Patent Application
20130053374
The present invention relates to an ophthalmic composition comprising a hyperbranched polymer. The hyperbranched polymer of the present invention may be any hyperbranched polymer which is pharmaceutically acceptable, e.g., a hyperbranched polymer with a Polyethyleneimine, Polypropyleneimine or Polyester.
COSOPT® and TRUSOPT® are commercially available topical ophthalmic solutions developed by Merck for treating an eye disease called glaucoma. In the case of TRUSOPT®, the active ingredient is Dorzolamide exclusively. In the case of COSOPT®, the active ingredients are Dorzolamide and Timolol (beta blocker). Dorzolamide is a carbonic anhydraze inhibitor with the aqueous solubility of 40 mg/mL at pH 4.0-5.5. It is a white to off-white, crystalline powder, which is soluble in water and slightly soluble in methanol and ethanol.
However, these formulations contain 2% (w/v) Dorzolamide, and are prepared at pH 5.65, due to the limited aqueous solubility of Dorzolamide at physiological pH. Consequently, the COSOPT® and TRUSOPT® formulations can lead to local irritation, due to the low pH. Dorzolamide has two pKa values of 6.35 and 8.5, which correspond to the protonated secondary amine group and the sulfonamide group, respectively. Dorzolamide is mainly in its hydrophilic cationic form at pH below 6.4, and in its hydrophilic anionic form above pH 8.5.
Thus, Dorzolamide has a relatively low aqueous solubility in solutions with pH between 6.4 and 8.5, mainly because of Dorzolamide's non-ionic behavior in that physiological pH range.
AZOPT® (Brinzolamide ophthalmic suspension) 1% is a sterile, aqueous suspension of Brinzolamide, which has been formulated to be readily suspended and slow settling, following shaking. AZOPT® is developed by Alcon and contains Brinzolamide as active ingredient. The formulation has a pH of approximately 7.5 and an osmolality of 300 mOsm/kg. It is instilled for the reduction of elevated intraocular pressure in patients with open-angle glaucoma or ocular hypertension. Brinzolamide's pKa values are 5.9 (amine) and 8.4 (primary sulfonamide), allowing it to act as an acid or a base (ampholyte) depending upon the pH. It is mainly in its hydrophilic cationic form at pH below 5.9 and hydrophilic anionic form above pH 8.4. It is clear that Brinzolamide is significantly less protonated (<10%) at physiological pH. Thus, Brinzolamide has relatively low aqueous solubility in solutions with pH between 5.9 and 8.4, mainly because of Brinzolamide's nonionic (lipophilic) behavior in that pH range.
Dendritic polymers are tree-like polymers that can be classified into two main types based on their branching architecture as “perfectly branched” (dendrimers) and “imperfectly branched” (hyperbranched polymers or HP). Hyperbranched polymers are molecular constructions having a branched structure, generally around a core. Unlike dendrimers, the structure of hyperbranched polymers generally lacks symmetry, as the base units or monomers used to construct the hyperbranched polymer can be of diverse nature and their distribution is non-uniform. The branches of the polymer can be of different natures and lengths. The number of base units, or monomers, may be different depending on the different branching. While at the same time being asymmetrical, hyperbranched polymers can have: an extremely branched structure, around a core; successive generations or layers of branching; a layer of end chains. Hyperbranched polymers are generally derived from the polycondensation of one or more monomers ABx, A and B being reactive groups capable of reacting together, x being an integer greater than or equal to 2. However, other preparation processes are also possible. Hyperbranched polymers are characterized by their degree of polymerization DP=100−b, b being the percentage of non-terminal functionalities in B which have not reacted with a group A. Since the condensation is not systematic, the degree of polymerization is less than 100%. An end group T can be reacted with the hyperbranched polymer to obtain a particular functionality on the ends of chains.
Hyperbranched polymers are mainly identified by their core type and their terminal groups. Examples of a core type for a hyperbranched polymer are polyethylenimine, polypropylenimine, polyglycol, polyether, polyester, etc. A hyperbranched polymer with a polyester core may be referred to as a hyperbranched polyester. Examples of terminal or surface functional groups of hyperbranched polymers are amine, hydroxyl, carboxylic acid, a fatty acid, polyethylene glycol (PEG), polyester, etc. See U.S. Pat. No. 6,432,423, U.S. Pat. No. 7,097,856, and U.S. Patent Publication 2006/0204472, the contents of which are incorporated herein by reference.
In contrast to the “structurally perfect” dendrimers prepared by multi-step synthesis, somewhat less perfect hyperbranched polymers can be synthesized in one-step reactions. Thus, unlike dendrimers, hyperbranched polymers are rapidly prepared with no purification steps needed for their preparation. Consequently, hyperbranched polymers are significantly less expensive than dendrimers. Thus it makes hyperbranched polymers amenable for large-scale in vivo trials and bringing highly branched polymers as candidates for drug delivery of even common drugs as ibuprofen (Kannan, R. M. et al., Biomedical Applications of Nanotechnology, 2007, John Wiley & Sons Inc., p. 105).
An object of the invention is to provide an improved ophthalmic composition, with improved aqueous solubility and corneal permeation of the active agent.
The present inventors have studied ophthalmic compositions comprising hyperbranched polymers. The present inventors have discovered that hyperbranched polymers are muco-adhesive polymers with a high force of bioadhesion, which provide strong electrostatic interactions between the negatively charged cornea mucin membrane and the cationic hyperbranched polymers.
The present inventors have discovered that hyperbranched polymers increase the aqueous solubility of carbonic anhydrase inhibitors such as Dorzolamide or Brinzolamide for glaucoma therapy. Additionally, the present inventors have discovered that the aqueous solubility of Dorzolamide or Brinzolamide increases linearly with an increase in the concentration of the hyperbranched polymer. Furthermore, the present inventors have discovered that hyperbranched polymers, such as Bis-MPA hyperbranched polyester with hydroxyl functional groups (2nd generation), can be safely employed up to 4% (w/v) with no cytotoxic or eye irritation, based on in vitro human corneal epithelial cell culture studies. Additionally, the present inventors have discovered that hyperbranched polymers increase the corneal permeation and partitioning of Dorzolamide and Timolol into intact cornea, and increase the partitioning of Dorzolamide and Timolol into the lipophilic cornea membrane.
Accordingly, the present invention provides:
The compositions of the present invention are topically administratable therapeutic compositions for treatment of conditions of the eye. Such conditions of the eye include glaucoma, and ocular diseases such as cataract, conjunctivitis, infection, inflammation or retinopathy.
A detailed description of the invention is provided below.
The present invention includes an ophthalmic composition comprising a hyperbranched polymer.
The hyperbranched polymer according to the present invention may be any hyperbranched polymer which is pharmaceutically acceptable, e.g., a hyperbranched polymer with a Polyethyleneimine, Polypropylenimine or a polyester core. The molecular weight of the hyperbranched polymer in the ophthalmic compositions of the present invention is in the range of from 1,000 to 750,000 Daltons, preferably in the range of 1,000 to 12,000 Daltons. The molecular weight is weight average molecular weight measured by dynamic light scattering. The concentration of the hyperbranched polymer in the ophthalmic compositions of the present invention is in the range from 0.001% to 10% (w/v), preferably in the range from 0.001% to 5% (w/v), more preferably in the range from 0.001% to 4% (w/v), more preferably in the range from 0.01% to 4% (w/v), more preferably in the range of 0.01% to 3% (w/v).
The ophthalmic composition discussed above may also comprise a carbonic anhydrase inhibitor. Carbonic anhydrase inhibitors are a class of pharmaceuticals that suppress the activity of carbonic anhydrase, and are known to be useful as anti-glaucoma agents. Examples of carbonic anhydrase inhibitors which may be present in the ophthalmic compositions of the present invention are Dorzolamide, Brinzolamide or Acetazolamide.
The ophthalmic composition discussed above may also comprise a non-ionic surfactant. The non-ionic surfactant may be any non-ionic surfactant which is known as a pharmaceutically acceptable additive, for example, Polysorbate 80, PEG 8000, HPMC or HEC.
The ophthalmic compositions of the present invention are advantageously used after being adjusted to a pH range which is conventionally adopted for topical application to the eye, and is normally employed after being adjusted to a pH of 3 to 8, preferably a pH of 5 to 8. For the pH adjustment, hydrochloric acid, acetic acid, sodium hydroxide, etc. can be used.
The ophthalmic compositions of the present invention may also comprise a beta-blocker. Beta-blockers are known to reduce the pressure within the eye (the intraocular pressure), and thus, are used to lessen the risk of damage to the optic nerve and loss of vision in patients with glaucoma. The beta-blocker in the ophthalmic compositions of the present invention may be any beta-blocker which is known as acceptable in ophthalmic compositions, such as Carteolol, Levobunolol, Betaxolol, Metipranolol, Timolol and Propranolol.
A first specific embodiment of the present invention is an ophthalmic composition comprising a hyperbranched polymer, Timolol, Dorzolamide, PEG 8000 and Polysorbate 80.
Such compositions preferably comprise about 0.001% to 10% (w/v) of the hyperbranched polymer, most preferably about 1 to 5% (w/v), and 0.05 to 1% (w/v) of Timolol, most preferably about 0.5% (w/v), and about 0.05 to 5% (w/v) of Dorzolamide, most preferably about 0.5 to 2% (w/v), and about 0.05 to 5% (w/v) of PEG 8000, most preferably about 0.5 to 4% (w/v), and about 0.05 to 5% (w/v) of Polysorbate 80, most preferably about 0.5 to 4% (w/v), and are to be administered once or twice a day to each affected eye.
A second specific embodiment of the present invention is an ophthalmic composition comprising a hyperbranched polymer, Timolol, Brinzolamide, PEG 8000 and Polysorbate 80.
Such compositions preferably comprise about 0.001% to 10% (w/v) of the hyperbranched polymer, most preferably about 1 to 5% (w/v), and 0.05 to 1% (w/v) of Timolol, most preferably about 0.5% (w/v), and about 0.05 to 5% (w/v) of Brinzolamide, most preferably about 0.5 to 2% (w/v), and about 0.05 to 5% (w/v) of PEG 8000, most preferably about 0.5 to 4% (w/v), and about 0.05 to 5% (w/v) of Polysorbate 80, most preferably about 0.5 to 4% (w/v), and are to be administered once or twice a day to each affected eye.
A third specific embodiment of the present invention is an ophthalmic composition comprising a hyperbranched polyester, Timolol, Dorzolamide, PEG 8000 and Polysorbate 80.
Such compositions preferably comprise about 0.1% to 10% (w/v) of the hyperbranched polyester, most preferably about 1 to 5% (w/v), and 0.05 to 1% (w/v) of Timolol, most preferably about 0.5% (w/v), and about 0.05 to 5% (w/v) of Dorzolamide, most preferably about 0.5 to 2% (w/v), and about 0.05 to 5% (w/v) of PEG 8000, most preferably about 0.5 to 4% (w/v), and about 0.05 to 5% (w/v) of Polysorbate 80, most preferably about 0.5 to 4% (w/v), and are to be administered once or twice a day to each affected eye.
A fourth specific embodiment of the present invention is an ophthalmic composition comprising a hyperbranched polyester, Timolol, Brinzolamide, PEG 8000 and PEG 8000.
Such compositions preferably comprise about 0.1% to 10% (w/v) of the hyperbranched polyester, most preferably about 1 to 5% (w/v), and 0.05 to 1% (w/v) of Timolol, most preferably about 0.5% (w/v), and about 0.05 to 5% (w/v) of Brinzolamide, most preferably about 0.5 to 2% (w/v), and about 0.05 to 5% (w/v) of PEG 8000, most preferably about 0.5 to 4% (w/v), and about 0.05 to 5% (w/v) of Polysorbate 80, most preferably about 0.5 to 4% (w/v), and are to be administered once or twice a day to each affected eye.
The ophthalmic compositions according to the present invention may comprise a pharmacologically acceptable carrier, excipient or diluent which is known per se and may be formulated by a method known per se for preparing ophthalmic compositions. The ophthalmic compositions of the present invention may be provided in any pharmaceutical dosage form that is conventionally used as an ophthalmic preparation, e.g., eye drops, emulsions, and eye ointments.
The eye drop formulation may, for example, be an aqueous formulation, such as ophthalmic solution which is clear solution, ophthalmic suspension, ophthalmic emulsion, as well as non-aqueous formulations, such as non-aqueous ophthalmic solution and non-aqueous ophthalmic suspension.
The ophthalmic solution formulation may contain various additives incorporated ordinarily, such as buffering agents (e.g., phosphate buffers, borate buffers, citrate buffers, tartarate buffers, acetate buffers, amino acids, Sodium acetate, Sodium citrate and the like), isotonicities (e.g., saccharides such as sorbitol, glucose and mannitol, polyhydric alcohols such as Glycerin, concentrated Glycerin, PEG and Propylene glycol, salts such as Sodium chloride), preservatives or antiseptics (e.g., Benzalkonium chloride, Benzethonium chloride, P-oxybenzoates such as Methyl p-oxybenzoate or Ethyl p-oxybenzoate, Benzyl alcohol, Phenethyl alcohol, Sorbic acid or its salt, Thimerosal, Chlorobutanol and the like), solubilizing aids or stabilizing agents (e.g., cyclodextrins and their derivative, water-soluble polymers such as polyvinyl pyrrolidone, surfactants such as tyloxapol, pH modifiers (e.g., Hydrochloric acid, Acetic acid, Phosphoric acid, Sodium hydroxide, Potassium hydroxide, Ammonium hydroxide and the like), thickening agents (e.g., HEC, Hydroxypropyl cellulose, Methyl cellulose, HPMC, Carboxymethyl cellulose and their salts), chelating agents (e.g., Sodium edetate, Sodium citrate, condensed Sodium phosphate) and the like.
The eye drop formulation in the form of an aqueous suspension may also contain suspending agents (e.g., Polyvinyl pyrrolidone, Glycerin monostearate) and dispersing agents (e.g., surfactants such as Tyloxapol, ionic polymers such as Sodium alginate) in addition to the additives listed above, whereby ensuring that the eye drop formulation is a further uniform microparticulate and satisfactorily dispersed aqueous suspension.
The eye drop formulation in the form of an aqueous suspension preferably contains Sodium citrate or Sodium acetate as a buffering agent, concentrated Glycerin and/or Propylene glycol as an isotonicity and Polyvinyl pyrrolidone as a suspending agent. A preferred dispersing agent is a surfactant and/or Sodium alginate. Such surfactant is preferably Tyloxapol.
The ophthalmic composition of the present invention may be administered to a mammal which is or may be suffering from an ophthalmic disease, such as glaucoma (e.g., a human, rabbit, dog, cat, cattle, horse, monkey).
While the administration route and the dose may vary depending on a symptom, age and body weight of a subject, the concentration of the active agent in the ophthalmic composition of the present invention is about 0.001 to 5 (w/v) %, preferably about 0.01 to 3 (w/v) % contained in an aqueous eye drop formulation when given to an adult, and is given preferably 1 to 8 times a day with a single dose being one to several drops.
Unless the intended purpose of use is affected adversely, the ophthalmic compositions of the present invention may contain or may be used together with other appropriate pharmacologically effective substances, for example, steroidal anti-inflammatory agents (Dexamethasone, Prednisolone, Loteprednolm Fluorometholone, Fluocinolone and the like), non-steroidal anti-inflammatory agents (Diclofenac sodium, Pranoprofen, Bromfenac, Ketorolac tromethamine, Napafenac, Flurbiprofen Sodium and the like), antiallergic agents (Tranilast, Ketotifen fumarate, Olopatadine hydrochloride, Sodium Cromoglicate, Potassium Pemirolast, Sodium Nedocromil and the like), antihistamic agents (Epinastine hydrochloride, Azelastine hydrochloride, Azalastine hydrochrilidem, Diphenhydramine hydrochloride and the like), glaucoma-treating agents (Pilocarpine hydrochloride, Physostigmine salicylate, Timolol, Isopropylunoprostone, Latanoprost, Betaxolol hydrochloride, Apraclonidine, Brimonidine Tartrate, Carbacol, Dipivefrin, Bimatoprost, Travoprost, Brimonidine tartrate and the like), antibiotics (Azithromycin, Gentamycin sulfate, Fradiomycin sulfate, Tobramycin, Sulbenicillin, Cefinenoxime, Erythromycin, Colistin, Oxytetracycline, Polymyxin B, Chloramphenicol, Micronomicin, Dibekacin, Sisomicin and the like), antibacterial agents (Sulfamethizole, Sulfamethoxazole, Ofloxacin, Norfloxacin, Lomefloxacin hydrochloride, Moxifloxacin hydrochloride, Enoxacin, Ciprofloxacin hydrochloride, Cinoxacin, Sparfloxacin, Tosufloxacin tosylate, Nalidixic acid, Pipemidic acid Trihydrate, Pipemidic acid, Fleroxacin, Levofloxacin, Gatifloxacin and the like), and antiviral agents (Idoxuridine, Acyclovir and the like), and antimycotic agents (Pimaricin, Fluconazole, Miconazole, Amphotericin B, Flucytosine, Itraconazole and the like), anti VEGF antibody (Pegaptanib and the like).
The ophthalmic compositions of the present invention may be produced by dissolving or dispersing the active agent(s), hyperbranched polymer and optionally the non-ionic surfactant in a solution appropriately containing pharmaceutically acceptable additives, such as isotonicity agents, buffers, preservatives, suspending agents, thickeners, stabilizers, pH adjusting agents, and the like.
The present inventors hereby incorporate by reference prior filed U.S. application Ser. No. 12/774,419, in its entirety. The present invention is further illustrated in detail by the following Experimental Examples. These Experimental Examples are merely illustrative, and are not intended to limit the scope of the present invention.
pH-solubility profile of Dorzolamide in aqueous solution containing different concentrations of Hyperbranched Polymer (HP) (Lupasol® G20, Lupasol® G 35, Lupasol® PS) and PEG.
Suspensions of Dorzolamide hydrochloride in 0.1% (w/v) phosphate buffer solution at pH 5.5, pH 6, pH 6.5, pH 7, pH 7, pH 8 and pH 8.5 were prepared. Similar suspensions were also prepared in aqueous solution containing different concentrations of different HP and PEG with a molecular weight of 8000. A combination of Polysorbate 80 and PEG 8000 was also attempted. The pH was measured accurately with micro-pH electrode (Thermo Scientific). The desired pH was adjusted using either 1 M NaOH or 1 M HCl. The suspension solutions were first stirred for 10 min at room temperature (with heating up to 60° C. for 5 minutes). After allowing the suspensions to equilibrate at room temperature for an additional 30 minutes, the suspension solutions were then sonicated for 10 minutes and finally filtered through 0.45 μm syringe filters. The filtrates were analyzed for Dorzolamide concentration using UPLC. Dorzolamide detection was performed using: a gradient 1% (v/v) Triethylamine (TEA) in water:acetonitrile method, performed at room temperature, with the flow rate of 0.7 mL/min, at 254 nm wavelength and 10 μL injection volume, on BEH C18 1.7 μm, 2.1×50 mm column. A calibration curve was prepared to find Dorzolamide concentration. The properties of polymers used are listed in Table 1.
As shown in
The present inventors discovered that the aqueous solubility of Dorzolamide increased with an increase in the concentration of HP and PEG. In the case of PEG, the solubility also increased linearly with an increase in the molecular weight of the PEG. Further, the Polysorbate 80 assists in dispersing the Dorzolamide molecules and inhibits the precipitation in water in presence of PEG.
From these results, it is concluded that HP significantly enhances the solubility of Dorzolamide. Additionally, hydrophilic polyethylene glycol was discovered to be a Dorzolamide solubility enhancer. The results demonstrate the advantages of using hyperbranched polymers and PEG as Dorzolamide solubility enhancing additives at pH values closer to physiological pH.
A simple rheological method for the in vitro assessment of mucin-hyperbranched polymer bioadhesive bond strength.
A simple viscometric method was adopted to quantify the mucin-polymer bioadhesive bond strength. In order to determine the muco-adhesive properties of commercially available HP called Lupasol® PS, the force of bioadhesion was calculated for different concentrations of HP with porcine gastric mucin at pH 7 in comparison with the market product COSOPT®. Porcine gastric mucin was used as a model mucin. However, since all mucins appear to share general physical, structural, and rheological properties, it is believed that porcine gastric mucin is a satisfactory model for primary evaluation of bioadhesive materials.
Brookfield Rotational L VDVE viscometer was employed for all measurements. Spindle with code number 18 was used for all viscosity measurements. A factor of 1.32 was used to convert rpm to shear rate (s−1) as per the manual. A solution of 15% (w/v) of gastric mucin was prepared in 0.1% (w/v) phosphate buffer at pH 7. The individual viscosities 0.5% (w/v) and 1% (w/v) of Lupasol® PS in phosphate buffer solution were measured. The viscosities of 15% mucin in phosphate buffer were also measured. The viscosity was measured at 20° C. at different shear rates D from 2.6 to 80 s−1 (Hassan, E. et al., Pharm Res. 5 (1990) 491) Five samples of 10 mL each were prepared with different concentrations of Lupasol® PS, PEG and with and without 15% (w/v) gastric mucin in 0.1% (w/v) phosphate buffer at pH 7.
Sample #7 is the original COSOPT® market product. The viscosity at 20° C. was measured at different shear rates. The force of bioadhesion was calculated using equations (1) and (2), discussed above.
The force of bioadhesion (F) was calculated as per the following equation (1):
F=η
bτ (1),
where τ is the rate of shear per second, and ηb is based on experimental measured values as per the following equation (2):
ηb=ηt−ηm−ηp (2)
where ηt is the viscosity coefficient of the system, and ηm and ηp are the individual viscosity coefficients of mucin and the bioadhesive polymer (e.g., HP and PEG 8000), respectively.
For equations (1) and (2) to be valid, ηt, ηm, and ηp should be measured at the same concentration, temperature, time, and rate of shear. The bioadhesive phenomenon plays a dominant role in the contact time of aqueous tear that substitute in the precorneal area.
As shown in
The force of bioadhesion was quantified based on the data available from
As shown in
In conclusion, data generated by the viscometric assessment method of bioadhesion suggests that the hyperbranched polymers are bio-adhesive additive materials that could strongly interact with ocular mucin. These bioadhesive forces between mucin and HP could eventually lead to enhancement of the ocular bioavailability of the drug.
Aqueous solubility of Dorzolamide in the presence of Timolol for a novel formulation containing HP (Lupasol® PS) and Polysorbate 80 or a combination of PEG and Polysorbate 80 at pH 5.65 and pH 7.
A suspension of Dorzolamide hydrochloride and 0.5% (w/v) Timolol in citrate buffer solution at pH 5.65 was prepared (Control sample). A similar suspension was also prepared in aqueous solution containing 2% (w/v) of HP in citrate buffer of pH 3. The final pH was adjusted to 5.65 with 1 M NaOH after addition of HP (sample 1). The combination of different molecular weight PEG and Polysorbate 80 at pH 5.65 as per Table 3 were also attempted. Table 3 shows all the different test samples suspensions to be prepared in 10 mM citrate buffer.
Similarly, the formulations were again prepared in 10 mM phosphate buffer (Table 3) for the formulations to be tested for Dorzolamide solubility at pH 7 in phosphate buffer rather than citrate buffer. The suspension solutions were first stirred for 10 min at room temperature (with heating up to 60° C. for 5 minutes). After allowing the suspensions to equilibrate at room temperature for an additional 30 minutes, the suspension solutions were then sonicated for 10 minutes and finally filtered through 0.45 μm syringe filters. The filtrates were analyzed for Dorzolamide and Timolol concentration using UPLC with the same condition as EXPERIMENTAL EXAMPLE 1.
In this experiment, the present inventors used HP, PEG, and Polysorbate 80 as solubility enhancer additives. Different combinations were attempted at pH 5.65 and pH 7. As shown in
While the market COSOPT® product has 2% (w/v) Dorzolamide at pH 5.65, the enhancement of solubility at pH 5.65 with more than 2% (w/v) Dorzolamide solubility by addition of HP or PEG will not have useful contribution to efficacy enhancement of drug by increasing the dosage. Thus, the solubility data at pH 7 is more vital, where Dorzolamide has poor solubility (less than 0.5% w/v solubility) in 10 mM phosphate buffer. It was also noted that the solubility of Timolol in the formulation samples (each containing exactly 0.5% w/v Timolol) did not change at pH 5.65 and pH 7 with the addition of additives. Since COSOPT® is formulated at pH 5.65, the Dorzolamide solubility in the presence of Timolol was quantified by the addition of different HP of different concentrations at pH 5.65 to the formulation sample. The result is presented in
The improvement in aqueous solubility of Dorzolamide in the presence of Timolol was significant with the additions of a HP or a combination of PEG and Polysorbate 80 at pH 5.65. In this case, the Polysorbate 80 helped in dispersing Dorzolamide molecules and inhibited the precipitation in water in the presence of PEG. A combination of HP and Polysorbate 80 was the best combination for enhancement of Dorzolamide solubility in presence of Timolol at pH 7. From the results, it can be concluded that HP and Polysorbate 80 significantly enhance the solubility of Dorzolamide in the presence of Timolol at pH 7. Hydrophilic PEG also turned out to be Dorzolamide solubility enhancer. Furthermore, a combination of low concentrations of Polysorbate 80 and PEG 8000 also proved to be a very useful additive for enhancement of solubility of Dorzolamide. Overall, a formulation at pH 7 with optimized concentration of HP and Polysorbate could be very useful for increasing the ocular bioavailability.
The results clearly indicate the advantages of using HP and Polysorbate 80 as Dorzolamide solubility enhancing additives at pH values closer to physiological pH that are more conducive for penetration of close to 1% (w/v) Dorzolamide through cornea membrane. Polysorbate 80 also proved to be an effective emulsifier, suppressing the precipitation of poorly soluble Dorzolamide at pH 7 in the presence of a HP.
In vitro corneal permeation study of Dorzolamide and Timolol for novel topical formulations containing HP (Lupasol® PS) and Polysorbate 80.
In vitro experiments on the corneal permeation of Dorzolamide and Timolol (active ingredients of COSOPT®) were carried out to investigate the effect of the addition of 0.5% (w/v) HP, or the addition of 0.5% (w/v) HP and 1% (w/v) Polysorbate 80, in comparison to the original market topical formulation (only active ingredients) at pH 5.65.
Experimental Method
1. Formulation Preparation: The following three solutions in 10 mM citrate buffer were formulated for examining the in vitro corneal permeation of Dorzolamide and Timolol, as well as determining the corneal hydrolysis effect.
The samples were filtered by 0.45 μm filter syringe. The initial concentration of both the samples was determined by UPLC analysis. From the experimental data, the following inferences were made:
2. In Vitro Rabbit Corneal Permeation Experiment
Three male rabbits (New Zealand) weighing 3-4 pounds. The age of the rabbits was 11-12 weeks. Immediately after sacrifice by an overdose of carbon dioxide gas, the eyes were enucleated, saline washed, and the corneas were separated for the use in permeation experiments. Each cornea was rinsed with freshly prepared receptor solution (Table 4) to remove excess stain. The six intact and fresh corneas were fixed between clamped donor and receptor compartments of an all glass side-by-side diffusion cell in such a way that its epithelial surface faces the donor compartment.
The corneal area available for permeation was 0.211 cm2. The receptor compartment was filled with freshly prepared receptor solution at pH 7.2, as per the composition described in Table 4. An aliquot (5 mL) of sample #1 was placed on the two intact corneas, and the opening of the donor cells was sealed with a glass cover slip. After 10 minutes of applying sample #1, an aliquot (5 mL) of sample #2 was applied on the next two intact corneas. Again, after 10 minutes, sample #3 aliquot (5 mL) was applied on the remaining two intact corneas. The receptor fluid (5 mL in each receptor cell) was kept at constant temperature of 34° C. using constant stirring through water jacket in all the six cases. At predetermined time intervals of 10, 20, 40, 60, 80, 100, 120, 140, 160, and 180 minutes, 200 μL samples were withdrawn from the receptor solution. Thereafter, the same amount of the phosphate buffer solution was added to the receptor cell. The drug concentrations were assayed by UPLC.
3. Analysis
The Dorzolamide and Timolol maleate detection conditions were a gradient 1% (v/v) Triethylamine (TEA) in water: acetonitrile method, performed at room temperature, with the flow rate of 0.7 mL/min, at 254 nm and 298 nm wavelength and 1 μL injection volume, on BEH C 18 1.7 μm, 2.1×50 mm column.
4. Corneal Permeation Parameters Calculation
At the end of the experiment, each cornea (free from adhering sclera) was weighed after soaking in de-ionized water. The wet cornea was dried overnight in oven, and reweighed. From the difference of weights, corneal hydration was calculated. The final results of drug permeation were expressed as cumulative amount permeated (Q). The parameters that were calculated are as follows:
The initial concentrations of Dorzolamide and Timolol determined by UPLC are given in Table 5.
The corneal hydration was measured based on the net wet weight and dry weight of cornea. Typically, the % (w/w) hydrations for cornea in normal mammalian are in the range of 75-80%. Overall, there was no significant change in the % hydrations for all the test samples, and they were within the desired range in all the cases. Thus, the HP or Polysorbate 80 did not have impact on corneal hydration.
The diffusion coefficient of Dorzolamide and Timolol, which is inversely proportional to the lag time, did not change significantly by the addition of HP and Polysorbate 80 (see
HP promotes encapsulation of Timolol and Dorzolamide, and thus enhances the partitioning of Timolol into corneal epithelium. This theory is also supported by the data in
The addition of 0.5% (w/v) HP and 1% (w/v) Polysorbate 80 enhanced the corneal permeation rate of Dorzolamide and Timolol by about 25-30%. The presence of HP increased the partitioning of Dorzolamide and Timolol at pH 5.65 into the corneal membrane. There was insignificant change in the corneal diffusion rate and corneal hydration rate by the addition of HP and Polysorbate 80, suggesting that these additives did not have a harmful impact on the cornea surface. The corneal permeability coefficients of Dorzolamide and Timolol were relatively higher in the presence of HP, suggesting the significance of HP as an effective drug carrier additive. Thus, the present inventors discovered a novel formulation with enhanced corneal permeation compared to the current market product. The corneal permeation could be further enhanced by increasing the concentration of HP.
The cumulative amount permeated of Dorzolamide and Timolol at pH 5.65 in the presence of additives such as HP was relatively high, compared to the control formulation with no additives (COSOPT® active ingredients formulation). The 0.5% (w/v) HP and 1% (w/v) Polysorbate 80 addition to the formulation enhanced the corneal permeation rate of Dorzolamide and Timolol by about 25-30%. The partitioning of active ingredients into the corneal epithelium increases in presence of HP. Thus, the combination of HP and Polysorbate 80 could be very effective for increasing the ocular bioavailability of COSOPT® active ingredients.
Solubility enhancement of Brinzolamide in aqueous solution containing HP (Lupasol® PS) or a combination of HP and Polysorbate 80, or PEG and Polysorbate 80 combinations at pH 7 in phosphate buffer.
The aqueous solubility of Brinzolamide in the presence of Timolol at pH 7 in 10 mM phosphate buffer was studied.
A suspension of Brinzolamide in phosphate buffer containing 1% (w/v) was prepared for the control sample. Similar suspensions containing excess of Brinzolamide (>1% (w/v)) were also prepared in aqueous solution (10 mM phosphate buffer) containing different combinations of HP, PEG and Polysorbate 80 as per Table 7 above. The final pH was adjusted to 7 with 1 M NaOH. The suspension solutions were first stirred for 10 min at room temperature (with heating up to 60° C. for 5 minutes). After allowing the suspensions to equilibrate at room temperature for additional 30 minutes, the suspension solutions were then sonicated for 10 min and finally filtered through 0.45 μm syringe filters. The filtrates were analyzed for Brinzolamide concentration using UPLC with the same condition as EXPERIMENTAL EXAMPLE 1.
In this study, the present inventors used HP, PEG, and Polysorbate 80 as solubility enhancer additives. Different combinations were attempted at pH 7. In
As shown in
The addition of Polysorbate 80 to HP increases the Brinzolamide solubility by preventing the precipitation. Polysorbate 80 may act as a surfactant thereby reducing the aggregation of Brinzolamide after phase separation in presence of HP. A combination of 0.5% (w/v) HP and 1% (w/v) Polysorbate could be very effective in the presence of 0.5% (w/v) Timolol formulation at pH 7.
The improvement in aqueous solubility of Brinzolamide in presence of Timolol was significant with the additions of HP or a combination of PEG and Polysorbate 80 at pH 7. The Polysorbate 80 helps in dispersing the Brinzolamide molecules and inhibits the precipitation in water in presence of PEG. A combination of HP and Polysorbate 80 could be good combination for enhancement of Brinzolamide solubility in presence of Timolol at pH 7. From the results, it can be concluded that HP and Polysorbate 80 significantly enhance the solubility of hydrophobic Brinzolamide in presence of Timolol at pH 7. Hydrophilic PEG also turned out to be a Brinzolamide solubility enhancer. Furthermore, a combination of low concentrations of Polysorbate 80 and PEG 8000 also proved to be a very useful additive for enhancement of solubility of hydrophobic Brinzolamide. Overall, a formulation at pH 7 with optimized concentration of HP and Polysorbate 80 could be very useful for increasing the ocular bioavailability.
The results clearly indicate the advantages of using HP and Polysorbate 80 as hydrophobic Brinzolamide solubility enhancing additives at pH values closer to physiological pH. Polysorbate 80 also proved to be an effective emulsifier suppressing the precipitation of poorly soluble Brinzolamide at pH 7 in presence of HP. Timolol may have an effect on the solubility of Brinzolamide by changing the ionic strength of the solution.
In vitro corneal permeation study of Dorzolamide and Timolol containing a HP with terminal hydroxyl groups.
In vitro experiments on corneal permeation of Dorzolamide and Timolol (active ingredients of COSOPT®) were carried out to investigate the effect of the addition of a hyperbranched polyester with hydroxyl functional groups in comparison to the original market topical formulation (only active ingredients).
A novel formulation containing the commercially available HP called Boltorn® H20. The generic definition of Boltorn® H20 is a HP with polyester core and 16 terminal hydroxyl functional groups. It enhances the solubility of non-ionic (lipophilic) Dorzolamide that is formulated at pH 7 or pH 7.4.
The properties of HP used in this example are listed in Table 8. It has 16 primary hydroxyl groups per molecule. The solid content is 100% (w/v).
The in vitro transcorneal permeation of Dorzolamide and Timolol was determined from a novel formulation containing up to 2% (w/v) HP. The effect of the concentration of HP on the active ingredients was also determined. A standard solution containing COSOPT® active ingredients at pH 7.4 was used as a control sample.
Formulation Preparation
The following three solutions in 0.1% (w/v) phosphate buffer (Table 9) were formulated for examining the in vitro corneal permeation of Dorzolamide and Timolol, as well as for determining the corneal hydrolysis effect.
First, the 10 mM phosphate buffer was added to the appropriately weighed mass of solid active ingredients and stirred thoroughly for 15 minutes. Secondly, the effective volume of 5% (w/v) HP suspension solution was added to Test Sample 2 and Test Sample 3 to make up the exact concentrations described in Table 9.
The three test solutions were then stirred for 10 minutes at room temperature (with heating up to 60° C. for 5 minutes). After stirring, the solution was sonicated for 5 minutes. Apart from the control solution (Control Sample #1), the solutions with HP were white slurry suspensions before adjusting the pH. After allowing the complete dilution of all the active and non-active ingredients, the pH was adjusted to 7.4 by using 1 M NaOH or 1 M HCl, and additional buffer was added to make up the exact composition as in Table 8. With the adjustment of pH, suspension solutions were formed in all cases, which were equilibrated by stirring for an additional 15 hours or more at room temperature. The pH of all the sample solutions was measured again to confirm the final desired pH.
These suspension solutions were used directly as sample donor solutions for the cornea permeation study. In order to determine the solubility, the suspensions were filtered through 0.45 μm syringe filters. The filtrates were analyzed for Dorzolamide and Timolol concentrations using UPLC, after diluting each sample with ultrapure water (dilution factor=1000). The in vitro cornea permeation profile results were also compared to the data obtained at pH 5.65 for the control sample containing active ingredients from EXPERIMENTAL EXAMPLE 4.
Three male rabbits (New Zealand) weighing 2 to 3 kg. Immediately after sacrifice by an overdose of inhaler isoflurane, the eyes were enucleated, and the corneas were separated for use in the permeation experiments. The details of the experimental procedure are described in previous EXPERIMENTAL EXAMPLE 4.
The calculated parameters that were calculated are as described in EXPERIMENTAL EXAMPLE 4, with Cd [ng/mL] being the initial drug concentration of active pharmaceutical ingredient in donor solution (Table 10).
The initial concentrations of Dorzolamide and Timolol determined by UPLC are given in Table 10. The percentage corneal hydration calculations are given in Table 10.
The corneal hydration was measured based on the net wet weight and dry weight of the cornea. Typically, the % hydrations for a cornea in a normal mammal are in the range of 75-80%. Overall, there was no significant change in the % hydrations for all the test samples, and they were within the desired range in all the cases. Thus, the HP did not have impact on corneal hydration.
Overall, the addition of a HP with hydroxyl functional groups enhances the corneal permeation rate of Dorzolamide and Timolol significantly, with an increase in the concentration of HP. Thus, HP improved the corneal penetration of active ingredients, when compared to the market products known as COSOPT® or TRUSOPT® or AZOPT®, which are used for glaucoma treatment.
The diffusion coefficient of Dorzolamide and Timolol, which is inversely proportional to the lag time, did not change significantly by the addition of HP (see
HP promotes the encapsulation of Timolol and Dorzolamide, and thus enhances the partitioning of Timolol and Dorzolamide into the corneal epithelium. This theory is also supported by the data suggested in
The cumulative amount permeated of Dorzolamide and Timolol at pH 7.4 in the presence of HP additives, such as commercially available Boltorn® H20 with hydroxyl functional group, was relatively high, compared to the control formulation with no additives. The increase in concentration of such HP in the formulation enhanced the corneal permeation rate of Dorzolamide and Timolol significantly. The corneal permeability coefficients of Dorzolamide and Timolol were relatively higher in the presence of HP. The partitioning of active ingredients into the corneal epithelium increased in the presence of HP. Thus, an HP with hydroxyl functional groups could be very effective for increasing the ocular bioavailability of COSOPT® active ingredients.
The aqueous solubility of carbonic anhydrase inhibitors, such as Dorzolamide and Brinzolamide, in the presence of HP and Timolol at pH 7.4 in 10 mM phosphate buffer was studied. A HP called Boltorn® W3000 was used. The terminal functional groups of this HP are PEG (hydrophilic) and unsaturated long chain fatty acids. The model of the hyperbranched polyester used in this example is shown below.
The properties of HP used in this experiment are described in Table 12. The HP has 50 primary hydroxyl groups per molecule, and the solid content is 55% (w/w).
Table 13 shows the different test samples formulations which were prepared in 10 mM phosphate buffer at pH 7.4.
The emulsion was prepared by slowing dispersing water to the weighed amount of waxed Boltorn® W3000 to make 5% (w/v) emulsion with continuous stirring and heating at 60-70° C. for at least 30 minutes, followed by continuous vigorous stirring for an additional 15 hours, to obtain a homogeneous emulsion mixture in a flask.
10 mM phosphate buffer was added to the appropriately weighed mass of solid active ingredients and stirred thoroughly for 15 minutes. Secondly, the effective volume of 5% (w/v) HP emulsion solution was diluted appropriately to make up the exact concentrations described in Table 13. The sample test emulsion solutions were then stirred for 10 minutes at room temperature (with heating up to 60° C. for 5 minutes). After stirring, the emulsion solution was sonicated for 5 minutes. After allowing the complete emulsion of all the active and non-active ingredients, the pH was adjusted to 7.4 by using 1 M NaOH, and additional buffer was added to make up the exact compositions in Table 13. With the adjustment of the pH, the emulsion solutions were further equilibrated by stirring for an additional 15 hours or more at room temperature. The pH of all the sample emulsion solutions was measured again to confirm the final desired pH of 7.4. The filtrates were analyzed for CAI concentration using UPLC with the same condition as EXPERIMENTAL EXAMPLE 1.
In this experiment, an amphiphilic self emulsifying HP was used as a solubility enhancer additive. Different concentrations of the HP were attempted at pH 7.4.
In
In vitro corneal permeation study of Dorzolamide and Timolol containing HP with amphiphilic functional groups (Boltorn® W3000).
In vitro experiments on corneal permeation of Dorzolamide and Timolol (active ingredients of COSOPT®) were carried out to investigate the effect of the addition of a HP with amphiphilic functional groups in comparison to the original market topical formulation (only active ingredients).
A new topical formulation containing Boltorn® W3000 (hyperbranched polyester) with non-ionic PEG as hydrophilic functional groups and unsaturated fatty acid as hydrophobic functional groups (commercially available), thus making it amphiphilic. The solubility of Dorzolamide was increased from 4.3 to 15 mg/mL by adding 2% (w/v) of this HP at pH 7.41. In this experiment, the in vitro transcorneal permeation of Dorzolamide and Timolol was determined from a novel formulation containing up to 2% (w/v) HP that was comparable to COSOPT®. The effect of concentration of HP on the active ingredients was also determined. A standard solution containing COSOPT® active ingredients at pH 7.4 was used as a control sample.
1. Formulation Preparation
The following three solutions in 0.1% (w/v) phosphate buffer (Table 14) were formulated for examining the in vitro corneal permeation of Dorzolamide and Timolol, as well as determining the corneal hydrolysis effect.
10 mM phosphate buffer was added to the appropriately weighed mass of the solid active ingredients, and stirred thoroughly for 15 minutes. Secondly, the effective volume of 5% (w/v) HP suspension solution was added to Test 2 to make up the exact concentrations as in Table 14. The three test solutions were then stirred for 10 minutes at room temperature (with heating up to 60° C. for 5 minutes). After stirring, the solution was sonicated for 5 minutes. After allowing the complete dilution of all the active and non-active ingredients, the pH was adjusted to either 7.4 or 5.65 by using 1 M NaOH or 1 M HCl, and additional buffer was added to make up the exact compositions in the Table 15. With the adjustment of pH, the appearance was noted as per Table 14, and the formulations were further equilibrated by stirring for an additional 15 hours or more at room temperature. The pH of the all sample solutions was measured again to confirm the final desired pH. These formulations were used directly as sample donor solutions for the cornea permeation study. In order to determine the solubility, the suspension/emulsions were filtered through 0.45 μm syringe filters. The filtrates were analyzed for Dorzolamide and Timolol concentration using UPLC after diluting each sample with ultrapure water (dilution factor=1000).
2. In Vitro Rabbit Corneal Permeation Experiment
The experimental procedure and analysis to be performed are described in detail in previous EXPERIMENTAL EXAMPLE 4. The parameters that were calculated are those described in EXPERIMENTAL EXAMPLE 4, where Cd [ng/mL] is the initial drug concentration of active pharmaceutical ingredient in donor solution (from Table 15).
The initial concentrations of Dorzolamide and Timolol determined by UPLC are given in Table 15. The percentage corneal hydration calculations are given in Table 16.
The corneal hydration was measured based on the net wet weight and dry weight of the cornea. Typically, the % (w/w) hydrations for a cornea in a normal mammal are in the range of 75-80%. Tests 1 and 3 are both above 80%. However, there is no difference in the calculated values of partition and permeability coefficient, suggesting that there could not be any corneal damage due to higher hydration %. Overall, there was no significant change in the % hydrations for all the test samples, which were within the desired range in all the cases. Thus, the HP did not appear to have an impact on corneal hydration.
Furthermore, the Timolol permeation significantly increased in the presence of HP for Test 2 compared to Tests 1 and 3 having similar aqueous solubility, unlike Dorzolamide (see
Overall, the addition of HP with amphiphilic functional group enhances the corneal permeation rate of Dorzolamide and Timolol significantly, with an increase in the concentration of HP. Thus, a dendritic polyester HP with amphiphilic functional groups improves the corneal penetration of active ingredients compared to the market products, known as COSOPT® or TRUSOPT® or AZOPT®, which are used for glaucoma treatment.
The diffusion coefficient of Dorzolamide and Timolol, which is inversely proportional to the lag time did not change significantly by the addition of HP (see
The Timolol and Dorzolamide partition coefficients to the corneal surface for Test 2 were higher than Tests 1 and 3, thus indicating the improvement in partitioning of Timolol and Dorzolamide into lipophilic corneal membrane in the presence of a highly functional (amphiphilic) HP. Thus, the improved permeation in the presence of HP is mainly because of improved partitioning to the epithelium. The permeation can be further enhanced by increasing the concentration of HP in the formulation solution from 2% to 5% (w/v).
The cumulative amount permeated of Dorzolamide and Timolol at pH 7.4, in the presence of HP additives, such as commercially available Boltorn® W3000 with amphiphilic functional group (2% w/v), was almost 2 times higher compared to the control formulation at similar pH values, with no additives. The increase in concentration greater than 2% (w/v) of such HP in the formulation could further enhance the corneal permeation rate of Dorzolamide and Timolol significantly compared to the market product at pH 5.65. The novel topical formulation is prepared at pH 7.4, thus making it more conducive and comfortable for the patients. The partitioning of active ingredients into the corneal epithelium increased in presence of HP. Thus, HP with amphiphilic functional groups could be very effective for increasing the ocular bioavailability of COSOPT® active ingredients.
Solubility enhancement of carbonic anhydrase inhibitor (CAI) by Bis-MPA polyester hyperbranched polymer (BMPA-HP) or a combination of PEG and BMPA-HP.
The influence of functionalized hyperbranched polymers on the aqueous solubility of a CAI, such as Dorzolamide and Brinzolamide, in the presence of Timolol at pH 7.4 in 10 mM phosphate buffer was studied. The generic definition of BMPA-HP is a hyperbranched polymer with dimethylolpropionic acid (Bis-MPA) polyester core and terminal hydroxyl (OH) functional groups. The number of terminal hydroxyl functional groups depends on the generation of the hyperbranched polyester. The generation is defined by the number of branching layers or the extent of branching from the core to the terminal functional groups. For example, the 2rd generation BMPA hyperbranched polyester contains 16 hydroxyl groups while the 3rd generation contains 32 hydroxyl groups. The structure of BMPA-HP is shown below.
The properties of the polyester HPs of different generations used in this research study are listed in Table 17.
Table 18 shows the different test sample emulsions, except the control solution, to be prepared in 10 mM phosphate buffer at pH 7.4.
10 mM phosphate buffer was added to the appropriately weighed mass of solid active ingredients and stirred thoroughly for 15 minutes. Secondly, the effective volume of 5% (w/v) BMPA-HP solution was diluted appropriately to make up the exact concentrations described in Table 18. The sample test solutions were then stirred for 10 minutes at room temperature (with heating up to 60° C. for 5 minutes). After stirring, the solution was sonicated for 5 minutes. After allowing the complete suspension of all the active and non-active ingredients, the pH was adjusted to 7.4 using 1 M NaOH, and additional buffer was added to make up the exact compositions described in Table 18. With the adjustment of pH, suspension solutions were further equilibrated by stirring for an additional 15 hours or more at room temperature. The pH of the all sample solutions was measured again to confirm the final desired pH of 7.4. The filtrates were analyzed for CAI using UPLC with the same condition as EXPERIMENTAL EXAMPLE 1.
In this experiment, 2nd BMPA-HP or 3rd BMPA-HP were employed as solubility enhancer additives, and different concentrations of each were tested at pH 7.4. It is important to develop a lipophilic Brinzolamide or Dorzolamide drug with enhanced solubility close to pH 7.4 (pH of tear fluid is 7.4) in order to enhance ocular bioavailability and to decrease eye irritation appearance of CAIs. The solubility of Dorzolamide and Brinzolamide in the presence of 0.5% Timolol with 5% 3rd BMPA-HP is slightly about 1% (w/v) of CAI. With lower generation 2nd BMPA-HP, the solubility of CAI decreased by less than 10% in comparison to 3rd BMPA-HP for similar concentration of 2nd BMPA-HP used as an additive for all the samples.
When the combination of 2% (w/v) PEG 8000 and HP is used, the solubility of CAI increases dramatically (4 times more than the control). With the addition of 2% (w/v) PEG 8000 to 2% (w/v) BMPA-HP, the solubility of CAI almost doubled (see
While the market Azarga® suspension product has 1% Brinzolamide at pH 7.4 and COSOPT® has 2% (w/v) Dorzolamide at pH 5.65, the enhancement of solubility at pH 7.4 by the addition of BMPA-HP will have useful contribution to efficacy enhancement of the drug, by increasing the dosage to greater than 1% (w/v).
The results clearly indicate the advantages of using BMPA-HP as a hydrophobic CAI solubility enhancer at pH values closer to physiological pH. The addition of PEG to the solution containing BMPA-HP further improved the solubility of CAI.
Human Corneal Epithelium (HCE) tissue culture study of determining the eye irritancy of Bis-MPA hyperbranched polyester and the optimized sample application time for ophthalmic study based on the cytotoxicity of the cells.
The samples were prepared in accordance with Table 19 below, in saline phosphate buffer (with the exception of AZOPT®) and sterilized using a 0.2 μm Sterile syringe filter. 0.02% BAK was used as a positive control. The reconstructed human corneal epithelium was purchased from Skin Ethics laboratory (France).
The Cell Culture Method is described below:
The percentage viability of each of the treated cultures was calculated from the percentage MTT conversion in the test chemical treated cultures relative to the corresponding negative controls (100% viable).
The following equation was used:
Percentage viability=[individual ODchemical/mean ODnegative control]×100.
HCE viability classification prediction model: NI (viability≧60%), I (viability<60%), i.e., the product is classified as an irritant (according to in vivo classifications) if the percentage of viability compared to the negative control obtained for the test product is <60%.
As shown in
Combining the results from previous studies, the cytotoxicity of Bis-MPA commercial hyperbranched polyester for different concentrations is revealed in
The rate of epithelial cell death increased with the increase in the concentration dose and the generation (molar mass and extent of branching). In the case of the AZOPT® market product, it causes eye irritation with less than 50% cell viability, possibly due to 0.01% (w/v) of BAK with exposure time of one hour.
Solubility enhancement of CAI containing BMPA-HP or a combination of non-ionic surfactants and BMPA-HP.
The aqueous solubility and stability of Dorzolamide was studied in the presence of Timolol at pH 7.4 in 10 mM phosphate buffer.
First, the 10 mM phosphate buffer was added to the appropriate weighed mass of solid active ingredients and stirred thoroughly for 15 minutes. After complete dissolution of active pharmaceutical ingredient, hyperbranched 2nd BMPA-HP was added to the solution. After HP was dissolved, PEG 8000 was added as per the formulation concentration needed. The sample test solutions were then stirred for 10 minutes at room temperature (with heating up to 60° C. for 5 minutes). After stirring, the solution was sonicated for 20 minutes. After allowing the complete dissolution of all the active and non active ingredients, the pH was adjusted to 7.4 by using 1 M NaOH and additional buffer was added to make up the exact compositions as per Tables 20 and 21. With the adjustment of pH, the formulations were further equilibrated by stirring for additional 15 hours or more at room temperature. The samples were filtered through 0.45 um syringe filter. Polysorbate 80 was added to the final formulation. In case of sample number 3, the formulation is equilibrated at 60° C. (24 hours) after pH adjustment, then Polysorbate 80 was added. The pH of all the sample solutions was measured again to confirm the final desired pH of 7.4. All samples were stored for 14 days at 25 and 60° C., and the filtrates were analyzed for Dorzolamide and Timolol using UPLC after diluting each sample with ultrapure water (dilution factor=1000).
In this study, 2nd BMPA-HP was studied as a solubility enhancer additive. Different combinations with non-ionic surfactants, such as PEG and Polysorbate 80, were attempted at pH 7.4. All the formulations were clear solutions at room temperature after sample preparation. In
Thus, higher concentration of Polysorbate 80 prevents the precipitation and helps stabilize the new formulation. Negligible change was observed in the pH of all the formulations over a period of 2 weeks (See Table 20 and Table 21). The change in Timolol concentration was insignificant over a period of two weeks. While the market AZARGA® suspension product has 1% (w/v) Brinzolamide at pH 7.4, and COSOPT® has 2% Dorzolamide at pH 5.65, the enhancement of solubility at pH 7.4 by addition of HP and non-ionic surfactants will have useful contribution to efficacy enhancement of drug by increasing the dosage to greater than equal to 1% (w/v).
The results clearly demonstrate the advantages of using 2nd BMPA-HP in combination with PEG 8000 and Polysorbate 80 as CAI solubility enhancer at pH values closer to physiological pH.
A study to determine the topical formulation at pH 7.4, based on solubility and stability of carbonic anhydrase inhibitor (Dorzolamide and Brinzolamide) in the presence of Timolol in an aqueous solution containing different combinations of Hyperbranched bis-MPA polyester-16-hydroxyl, generation 2 (2nd BMPA-HP), PEG 8000 and Polysorbate 80 in phosphate buffer was performed.
Table 22 shows all the different test samples to be prepared in 10 mM phosphate buffer at pH 7.4.
First, the 10 mM phosphate buffer was added to the appropriately weighed mass of solid active ingredients and stirred thoroughly until the active pharmaceutical agent was dissolved. Secondly, solid BMPA-HP powder was added to the sample formulations 1, 2 and 3. After dissolution of the HP, the appearance was clear solution. The appropriate mass of non-ionic surfactants, such as PEG 8000, was added to the formulations to make up the exact concentrations as per the formulation content described in the above Table 22. All test solutions were then stirred for 10 minutes at room temperature (with heating up to 60° C. for 5 minutes). After stirring, the solution was sonicated for 5 minutes. After allowing the complete dissolution of all the active and non active ingredients, the pH was adjusted to 7.4 or 5.65 by using 1 M NaOH or 1 M HCl, and additional buffer was added to make up the exact compositions in accordance with the Table 22 sample compositions. With the adjustment of pH, test solutions were formed, which were equilibrated by stirring for an additional 24 hours or more at room temperature. For the samples that contain Polysorbate 80 and hyperbranched polyester (Table 2), Polysorbate 80 was only added after 24 hours of equilibration at 60° C. of the final formulation. The pH of the all the sample solutions was adjusted again to confirm the final desired pH.
The filtrates were analyzed for carbonic anhydrase inhibitors, Timolol and HP concentration using UPLC after diluting each sample with ultrapure water (dilution factor=1000). The optimal conditions obtained from the EXPERIMENTAL EXAMPLE 1 for CAI and Timolol detection were used. The appearance and pH of each formulation is recorded over a period of 1 month at room temperature and 60° C.
This study used a HP with hydroxyl terminal groups, PEG, and Polysorbate 80 as solubility enhancer additives. Different combinations were attempted at pH 7.4 in order to determine the best formulation based on the previous results.
Tables 23 and 24 demonstrate the stability test results of Dorzolamide and Timolol over a period of 4 weeks for all the formulation samples. It is evident from the Table that sample #1 and sample #2 are relatively stable and clear solutions after 1 month. The presence of PEG in sample #1 could be avoided since sample #2 without PEG gives similar results.
The results clearly indicate the advantages of using HP with hydroxyl terminal functional groups in combination with surfactants such as Polysorbate 80 and PEG 8000. The surfactant behavior of Polysorbate 80 is very helpful for increasing the solubility of CAI by preventing the precipitation. The good formulation based on the solubility and 1 month stability results is a formulation containing active ingredients with a combination of 4% (w/v) 2nd BMPA-HP and 4% Polysorbate 80 only in phosphate buffer at pH 7.4.
In vitro corneal permeation study of Dorzolamide and Timolol for novel topical formulation containing Bis MPA hyperbranched polyester and Polysorbate 80.
The following three solutions in 0.1% (w/v) phosphate buffer (Table 27) were formulated for examining the in vitro corneal permeation of Dorzolamide and Timolol, as well as for determining the corneal hydrolysis effect.
First, the 10 mM phosphate buffer was added to the appropriately weighed mass of solid active ingredients and stirred thoroughly for 15 minutes. In case of sample numbers 2 and 3, 2nd BMPA-HP powder was added to the solution as per the composition in Table 25. The three test solutions were then stirred for 10 min at room temperature (with heating up to 60° C. for 5 minutes). After stirring, the solution was sonicated for 5 minutes. After allowing the complete dissolution of all the active and non active ingredients, the pH was adjusted to 7.4 or 5.65 by using 1 M NaOH or 1 M HCl and additional buffer was added to make up the exact composition as per the Table sample compositions. With the adjustment of pH, test solutions were formed which were equilibrated by stirring for additional 24 hours or more at room temperature for samples 1 and 2. In sample numbers 2 and 3, the equilibration was conducted for 24 hours at 60° C. Polysorbate 80 (4% (w/v)) was then added followed by another pH adjustment. The pH of the all the sample solutions was measured again to confirm the final desired pH.
These clear solutions were used directly as sample donor solutions for the cornea permeation study. In order to determine the solubility, the test solutions were filtered through 0.45 μm syringe filters. The filtrates were then analyzed for Dorzolamide and Timolol concentration using UPLC after diluting each sample with ultrapure water (dilution factor=1000). The details of the materials and equipment used, as well as the rabbit cornea study procedure are given in EXPERIMENTAL EXAMPLE 4.
The initial concentrations of Dorzolamide and Timolol determined by UPLC are given in Table 26.
The corneal hydration was measured based on the net wet weight and dry weight of the cornea. Typically, the % (w/w) hydrations for cornea in normal mammalian are in the range of 75-85%. Overall, there was no significant change in the % hydrations for all the test samples, and all were within the desired range, as shown in Table 27. Thus, the HP and Polysorbate 80 did not have impact on corneal hydration.
The slopes from
Overall, the data from
HP and Polysorbate 80 promote encapsulation of Timolol and Dorzolamide, and thus enhance the partitioning of Timolol into corneal epithelium. This theory is also supported by the data in
The cumulative amount permeated of active pharmaceutical agent at pH 7.4 in the presence of HP additives such as commercially available dendritic Bis-MPA HP and Polysorbate 80 was almost comparable to the market product in case of Dorzolamide and more than 2 times higher for Timolol permeation. This novel topical formulation is prepared at pH 7.4, thus making it more conducive for lipophilic epithelial cornea penetration and comfortable for the patients. Thus, HP with hydroxyl terminal groups and Polysorbate 80 could be very effective for increasing the ocular bioavailability of COSOPT® active ingredients.
According to the present invention, an ophthalmic composition comprising a HP, which shows increased aqueous solubility of carbonic anhydrase inhibitors, such as Dorzolamide or Brinzolamide, can be provided. The ophthalmic composition may also comprise a non-ionic surfactant and/or a beta-blocker. The ophthalmic compositions of the present invention result in increased permeation of the active agent into the cornea. Therefore, the overall ocular bioavailability and hence the therapeutic activity of the topical ophthalmic solution containing a carbonic anhydrase inhibitor and beta blocker (active ingredients) can be increased compared to current relevant ophthalmic market products available. The topical ophthalmic compositions presented in this invention provide more potent anti-glaucoma compositions that may increase patient compliance by increasing ocular bioavailability.
While some of the embodiments of the present invention have been described in detail in the above, those of ordinary skill in the art can enter various modifications and changes to the particular embodiments shown without substantially departing from the novel teaching and advantages of the present invention. Such modifications and changes are encompassed in the spirit and scope of the present invention as set forth in the appended claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/35147 | 5/4/2011 | WO | 00 | 11/5/2012 |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12774419 | May 2010 | US |
| Child | 13695930 | US |
