions allow PPARs to encourage lipid consumption in the production of ATP when there is a cellular demand for energy. Many PPAR modulators have poor aqueous solubility and/or bioactivity which may limit therapeutic utility.
Rosiglitazone is an exogenous agonist of PPAR-gamma and belongs to the thiazolidinedione family, which acts as insulin sensitizers. Rosiglitazone was originally used to counter insulin resistance in type 2 diabetes, has recently shown promise as a therapy in animal models of PD (Normando et al 2016). Rosiglitazone therapy is reported to promote an anti-inflammatory response, with attenuation of microglial activation, release of pro-inflammatory cytokines, oxidative stress, astrocytic gliosis and reversible inhibition of monoamine oxidase—a crucial enzyme for dopamine metabolism. The outcomes of clinical investigations of thiazolidinedione therapies for the treatment of PD have so far been complex and administration of these agents is reported to not slow disease progression.
More recently, curcumin, resveratrol and acylhomoserine lactones (AHLs) have each been reported to bind PPARs and this interaction has been found to contribute to their biological activity and/or therapeutic action, including: promotion of an anti-inflammatory response, with attenuation of microglial activation, release of pro-inflammatory cytokines and oxidative stress. AHLs are documented to compete with RSG binding to the same site on PPAR-gamma (Jahoor et al 2008; Cooley et al 2010). Like other PPAR modulators, these compounds suffer from poor aqueous solubility and/or bioavailability.
There is hence a need to provide improved formulations for PPAR modulators in order to improve bioavailability and/or bioactivity.
In a first aspect the present invention provides a pharmaceutical composition comprising a peroxisome proliferator activated receptor (PPAR) modulator incorporated within a polymeric nanocarrier component, wherein the polymeric nanocarrier component is capable of solubilising the PPAR modulators in an aqueous medium. PPAR modulators are known to have low solubility in water, requiring the use of solvents such as dimethyl sulfoxide (DMSO). However, the present inventors have found that polymeric nanocarriers can be used to solubilise PPAR modulators at physiological pH (about pH 4 to about pH 8). The compositions of the invention can therefore be prepared without the need to use potentially harmful solvents such as dimethyl sulfoxide (DMSO). Surprisingly, compositions of the invention have been observed to have neuroprotective effects in in vivo models of Central Nervous System Injury. Additionally, systemic administration of such compositions was observed to have a neuroprotective effect on the retina and CNS. Compositions of the invention have been shown to exhibit greater neuroprotective efficacy than administration of PPAR modulators alone.
The PPAR modulator may be an endogenous or exogenous molecule and includes compounds which modulate the activity of a PPAR such that net receptor activity is changed, i.e. the compounds may act as PPAR agonists or inhibitors. For example, activity of a PPAR may be modulated by PPAR agonists binding to the receptor or acting on downstream components of the pathway activated by the PPAR to induce similar activity. PPAR agonists may be superagonists, partial agonists or full agonists. PPAR modulators may have a binding affinity for the PPAR of 100 μM or less, preferably 10 μM or less, 5 μM or less, or more preferably 2 μM or less or 1 μM or less. In embodiments of the invention PPAR modulators may have a binding affinity for the PPAR of 100 nM or less or 10 nM or less.
The polymeric nanocarrier component as used herein refers to a component comprising a polymer. For example, the polymeric nanocarrier component may comprise a polyethylene glycol (PEG) group and/or a polymer based constituent, such as a poloxamer. Preferably the polymeric nanocarrier component is a surfactant or a synthetic derivative thereof.
The polymeric nanocarrier component may be a non-ionic surfactant and/or may be a micelle forming surfactant. Advantageously, non-ionic micelle forming surfactants form relatively soft/flexible micelles, which can enhance their transport across non-parenteral routes of administration, such as across mucosal membranes or biological membranes. Non-ionic surfactants may include polysorbates (Tweens), Triton X-100, polyethoxylated castor oil, and Solutol HS. In embodiments of the invention the micelle forming surfactant may be selected from one or more of D-α-tocopherol polyethylene glycol 1000 succinate (Vitamin E TPGS), PEGylated phospholipid derivatives (e.g. DSPE-PEG, DSPS-PEG etc.), poloxamers (e.g. Lutrol F68, Lutrol F127 etc.), poly(lactic-co-glycolic acid) (PLGA) or chitosan derivatives. Optionally, PEG derivatives may be further functionalised via addition of a “click-chemistry” reactive group such as maleimide-PEG for covalent conjugation to thiol groups or azide-PEG/alkyne-PEG for covalent conjugation to alkyne/azide functionalised targeting moieties, e.g. TPGS-PEG-MAL, DSPE-PEG-AZIDE etc. Said functionalised targeting moieties may include proteins or peptides, including phosphatidylserine binding proteins such as annexins, especially annexin V or functional fragments or functional derivatives thereof, which preferably comprise the annexin repeat. Alternatively, His-tagged proteins or peptides can be non-covalently associated with the particles surface using Nickel functionalised lipids (e.g. 18:1 DGS-NTA(Ni)).
In preferred embodiments of the invention the polymeric nanocarrier component comprises Vitamin E TPGS, optionally in combination with Lutrol F127, Solutol HS, chitosan or DSPE-PEG. Vitamin E TPGS is a non-ionic surfactant that forms stable micelles at concentrations of greater than 0.02% w/w, providing a low critical micelle concentration. The α-tocopherol component also has an endogenous nature and antioxidant properties, as well as P-glycoprotein antagonism, which can enhance the barrier crossing ability of formulations containing this agent. Polymeric nanocarrier compositions of the present invention may comprise Vitamin E TPGS at concentrations of about 0.02 mg/mL to about 100 mg/mL, preferably about 10 mg/mL to about 65 mg/mL, more preferably about 20 to about 55 mg/mL.
Lutrol F127 is a difunctional block copolymer surfactant consisting of a central hydrophobic polyoxypropylene group flanked by hydrophilic polyoxyethylene groups, and can sterically stabilise nanocarriers against aggregation. Polymeric nanocarrier compositions of the present invention may comprise Lutrol F127 at concentrations of about 0.2% w/v to about 30% w/v, preferably about 5% w/v to about 20% w/v, more preferably about 10% w/v to about 20% w/v. Lutrol F127 may be used alone or in combination with Vitamin E TPGS.
Solutol HS (2-hydroxyethyl 12-hydroxyoctadecanoate) is non-ionic solubilizer and emulsifying agent, with low toxicity. Polymeric nanocarrier compositions of the present invention may comprise Solutol HS at concentrations of about 100 mg/mL to about 200 mg/mL. Solutol HS is preferably used in combination with Vitamin E TPGS.
The polymeric nanocarrier component may be up to 100% micelle forming surfactant, for example, the polymer nanocarrier component may be up to 100% vitamin E TPGS or up to 100% Lutrol F127. Alternatively, the micelle forming surfactant may comprise a combination of surfactants, such as Vitamin E TPGS in combination with a PEGylated phospholipid derivative (e.g. DSPE-PEG), a non-ionic surfactant (e.g. Solutol HS) or a poloxamer (e.g. Lutrol F127).
The composition is preferably in the form of an encapsulated formulation, most preferably a micelle. Without being bound by theory, the inventors believe that encapsulation of the PPAR modulator may improve bioavailability of this component by providing sustained release of the PPAR modulator and protect PPAR modulators against hydrolytic degradation. Compositions of the invention have been observed to exhibit a greater neuroprotective effect than PPAR modulators administered in an unencapsulated form.
Micellar nanocarriers of the present invention can have a diameter of about 100 nm or less, preferably about 70 nm or less or about 50 nm or less. In preferred embodiments of the invention micellar nanocarriers have a diameter of about 30 nm or less. Micellar nanocarriers may have a minimum diameter of about 10 nm. Preferably the micellar nanocarriers have a diameter of about 20 nm. Liposomes on the absence of sizing (e.g. extrusion process) are heterogeneous in size, often ranging from 100 nm to 1000 nm in diameter. In contrast, micellar nanocarriers of the present invention are substantially homogenous in diameter. For example, at least 70% or at least 80% or at least 90% of the micellar nanocarriers in a composition of the present invention may have a diameter between about 10 nm and about 30 nm.
Solubilisation of the PPAR modulator preferably refers to encapsulation of the PPAR modulator, which may be quantified by encapsulation efficiency. When carried out at physiological pH encapsulation efficiency is preferably at least 5% or at least 10% or at least 15%. In embodiments of the invention encapsulation efficiency may be at least 20% or at least 25%. In embodiments of the invention encapsulation efficiency may be 50% or more, or 70% or more, or 80% or more and may be up to 100%.
Encapsulated formulations may comprise the PPAR modulator at concentrations of from about 0.1 mg/mL to about 100 mg/mL, preferably about 0.5 mg/mL to about 50 mg/mL, more preferably about 1 mg/mL to about 10 mg/mL.
In preferred embodiments of the invention the composition is in the form of a ternary system comprising an aqueous continuous phase, the PPAR modulator and polymeric nanocarrier component being predominantly present in a disperse phase distributed therein.
The PPAR modulator may be a PPAR-alpha modulator, a PPAR-gamma modulator, a PPAR-delta modulator, a dual PPAR modulator or a pan PPAR modulator. In preferred embodiments of the invention the PPAR modulator is a PPAR-gamma agonist or a compound having PPAR-gamma agonist activity. Without being bound by theory, the present inventors believe that PPAR-gamma agonists (or compounds having such activity) act on neurons and retinal ganglion cells (RGCs) to mitigate oxidative stress, reduce microglia activation and pro-inflammatory cytokine release and promote mitochondrial biogenesis. These pathways have been implicated in the pathogenesis of certain CNS disorders, including glaucoma and Parkinson's Disease, suggesting a mechanism of action in the treatment of such disorders as described herein.
The PPAR agonist may be a thiazolidinedione. In embodiments of the invention the PPAR agonist may be selected from one or more of pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, rivoglitazone, Phytocannabinoid 49-THCA and troglitazone.
Alternatively, as mentioned above, the PPAR modulator may be a compound that modulates the activity of a PPAR such that net receptor activity is increased, i.e. a compound that has the same net effect as an agonist. Such compounds include curcumin and resveratrol, which are known to have PPAR-gamma activity.
Alternatively, the PPAR modulator may be an acylhomoserine lactone (AHSL) compound. Such compounds have been shown to have PPAR modulation capability (Jahoor et al.). The AHSL may, for example, have an acyl group of 4 to 20 carbons in length. 3-oxo and 3-hydroxy derivatives may also be mentioned, as may the tetramic acid and tetronic acid derivatives of AHSLs. Exemplary AHSL compounds include 3-hydroxydodecanoyl homoserine lactone and 3-oxododecanoyl homoserine lactone. AHSL compounds have the additional advantage that they interact with tight junctions to increase permeability of biological barriers (Karlsson et al.). This can enhance delivery of the composition to the intended tissue in vivo.
For example, suitable polymeric nanocarrier compositions of the present invention may be micellar formulations of:
Compositions of the invention may comprise combinations of two or more PPAR modulators. Preferably at least one of the PPAR modulators is encapsulated as described above. Alternatively, both PPAR modulators may be encapsulated. When two or more PPAR modulators are encapsulated the type of encapsulation may be the same or different. For example, both PPAR modulators may be encapsulated in a single polymeric nanocarrier, or each PPAR modulator may be encapsulated in a separate polymeric nanocarrier which are combine prior to administration. In embodiments of the invention the composition may comprise a combination of resveratrol and curcumin.
The composition may be sterile and may comprise one or more pharmaceutically acceptable carriers or excipients. Suitable carriers and excipients will be familiar to the skilled person and may be optimised in line with the intended route of delivery. For example, compositions of the inventions may include buffers, binders, preservatives, thickeners or antioxidants, such as trehalose.
Preferably the composition is suitable for topical delivery, including ocular and nasal delivery, oral or dermal delivery. Local delivery of the composition may be advantageous due to reducing systemic exposure to the PPAR modulator, which have been linked to harmful side effects including increased risk of myocardial infarction and death.
Topical formulations are preferably in the form of a solution or suspension in an aqueous medium, such as a solution, lotion, gel, cream, ointment, gel or foam. Oral formulations may be in the form of solutions, suspensions, tablets, capsules, powders or granules. Parenteral administration may include intravenous, subcutaneous or intraperitoneal administration. Parenteral formulations in particular may be in the form of solutions or suspensions in aqueous media or may be provided as a lyophilised powder.
In embodiments of the invention the composition is suitable for intranasal delivery. Such formulations may be in the form of a solution, suspension or dry powder suitable for inhalation.
In general, and especially where the composition is to be administered in liquid form (via any of the above routes), the composition of the invention may be provided as a lyophilised powder. The compositions of the invention have been determined to be stable to lyophilisation, and can be reconstituted using, for example, normal saline or other (preferably aqueous) vehicles. It is preferred, when lyophilisation is to take place, to include a cryoprotectant material such as trehalose in the composition.
The composition of the invention may be used in therapy. In particular, the composition of the invention may be used in the treatment or prevention of a CNS disorder, such as a neurodegenerative disorder, a retinal disorder or a brain disorder.
In a further aspect the present invention provides a method for treating a CNS disorder, such as a neurodegenerative disorder, a retinal disorder or a brain disorder, the method comprising administering a composition of the invention to a patient. The patient is preferably a mammal, including a human, and may be a paediatric or geriatric patient.
The neurodegenerative condition may be Parkinson's Disease, Alzheimer's Disease or Huntington's Disease. Retinal disorders may include retinal degenerative conditions such as glaucoma, age-related macular degeneration (AMD), diabetic retinopathy and optic neuritis. Brain disorders may include traumatic brain injury, stroke, cerebral palsy, e.g. as caused by neonatal hypoxia, and cancer, including brain tumours. In embodiments of the invention the composition may be for use in the treatment of pre-symptomatic Parkinson's Disease.
The composition may be administered topically, such as ocularly, or intranasally, as described above. In embodiments of the invention the composition may be administered in combination with one or more additional therapeutic agents, such as insulin, metformin, dipeptidyl peptidase-4 (DPP-4) inhibitors (such as Alogliptin), glucagon-like peptide-1 (GLP-1) receptor agonists, antioxidants (such as resveratrol, Coenzyme Q10, Idebenone, Quercetin etc.), compounds of the vitamin D group, or derivatives thereof, vascular endothelial growth factor (VEGF) antagonists (such as ranibizumab, bevacizumab or functional fragments thereof), N-methyl-D-aspartate (NMDA) receptor antagonists, glutamate antagonists or memantine. The additional therapeutic agent may be administered simultaneously with the composition of the invention or may be administered sequentially. Where the additional therapeutic agent is administered simultaneously with the composition of the invention, it may be included in the composition of the invention, either in the same phase as the PPAR or in the continuous phase of a ternary composition. In a particular embodiment, the addition therapeutic agent has a hydrophobicity such that both it and the PPAR are present in the disperse phase (i.e. co-encapsulated). In embodiments of the invention the composition comprises curcumin and an antioxidant, such as resveratrol.
In a further aspect the present invention provides a method for preparing a PPAR modulator micellar composition as described above, the method comprising: (i) dissolving one or more polymeric nanocarrier components in a first solvent mixture; (ii) dissolving a PPAR modulator in a second solvent mixture; (iii) combining the dissolved polymeric nanocarrier component and dissolved PPAR modulator and drying the combination to a form a film; (iv) rehydrating the film with buffer to form a micelle solution; (v) filtering the suspension to remove unencapsulated PPAR modulator. Preferably, the first solvent mixture is a short chain primary alcohol, such as ethanol. Compared to other solvents such as chloroform/methanol, ethanol is less toxic, meaning that residual solvent which may be present in the composition is unlikely to be problematic. In embodiments of the invention the first and second solvent mixtures may be the same. In preferred embodiments of the method the PPAR modulator is curcumin, resveratrol or AHSL, which have been shown to be poorly soluble in other solvent mixtures. Preferably the suspension is filtered through a membrane filter having a pore size of about 0.22 μm, which can additionally remove any potential biological contaminants.
In an additional aspect the present invention provides a pharmaceutical composition comprising an active pharmaceutical ingredient (API) and an AHSL compound. The AHSL may, for example, have an acyl group of 4 to 20 carbons in length. 3-oxo and 3-hydroxy derivatives may also be included, as may the tetramic acid and tetronic acid derivatives of AHSLs. Exemplary AHSL compounds include 3-hydroxydodecanoyl homoserine lactone and 3-oxododecanoyl homoserine lactone.
As explained above, AHSL compounds interact with tight junctions to increase permeability of biological barriers. These compounds are known to be used by certain bacteria as part of the tissue invasion process during the establishment of an infection of a host. However, the potential of these compounds as a means for delivering an API into target tissues has not been recognised previously, and the present inventors have determined that such an approach may be used with a large range of APIs, including aforementioned PPAR modulators (curcumin, resveratrol etc) and those with high molecular weights, such as peptides or antibodies.
In a preferred embodiment, the composition of this aspect further includes an polymeric nanocarrier component encapsulating the AHSL and/or the API in liposomes or micelles. The polymeric nanocarrier component is preferably in the form of a liposome, which may include one or more phospholipids and can also include a sterol such as cholesterol and/or a vitamin E derivative such as TPGS. Suitable phospholipids include, for example, those based on phosphatidylcholine, phosphatidylserine and phosphatidylethanolomine. In more detail, phospholipids for use in compositions of the invention may include natural phospholipid derivatives or synthetic phospholipid derivatives. Natural phospholipid derivatives may include one or more of egg phosphatidylcholine, hydrogenated egg phosphatidylcholine, soy phosphatidylcholine, hydrogenated soy phosphatidylcholine or sphingomyelin, such as 1-Myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine. Synthetic phospholipid derivatives may include one or more of 1,2-Didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-Dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-Dilauroyl-sn-glycero-3-phosphoserine (DLPS), 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-Dimyristoyl-sn-glycero-3-phosphoserine (DMPS), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and 1,2-Distearoyl-sn-glycero-3-phosphoserine (DSPS). In embodiments of the invention the surfactant or lipid may be conjugated to polyethylene glycol (PEG), e.g. PLGA-PEG. Alternatively, the polymeric nanocarrier component may be as defined in relation to the first aspect of the invention.
Some AHSLs are capable of forming micelle-like structures in aqueous media, and hence are able to act as a delivery system for an API without addition of further components. However, the stability of the composition can be improved by the inclusion of an polymeric nanocarrier component, e.g. as defined above.
The API to be included in the composition of this aspect is not particularly limited, and the skilled person would readily be able to determine which APIs could potentially be employed. As examples, however, the various API-types mentioned above in relation to the first aspect may be considered.
In a related aspect, the invention provides the use of an AHSL for enhancing delivery of an API. In particular, delivery across biological barriers (such as the blood-brain barrier, blood-retinal barrier etc) is enhanced.
The invention will now be described in detail, by way of example only, with reference to the figures.
Micelles comprising TPGS and Lutrol F127 with up to 5 mg/mL of curcumin or Resveratrol have been prepared and tested for stability. Micelles have also been prepared using PEG-Cholesterol with Lutrol F127.
As shown in
Curcumin micelles were formed by a thin-film rehydration method (see
Unencapsulated curcumin was removed by filtration as shown in
Freeze-drying protocol involved three stages. 1° (Primary), 2° (Secondary); h (hours).
Cryoprotectant later replaced with 10 mg/mL to 100 mg/mL Trehalose (with 10 mM HEPES, pH 7.4) which showed better stability as shown in following formulations.
Curcumin micelle stability was good at room temperature and after freeze-drying in the presence of a trehalose cryoprotectant (10 mg/mL to 100 mg/mL trehalose). Micelles were prepared at both pH 4.5 and pH 7.4. See
Resveratrol micelle formulation (later co-administered with curcumin micelles in vivo) showed good stability using the same micelle formulation (see Table 5).
Preferred buffer composition is with Trehalose (10 mg/mL to 100 mg/mL) as this antioxidant prevents degradation of the product on storage over time versus PBS which causes a progressive discolouration of the product (see
Samples showed good stability for up to 10 weeks (See
The curcumin micelle formulation was found to be significant neuroprotective in vitro (see
Topical curcumin micelle eye drops (twice a day for three weeks) and curcumin/resveratrol micelle co-therapy (two drops of each per day) were found to be significantly neuroprotective in a rat model of optic neuropathy (partial optic nerve transection) (
Curcumin treatment had no effect on TOP suggesting the neuroprotective effects were TOP independent (see
Curcumin, D-α-tocopherol polyethene glycol 1000 succinate (TPGS) and Pluronic F127 were obtained at the highest available purity from Sigma-Aldrich (Kent, UK). Curcumin-loaded nanocarriers (CN) were prepared using an adaptation of the thin-film hydration technique described previously (Davis et al. 2014). Curcumin, TPGS, and Pluronic F127 were dissolved in ethanol to a concentration of 5 mg/mL, 10 mg/mL and 20 mg/mL respectively; with 10 min of gentle heating and bath ultrasonication to clarity. Solutions were aliquoted in the desired molar ratio (22.55 mM, 12.22 mM 7.94 mM of TPGS, curcumin, and Pluronic F127 respectively) into a round bottom flask, mixing well. The solvent was removed by rotary evaporation (50 mBar, 65° C., 2 h) using a Rotavapor R210 with a V850 Vacuum controller (Buchi, Switzerland) while protecting from light. After this time, the thin-film was rehydrated (50° C., 0.5 h) in the desired buffer (distilled water, phosphate buffered saline (pH 7.4) or HEPES trehalose buffer (10 mM HEPES, 50 mg/mL trehalose, pH 7.4). Unencapsulated curcumin was then removed from the formulation by 0.22 μm filtration (33 mm Millex filter, Merck Millipore, USA) as shown in
Lyophilisation of CN formulations in HEPES trehalose buffer was achieved by equilibrating 1 mL aliquots of nanocarriers in 7 mL screw neck squat form glass vials (CamLab, Cambridge UK) at 25° C. before freezing at −60° C. for 2 h at 760 Torr. Primary drying of samples was completed at −38° C. at 200 mTorr for 24 h, followed by a secondary drying phase at 25° C. and 200 mTorr for 2 h. Samples were capped immediately after cessation of secondary drying before storing at 25° C. while protecting from light until required. For stability assessment, samples were rehydrated for 30 minutes by addition of 1 mL of 0.22 μm filtered distilled water with gentle mixing.
The moisture content of formulations was determined using thermogravimetric analysis (TGA). Freeze dry samples were placed in an aluminium pan and analysed by a Discovery TGA (TA instruments, USA). Samples were purged with a flow rate of 25 mL/min nitrogen gas and heated from 30 to 200° C. with 10° C./min rate. The percent mass loss was calculated by TA Instruments Trios software at 120° C. for water content. Three freeze dry formulations were measured three times for each sample.
The loading efficiency of CNs was determined spectroscopically and results confirmed using HPLC. Spectroscopic determination of curcumin loading was achieved by diluting in DMSO 1:500 at 435 nm normalised to empty nanocarriers. The concentration of curcumin in each formulation was then determined using the molar extinction coefficient of curcumin (
where [C]s is the concentration of curcumin originally added to the formulation (typically 4.5 mg/mL) and [C]E is the concentration of curcumin detected spectroscopically within the nanocarriers after 0.22 μm filtration to remove unencapsulated material. Results were confirmed using an adaptation of an established HPLC technique (Guddadarangavvanahally et al.). Briefly, curcumin containing samples were diluted in methanol before 20 μL volumes were injected at 25° C. onto a Phenomenex® Synergi (4 μm Polar—RP 80 μL with size of 250×4.60 mm) column with an Acetonitrile: 0.1% trifluoroacetic acid 50:50 solvent system at a flow rate of 1 mL/min connected to a Agilent Technology 1260 Infinity HPLC system. Absorbance was recorded at 420 nm and the area under the curcumin elution curve compared to a standard curve of known curcumin concentrations.
Particle size was determined using a Malvern Zetasizer. Measurements of particle diameter and polydispersity index were recorded from a minimum of three formulations for each experimental condition or time point after manufacture. Nanocarriers were diluted 1 in 10 in the appropriate buffer prior to recording.
Nanocarrier suspensions were processed using carbon grids to absorb particles from suspension before staining with 1% uranyl acetate for 1 min and drying. Specimens were observed using a Joel-1010 Transition Electron Microscope operated at 100 kV with images acquired using a Gatan Orius digital camera.
X-ray diffraction graphs of drug alone, empty nanoparticles or CN were prepared from X-ray diffractometer (Rigaku MiniFlex 600) and the 2-thea angle was set from 5° to 65° with an angular increment of 0.05°/second. The measurements were performed at a voltage of 40 kV and 15 mA. The FT-IR spectrum of free curcumin, empty nanoparticles and CN were recorded using a PerkinElmer Spectrum 100 FT-IR spectrometer at 4 cm-1 resolution, with 4 scans between 4000 cm-1 and 650 cm-1.
In vitro curcumin release was assessed using an adaptation of a previously described protocol (Wang et al. 2012). Briefly, free curcumin (dissolved in 95% ethanol) or CNs containing 4.5 mg/mL of curcumin was loaded into a 1 mL Spectra-Por Float-A-Lyzer dialysis cassette (Sigma-Aldrich) with 3.5-5 kDa molecular weight cut-off. Samples were dialysed against 200 mL of PBS containing 10% Tween-80 to act as a sink for released curcumin maintained at 37° C. with stirring at 50 rpm. At the specified time points, samples were removed from the mixture and replaced with fresh buffer. The concentration of curcumin was determined as described above. Results from three experimental replicates were fit to a single phase association (equation 2);
Y=Y0+(Plateau−Y0)*(1−exp(−K*x)) (2)
Where Y0=zero, Plateau is the maximal release and K is the rate of curcumin release (h−1) from which half-life (t1/2) was calculated (t1/2=ln 2/K).
R28 cell line (Kerafast, Boston, Mass.) were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Paisley, UK) supplemented with 5% foetal bovine serum (Invitrogen, UK), 100 U/ml penicillin, 100 m/ml of streptomycin and 0.292 mg/mL glutamine (Gibco, UK), 7.5% sterile dH20 and 1.5 mM KCl (Sigma-Aldrich, UK). The medium was changed every other day and cultures were passaged at 90% confluence.
R28 cells were plated at 4,000 cells/well in 96-well plates for 24 h before treatment with varying concentrations of curcumin (0 to 20 μM) or an equivalent concentration of TPGS/Pluronic F127 only nanocarriers (vehicle control) in conjunction with varying concentrations of cobalt chloride or glutamate insults for a further 24 h. Cell viability was assessed in each case using the Alamarblue (Invitrogen, UK) assay according to manufacturer's instructions. Briefly, the Alamarblue solution was added to each well-plate and incubated for 4 hours before recording the fluorescence using a Safire plate reader excitation of 530 nm and emission of 590 nm 91.
All animal experiments were performed with procedures approved by the U.K. Home Office and in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For in vivo assessment of experiments: in total 48 Adult male Dark Agouti (DA) rats (Harlan Laboratories, UK) weighing 150 to 200 g were housed in an air-conditioned, 21° C. environment with a 12 h light-dark cycle (140-260 lux), where food and water were available ad libitum. 13 animals served as naïve controls which were not subject to further interventions before immunohistochemistry.
Ocular hypertension was surgically induced in the left eye of 18 DA rats (5 OHT only, 5 OHT+CN, 8 OHT+FC) as described previously (Morrison et al. 1997). Procedures were conducted under general anaesthesia using a mixture of 37.5% Ketamine (Pfizer Animal Health, Exton, Pa.), 25% Dormitol (Pfizer Animal Health, Exton, Pa.) and 37.5% sterile water, at 2 mL/kg administered intraperitoneally. Briefly, 50 μL of hypertonic saline solution (1.8 M) was injected into the two episcleral veins using a syringe pump (50 μL/min; UMP2; World Precision Instruments, Sarasota, Fla., USA). A propylene ring with a 1 mm gap cut from the circumference was placed around the equator to prevent injected saline outflow from other aqueous veins. The IOP from both eyes of each rat was measured at regular intervals using a TonoLab tonometer (Tiolat Oy, Helsinki, Finland) under inhalational anaesthesia (0.4% isoflurane in oxygen). Daily administration of topical CNs was performed in 5 DA rats (two 35 μL drops/day 5 min apart at 10 am each day) starting two days prior to model induction and continuing until model termination (21 days post IOP elevation) with 5 rats serving as OHT only controls. An additional 8 rats received free-curcumin (FC) prepared using the same protocol as CN curcumin without the addition of TPGS or Pluronic F127. FC was administered to OHT animals using the same dosing regime as described for CN. Animals were sacrificed three weeks after unilateral IOP elevation and retinas flat-mounted prior to Brn3a immunohistochemistry.
Partial optic nerve transection was conducted in the left eye of 17 DA rats, using a previously described technique (Levokovitch-Verbin et al. 2003). Under general anaesthesia, an incision was made in the superior conjunctiva, and the ON sheath was exposed. A longitudinal slit was next formed in the dura mater to expose the ON and a 0.2-mm cut was made in the dorsal ON, 2 mm behind the eye using an ophthalmic scalpel with steel cutting guard. Damage to major ophthalmic blood vessels was avoided and verified at the completion of surgery by ophthalmoscopy. Daily administration of topical CNs was conducted in 9 DA rats after induction of the pONT model using the same treatment regimen as described previously with the remaining 8 serving as pONT only controls.
Brn-3a labelling of RGCs in retinal whole-mounts was completed as described previously (Davis et al. 2016). Briefly, eyes were enucleated upon sacrifice and fixed in 4% paraformaldehyde at 4° C. overnight before dissecting retinal whole mounts. Whole mounts were stained for the RGC specific nuclear-localised transcription factor Brn3a using an anti-mouse mAb (1:500, Merck Millipore, Darmstadt, Germany) and examined under confocal microscopy (LSM 710, Carl Zeiss MicroImaging GmbH, Jena, Germany). Each retinal whole mount was imaged as a tiled z-stack at ×10 magnification which was used to generate a single plane maximum projection of the RGC layer in each retina for subsequent analysis. Each whole mount image was manually orientated so that the superior retina was towards the top of the image using in vivo cSLO imaging of retinal vasculature as a reference. Retinal image acquisition settings were kept constant for all retinas imaged, allowing comparison of Brn3a expression in each experimental group as previously described. 94 Automated quantification of Brn3a labelled RGCs in retinal whole-mounts was completed as described previously (Davis et al. 2016).
All data were analysed with the Student's t-test, ANOVA or with appropriate post hoc testing using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, Calif., USA) as appropriate. Data were presented as means±SE and p<0.05 was considered significant. Molecular structures were drawn using ACD/ChemSketch 2015 and all images were taken by the authors (BMD).
On dilution in dimethyl sulfoxide (DMSO) curcumin had an absorbance peak at 435 nm (
Spectroscopic determination of curcumin concentration in nanocarriers can also be used to give indication of the extent of curcumin degradation. Curcumin undergoes keto-enol tautomerization (
Initially, curcumin loaded nanocarriers were prepared by incorporation it into TPGS nanocarriers. TPGS was chosen due to the low critical micelle concentration of this excipient (0.02% w/w), the endogenous nature and antioxidant properties of the α-tocopherol component and P-glycoprotein antagonism, which enhances the barrier crossing ability of formulations containing this agent. TPGS is present in existing ophthalmic formulations and both curcumin and TPGS can be readily solubilized in ethanol, a solvent which is present at concentrations of 0.8% in commercially available eye drop formulations (i.e. Optrex ActiMist 2in 1 Eye Spray for Dry Irritated Eyes) so reducing risks associated with residual solvents from the manufacturing process. Furthermore, as the use of TPGS to enhance the bioavailability of orally administered drugs is well documented. This, in combination with recent interest in the use of Pluronic F127 food-research applications may suggest that the novel curcumin formulation described herein may also be suitable for oral administration.
Formulation of curcumin with TPGS micelles was found to produce nanocarriers with 16 nm diameter as determined by dynamic light scattering (data not shown). Unfortunately, these formulations rapidly aggregated at 25° C., resulting in the formation of sediment within hours of resuspension which may be indicative of Ostwald ripening processes. Stabilisation of curcumin loaded TPGS nanocarriers was achieved by the addition of the polymeric stabiliser Pluronic F127 (a triblock copolymer of polyoxyethylene and polyoxypropylene), which has previously been used to sterically stabilise nanocarriers against aggregation.
Curcumin-loaded nanocarriers (CN) were prepared according to the methods described, with encapsulation efficiency and average particle size determined (
The encapsulation efficiency and particle size of CN formulations were assessed over time after storage at 25° C. while protecting from light. The CN formulation was found to exhibit excellent stability for 9 weeks at 25° C., with no reduction in formulation EE % (
Several groups have previously attempted to prepare curcumin loaded nanoparticle formulations, including; PLGA-nanocarriers, solid lipid nanocarriers, liposomes and exosomes. Existing nanoparticulate formulations of curcumin possess limited stability (not assessed beyond 72 h in any study cited), only moderate curcumin loading has been achieved (<0.77 mg/mL) and most protocols would be difficult to translate to the clinic owing to complex, multi-step manufacture protocols requiring organic solvents. The TPGS/Pluronic F127 curcumin formulation described here compares favourably with those in the existing literature.
XRD and FT-IR spectra were acquired to ascertain the nature of curcumin once incorporated into nanocarriers (
Formulation of curcumin into nanocarriers substantially reduced the rate of drug release compared to free drug (t1/2=22.6 h versus 0.15 h respectively,
Glutamate excitotoxicity represents a potential mechanism leading to RGC loss in glaucoma. Using an AlamarBlue cell viability assay, co-incubation of immortalised R28 cells with both CNs and empty nanoparticles was found to be significantly protective (one-way ANOVA with Tukey post-test, p<0.001) against glutamate induced toxicity (
Upregulation of hypoxia-related factors such as Hypoxia Inducible Factor 1α (HIF-1α) has been suggested to implicate hypoxia in glaucoma pathology. Cobalt chloride (CoCl2) is a hypoxia mimetic and inducer of HIF-1α 75 used as an in vitro glaucoma model. The IC50 of R28 cells exposed to CoCl2 for 24 h (
Topically administered curcumin nanocarrier therapy protects RGCs in rodent models of ocular hypertension and optic nerve injury Having established the neuroprotective activity of CNs in vitro in relation to vehicle only treatments, we next assessed the neuroprotective effects of this formulation on RGC health using an established in vivo rodent model of RGC loss. We anticipate that topically applied curcumin loaded nanoparticles will reach the retina via a combination of topical and systemic absorption routes. In support of this hypothesis, Sigurdsson et al reported that their formulation of dexamethasone, which is a similar molecular weight to curcumin (392 versus 368 Da respectively), entered the retina 60% via topical penetration and 40% by systemic absorption route. We anticipate that the well-documented P-gp inhibition activity of tocopherols and curcumin, in conjunction with enhanced corneal penetration activity previously reported for PEGylated-micelle formulations will enhance curcumin delivery to the retina by the topical absorption route.
Optimum time points post model induction (maximal RGC loss in shortest time after induction) were chosen based on our previous work characterising the natural history of the OHT and pONT models where multiple time points were assessed after model induction. We recently reported that administration of TPGS containing micelles did not themselves have a neuroprotective effect in vivo, which in conjunction with our in vitro observations, suggest that any neuroprotective efficacy observed was a result of curcumin treatment. Rats received topical CNs according to the dosing regimen illustrated in
To further investigate the neuroprotective potential of topically applied CNs, whole-retinal brn3a labelled RGC population assessments were made in the pONT model (
The possibility of TPGS mediated neuroprotection via inhibition of glutamate excitotoxicity is intriguing and may contribute to the neuroprotective effect of our formulation in vivo. In support of this hypothesis and our present in vitro findings, Nucci et al previously reported that intraocular administration of a total of 10 μL of 0.5% (w/v) TPGS (equivalent to a total dose of 0.5 mg TPGS) was neuroprotective against ischemia/reperfusion injury in the rat. Previously, we reported that topical administration of TPGS at the same concentration did not have a neuroprotective effect in vivo. This discrepancy is likely to the lower concentration reaching the retina compared to invasive application, typically estimated to be ˜3% of the topically applied dose. Although our previous work with this model suggests that administration of TPGS only did not appear to have a neuroprotective effect in its own right, a synergism between curcumin and TPGS is extremely likely, if not via the neuroprotective effects of TPGS alone, then perhaps via TPGS mediated modulation of P-gp activity, enhancing curcumin transport across ocular barriers.
The neuroprotective effect of curcumin loaded nanocarriers observed in this study may be a result of treatment commencing two days before model induction, suggesting this therapy may be most effective for patients at risk of IOP spikes such as following phacoemulsification surgery or as a prophylactic to patients identified at high risk of developing glaucoma such as those with ocular hypertension or other glaucoma risk factors. Furthermore, with the development of new techniques such as DARC (detection of apoptotic retinal cells) with the potential to diagnose glaucoma earlier in the disease process (Cordeiro et al. 2017), therapies to slow or prevent RGC loss at earlier stages of disease progression will play a greater role in glaucoma management.
In conclusion, this study describes a novel nanocarrier formulation of curcumin in TPGS/Pluronic F127 that increases the solubility of this poorly soluble drug by a factor of almost 400,000. This formulation incorporates 4.3 mg/mL of curcumin with an encapsulation efficiency consistently >90% and excellent stability in liquid or lyophilized forms for at least two months when stored at room temperature, as determined by HPLC and spectroscopic techniques. This formulation was found to be neuroprotective against glutamate and cobalt chloride induced injury in retinal cultures in vitro and significantly preserved RGC density in two well-established rodent models of ocular injury. In conclusion, we demonstrate that curcumin loaded nanoparticles have exciting potential for overcoming ocular barriers and may facilitate the translation of curcumin based therapies to the clinic for the treatment of ocular conditions such as glaucoma.
Turning to
In further detail, and by way of example only, the compositions can be prepared as follows (note that Formulations 1 to 3 are reported in
Formulation 1. 3-OH—C12 HSL with TPGS or DSPE-PEG and Cholesterol Micelles
Note that in these exemplary compositions, 3-OH—C12 HSL can be substituted with any AHSL.
HCE-S cells (Immortalized Human Corneal Epithelial Cells) were cultured as a monolayer in 90% DMEM supplemented with 10% heat inactivated FBS and penicillin-streptomycin (100 U/mL). Cells were cultured at 37° C. in a humidified incubator with 5% CO2 and growth medium was replaced every three days. HCE-S populations were grown to 80% confluence before passage to 20% using 0.25% Trypsin-EDTA. Prior to each experiment cell populations were estimated using a haemocytometer and trypan blue exclusion assay.
HCE-S barrier models for transcytosis assays were prepared using an adaptation of a previously described method (Reichl (2008)). HCE-S cells (5×104 cells/well) were seeded on transwell inserts (polycarbonate, 3 μm pore size, 1.13 cm2) and maintained for 7 days refreshing the medium in the apical and basal chambers every second day. After this time cells were cultured for a further 10 days at an apical air interface to encourage development of a multilayer barrier. Prior to each experiment culture medium was replaced with phenol red free DMEM and barrier integrity measured via transepithelial resistance (TER). TER values were obtained in the range 300-600 Ωcm2 and only epithelial barriers with TERs >300 Ω·cm2, the accepted threshold for tight epithelial barriers, were used for transcytosis experiments (Becker et al.).
Although the HCE-S transwell model used here is a model of the corneal epithelium, the method of acylhomoserine lactone-mediated reversible barrier disruption is via interaction with IQGAP1 [Karlsson et al.]. A similar effect will therefore be observed in other biological barriers (i.e. blood-brain, blood retina, intestinal and dermal barriers).
To date the ability of acylhomoserine lactones to disrupt biological barriers has focused solely on the ability of these agents (which are produced by bacteria, and 3-OH—C12 HSL is a synthetic analogue of 3-oxo-C12 HSL) to permit bacteria to invade host tissues. This work presents the first demonstration of the use of these agents to facilitate the delivery of therapeutics (including macromolecules such as polysaccharides and large proteins (
To study the ocular tolerance in vitro the HETCAM® test was developed as described in the INVITTOX no15 protocol (Warren et al. 1990).
This test is based on the observation of the irritant effects (bleeding, vasoconstriction and coagulation) in the chorioallantoic membrane (CAM) of a 10 days embryonated egg induced by application of 0.3 ml of each of the studied formulations for the first 5 min of its application.
These eggs (from the farm G.A.L.L.S.A, Tarragona, Spain) were kept at a temperature of 12±1.0 for at least 24 h before placing them in the incubator with controlled temperature (37.8° C.) and humidity (50-60%) during the incubation days.
A series of controls were performed: SDS 1% (positive control for slow irritation), 0.1 N NaOH (positive control for fast irritation), NaCl 0.9% (negative control).
Data were analysed as the media±SD of the time at which the injury occurred (n=3/group). Scores of irritation potential can be grouped into four categories (Table 8).
The formulations analysed showed to be non-irritant in vitro (Table 9). Interestingly, in all the cases the only phenomena that appeared was a slight vasoconstriction process (
In vivo ocular tolerance was assessed using the Draize irritation test. It was performed using New Zealand albino male rabbits of 2.5 kg middle weight from San Bernardo farm (Navarra). This test was performed according to the Ethical Committee for Animal Experimentation of the UB and current legislation (Decret214/97, Gencat).
The sample was placed in the conjunctival sac of the left eye and a gentle massage was applied to assure the proper circulation (Nobrga et al. 2012). The appearance of irritation was observed both at the time of administration and after 1 h, using the right eye as a negative control (n=3/group).
The evaluation was performed by direct observation of the anterior segment of the eye, noting the possible injury of the conjunctiva (inflammation, chemosis, redness or oozing), iris and cornea (opacity and affected surface). Ocular irritation index (OII) was evaluated according to the observed injuries (Tables 10 and 11).
None of the formulations was irritant (011=0). The animals did not show any sign of irritation in vivo at the time of the application or after one hour (
A formulation of 4.5 mg/ml curcumin encapsulated in micelles formed from 25 mg/ml TPGS and 150 mg/ml solutol was prepared using the previously described thin-film rehydration technique.
The formulation demonstrated high encapsulation efficiency and stability over 90 days (
The formulation was then tested in the 3xTg-AD mouse model of Alzheimer's disease and was administered intranasally, 5 days per week for 3 months. The curcumin nanoparticles decreased the DARC count (see e.g. WO 2011/055121 for further details of DARC count) in the retina when compared to vehicle alone, indicating that cell death was reduced (
A formulation of 15 mg/ml resveratrol encapsulated in micelles formed from 25 mg/ml TPGS and 150 mg/ml solutol was prepared using the previously described thin-film rehydration technique.
The formulation demonstrated high encapsulation efficiency (>70%) and stability over 90 days (
R28 cells were cultured as described above before being treated with resveratrol (20 μm) containing micelles or an equivalent concentration of TPGS/solutol only (i.e., empty) micelles, in conjunction with varying concentrations of cobalt chloride or glutamate insults.
The resveratrol containing micelles were observed to be neuroprotective against glutamate excitotoxicity (
The formulation was then tested in the 3xTg-AD mouse model of Alzheimer's disease and was administered intranasally, 5 days per week for 3 months. The resveratrol nanoparticles decreased the DARC count in the retina when compared to vehicle alone, indicating that cell death was reduced (
| Number | Date | Country | Kind |
|---|---|---|---|
| 1810492.7 | Jun 2018 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2019/051812 | 6/27/2019 | WO | 00 |
