BIMATOPROST 0.01% SOLUTION COMPOSITIONS FOR THE TREATMENT OF OCULAR HYPERTENSTION

Abstract

The invention relates to topical ophthalmic preparations in eye drops containing bimatoprost as an active ingredient at the concentration of 0.1 mg/ml, wherein the presence of benzalkonium chloride is limited to the indispensable minimum commonly required for its activity as an antimicrobial preservative, i.e. from 50 at 80 ppm, and the bioavailability of the product is ensured by the addition of specific non-ionic surfactants, i.e., poloxamers.

Claims

1. An eye drops ophthalmic composition for use in the treatment of ocular hypertension and glaucoma comprising from 0.008% to 0.012% w/v of bimatoprost as active ingredient, from 0.005% to 0.1% w/v in total of one or more poloxamers, and an antimicrobial agent consisting of benzalkonium chloride, at a concentration of from 50 to 80 ppm, in an aqueous vehicle.

2. The eye drops ophthalmic composition for the use according to claim 1, further comprising from 0.05 to 0.15% w/v of EDTA.

3. The eye drops ophthalmic composition for the use according to claim 2, wherein the concentration of said EDTA is 0.1% w/v.

4. The eye drops ophthalmic composition for the use according to claim 1, wherein said one or more poloxamers are the only polymeric excipients of the composition.

5. The eye drops ophthalmic composition for the use according to claim 4, wherein said one or more poloxamers are present at a total concentration of 0.01% w/v, and are selected from poloxamer 188, poloxamer 407 and mixtures of the same.

6. The eye drops ophthalmic composition for the use according to claim 1, further comprising from 0.01 to 0.15% w/v of a polysorbate, or of a mixture of two or more polysorbates.

7. The eye drops ophthalmic composition for the use according to claim 6, wherein said polysorbate is polysorbate 20.

8. The eye drops ophthalmic composition for the use according to claim 1, wherein said bimatoprost is present at a concentration of 0.01% w/v, and said benzalkonium chloride is present at a concentration of 50 ppm.

9. The eye drops ophthalmic composition for the use according to claim 1, also comprising a buffer system, preferably based on disodium phosphate heptahydrate and citric acid, an isotonizing agent, such as sodium chloride, said composition being brought to a pH between 7.0 and 7.5 by adjustment with NaOH/HCl.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0057] The specific features of the invention will be more evident with reference to the accompanying drawings, in which:

[0058] FIGS. 1 and 2 respectively show in histogram form the apparent permeability through the cornea (unit: 10.sup.−6 cm/s) and the resistance value (unit: Ohm) of the various bimatoprost formulations tested in the experimental study reported below, which formulations include the compositions according to the present invention;

[0059] FIGS. 3 and 4 show in histogram form the apparent permeability through the cornea (unit: 10.sup.−6 cm/s) of different bimatoprost formulations, including the compositions of the present invention, for the purpose of comparing the effects of, respectively, the addition of different surfactants or complexing agents and the addition of EDTA.

EXPERIMENTAL STUDY

[0060] The aim of the study was to obtain experimental data to create new ophthalmic formulations for topical application containing 0.01% bimatoprost as an active ingredient that were capable of presenting a therapeutic activity similar to that of Lumigan® 0.01% but with a low concentration of benzalkonium chloride, of the order of 50-80 ppm, with the simple function of preservative.

[0061] As known, the reference product Lumigan® 0.01% eye drops (Allergan) contains 200 ppm of benzalkonium chloride, which in this concentration exercises two different actions: it preserves the solution from microbial contamination and acts as a promoter for the permeation of bimatoprost through the cornea.

[0062] To support the study of a new formulation of bimatoprost in 0.01% solution with about 50-80 ppm of benzalkonium chloride, permeation studies were conducted through EpiCorneal corneal tissues (COR-100, Mattek) and transcorneal permeation studies ex vivo through isolated rabbit corneas, to verify the ocular absorption of bimatoprost.

[0063] Main excipients chosen for all formulations [0064] a) Buffer solution: a buffer solution based on disodium hydrogen phosphate (Emprove®, Merck) with citric acid (Citric Acid, SA Citrique Beige N.V.) was chosen, with a pH between 7.2 and 7.4. The phosphate concentration corresponded to that used in the reference product. [0065] Buffer composition: Na.sub.2HPO.sub.4 heptahydrate (10 mM) and citric acid monohydrate (PBC). [0066] b) Isotonizing agent: Sodium chloride (Emprove®, Merck) was chosen as an isotonizing agent for all formulations. [0067] The concentration of NaCl was in the range between 0.83% w/v and 0.65% w/v. [0068] c) Antibacterial agent: benzalkonium chloride (BAK, Novo Nordisk Pharmatech A/S) was used at different concentrations depending on the type of experimental procedure. In particular, the formulation with 0.01% of BAK (100 ppm) was used for the ex vivo corneal permeation studies, and concentrations of 0.008% w/v (80 ppm) or lower (up to 0.005%, 50 ppm) were used in all formulations for trans-corneal permeation studies in vitro through the COR-100 epicorneal membranes (Mattek). [0069] d) Stabilizing agent: the inclusion of a stabilizing agent could be useful to reduce the oxidative degradation that can be accelerated by the presence of metallic impurities: for this purpose, sodium EDTA was chosen (EDTA, Emprove® Merck).

Bimatoprost Permeation Study (BMTP) Through EpiCorneal Tissues (COR-100 Mattek)

Experimental Procedure

[0070] Study formulations: all the formulations in the study were prepared in phosphate/citrate buffer and contained, in addition to the excipients, 0.005% w/v (50 ppm) or 0.008% w/v (80 ppm) of benzalkonium chloride (BAK) and 0.01% w/v of bimatoprost. NaCl was chosen as an isotonizing agent and the pH and osmolality values have been measured immediately after the preparation of the formulations.

[0071] Commercial preparations Lumigan 0.01% and Lumigan 0.03% used as reference products and two BMTP reference solutions based exclusively on bimatoprost (0.01%) in phosphate/citrate buffer, added with 0.005% of BAK (50 ppm) or 0.008% of BAK (80 ppm) were chosen as control solutions. The reference formulations contained 0.815% w/v of sodium chloride as an isotonizing agent. The buffer solution based on disodium hydrogen phosphate supplemented with citric acid with a pH between 7.2 and 7.4 was chosen. Buffer composition: 0.268% w/v of Na.sub.2HPO.sub.4 heptahydrate and 0.0128% w/v anhydrous citric acid. The formulations contained 0.815% w/v of sodium chloride as an isotonizing agent.

[0072] Evaluation of apparent permeability: An exactly weighted amount of BMTP corresponding to 0.01% w/v was dissolved in at least 85% of the theoretical amount of ultrapure water (MilliQ water, Millipore) required for each preparation, and then appropriate amounts of the other key excipients have been added: hydrogen phosphate disodium, citric acid, benzalkonium chloride and sodium chloride. Finally, water was added to the mixture up to volume and 0.815% w/v of NaCl. The formulations were kept under constant agitation for 4-6 hours at room temperature.

[0073] The EpiCorneal COR-100 tissues were equilibrated overnight in the culture medium (COR-100 ASY medium) under standard cell culture conditions (SCC, 37° C., 5% CO.sub.2) following the detailed Mattek protocol as reported in the application note. Then, the tissues were transferred to two different 12-well plates and balanced with 500 μL of Krebs Ringer bicarbonate buffer (KRB, pH 7.4) for 30 minutes before the starting of each treatment.

[0074] After equilibrating the tissue for 30 minutes in 500 μL of KBR medium, 100 μL of each selected preparation, previously thermostated maintaining the solutions at 37° C. in a water bath, were applied on the surface of the tissue. Then, the well plates were incubated in SCC. A slight agitation of the well plates was used to reduce the possibility that the drug molecule permeated near the basolateral layer of EpiCorneal tissue could hinder drug permeation across the barrier. At predetermined time intervals (0.5, 1, 2, 3, 4 hours), 300 μL of KRB receiving soil were collected and replaced with the same volume of fresh medium.

[0075] At the end of the experiment, the donor phase of each well plate was completely withdrawn to analyse the amount of BMTP. Each preparation was evaluated in duplicate.

[0076] The steady state flow (J) of BMTP was calculated from the slope of the graphs of BMTP permeate quantity per unit area over time (μg.Math.cm.sup.−2h.sup.−1) and the apparent permeability (P.sub.app, cm.Math.s.sup.−1) was obtained according to the following mathematical relation:

P.sub.app=J/(3600×C.sub.donor)

[0077] In any case, the amount of BMTP used for the calculations of flow and apparent permeability of BMTP is the cumulative amount of permeate drug that takes into consideration the drug removed from the receiving compartment.

[0078] Evaluation of tissue resistance: resistance measurements on EpiCorneal tissues were performed to assess the integrity of the corneal barrier before the start of permeation studies and at the end of the experiments. The measurements were performed using the EVOM® epithelial voltmeter equipped with a fixed pair of silver/silver chloride rod electrodes. The resistance values have been read directly on the digital display of the instrumentation. The initial resistance (t=0 h; TEER-1) of two different samples in different EpiCorneal well plates was measured after pre-treatment, immediately after filling the wells with 300 μL (donor phase) and 500 μL (receiver phase) of KRB buffer.

[0079] Furthermore, tissue resistance was measured for each sample at the end of the permeation study (t=4 h; TEER-2). To perform these measurements at the end of the permeation studies, each well plate was transferred to a new well plate containing 500 μL and 300 μL of fresh KBR in the receiving compartment and in the donor compartment, respectively. The results of the experimentation are summarized in Table 1 below.

TABLE-US-00010 TABLE 1 Results of the permeation study through tissue EpiCorneal Apparent permeability TEER-2 Name of the Concentration (average ± d.s.) (average ± d.s.) formulation Excipients BAK (% w/v) (10.sup.6 cm/s) (Ohm) P188 0.1 BAK 50 Polox. 188 50 0.1 1.20 ± 0.68 632.0 ± 13.0 P188 0.01 BAK 50 Polox. 188 50 0.01 4.46 ± 0.91  650.2 ± 126.3 P188 0.005 BAK 50 Polox. 188 50 0.005 2.51 ± 0.01 657.5 ± 31.2 P188 0.01 BAK 80 Polox. 188 80 0.01  4.36 ± 0.261  528.0 ± 117.4 P407 0.1 BAK 50 Polox. 407 50 0.1  2.0 ± 0.90 660.0 ± 20.0 P407 0.01 BAK 50 Polox. 407 50 0.01  4.33 ± 0.951 534.5 ± 43.2 P407 0.005 BAK 50 Polox. 407 50 0.005  4.48 ± 1.217 601.8 ± 66.3 P407 0.01 BAK 80 Polox. 407 80 0.01  4.54 ± 0.078 541.5 ± 13.4 TW20 0.02 BAK 50 TW 20 50 0.02 1.23 ± 0.05 716.0 ± 22.3 TW20 0.1 BAK 50 TW 20 50 0.1 2.52 ± 0.20 813.3 ± 48.5 TPGS 0.5 BAK 50 TPGS 50 0.5 2.13 ± 0.23 604.5 ± 25.4 β-W7 1.0 BAK 50 β-W7 50 1.0 1.39 ± 0.44 696.0 ± 32.9 P188 0.1/TW20 Polox. 188 50 0.1 0.87 ± 0.05 832.0 ± 9.9  0.02 BAK 50 TW 20 0.02 P188 0.01/TW20 Polox. 188 50 0.01 1.98 ± 0.17 798.0 ± 8.3  0.02 BAK 50 TW 20 0.02 P188 0.01/TW20 Polox. 188 50 0.01 3.93 ± 1.85 766.5 ± 30.4 0.1 BAK 50 TW 20 0.1 P188 0.01/EL Polox. 188 50 0.01 1.81 ± 0.33 798.0 ± 8.3  0.01 BAK 50 EL 0.01 P188 0.01/EDTA Polox. 188 50 0.01 4.56 ± 1.16 805.0 ± 35.4 0.1 BAK 50 EDTA 0.1 P407 0.01/TW20 Polox. 407 50 0.1 4.25 ± 0.23 558.5 ± 68.6 0.1 BAK 50 TW 20 P407 0.01/EDTA Polox. 407 50 0.01 4.56 ± 1.16 611.5 ± 0.7  0.1 BAK 50 EDTA 0.1 P188 0.01/P407 Polox. 188 50 0.01  3.04 ± 0.106 596.5 ± 19.1 0.01 BAK 50 Polox. 407 0.01 P188 0.01/P407 Polox. 188 50 0.01 3.68 ± 0.49 552.5 ± 46.0 0.005 BAK 50 Polox. 407 0.005 P188 0.01/P407 Polox. 188 80 0.01  2.86 ± 0.544 572.5 ± 12.0 0.01 BAK 80 Polox. 407 0.01 P188 0.01/P407 Polox. 188 80 0.01  3.57 ± 0.382 555.0 ± 7.1  0.005 BAK 80 Polox. 407 0.005 P188 0.01/EDTA Polox. 188 80 0.01  3.84 ± 0.233 558.5 ± 68.6 0.1 BAK 80 EDTA 0.1 BMTP 0.01 BAK 80 50  2.81 ± 0.962  707.7 ± 105.8 BMTP 0.01 BAK 50 80  3.52 ± 0.601 634.5 ± 36.1 Lumigan 0.01% 200 4.696 ± 0.764  439.1 ± 117.3 Lumigan 0.03% 100 3.37 ± 0.59 665.5 ± 20.5

[0080] From the data of Table 1, which are generally represented in the form of histograms in FIGS. 1 (apparent permeability) and 2 (resistance values) it can be observed that, surprisingly, the P188 used at 0.01% and containing 50 ppm of BAK showed a higher permeability coefficient than that of the reference solution. Furthermore, the value of P.sub.app was of the same order of magnitude as that calculated for commercial Lumigan at 0.01%, which contains 200 ppm of BAK. The highest P.sub.app coefficient was measured for P188 0.01%: in fact, both concentrations higher (0.1%) and lower (0.005%) of P188 determined a reduction in the apparent permeability of bimatoprost.

[0081] The P.sub.app value for the formulations containing P188 0.01% and 80 ppm of BAK was in the same order of magnitude as that calculated for Lumigan 0.01%, which contains 200 ppm of BAK.

[0082] All the formulations tested showed higher resistance values than those measured in the presence of 0.01% Lumigan.

[0083] It should be noted that a high value of corneal resistance is a fundamental requirement for the ophthalmic formulations in the present study.

[0084] A high resistance value was measured for the EpiCorneal tissue before the permeation study (1340 Ohm, for white Epicorneal), while after 4 hours of bimatoprost permeation test, all the treated tissues showed resistance values lower, between 500 and 800 Ohm, except those treated with the commercial 0.01% Lumigan. This reduction in resistance does not appear to be associated with the selected excipients but appears to be caused by the presence of benzalkonium chloride (50 and 80 ppm). In fact, the EpiCorneal fabric treated with the commercial Lumigan 0.01% preparation which contained a higher concentration of benzalkonium chloride, showed the lowest resistance value, up to 35% (439.1 Ohm) of the initial value.

[0085] It should be noted that the addition of different concentrations of other non-ionic surfactants (TPGS, TW20) or complexing agents such as hydroxypropyl-8-cyclodextrin (β-W7) in the presence of BAK at 50 ppm did not lead to an increase in P.sub.app of BMTP with respect to the BMTP reference formulation.

[0086] In all cases the P.sub.app values were lower than those calculated for the reference BMTP of 0.01% with 50 ppm of BAK, and for both Lumigan 0.01% containing 200 ppm of BAK and Lumigan 0.03% containing 100 ppm of BAK, as shown more clearly in the histogram of FIG. 3.

[0087] As shown more clearly in the histogram of FIG. 4, adding EDTA to the formulation containing P188 0.01% and BAK 50 determines a P.sub.app value similar to that calculated for P188 0.01% BAK 50 in the absence of EDTA.

[0088] The addition of other types of poloxamers (e.g. P407 at 0.01% concentration) to the formulation based on 0.01% of P188 and BAK 50 ppm, which represents the most promising concentration capable of favouring the apparent permeability of BMTP, resulted in a reduction in BMTP P.sub.app values even in the presence of different BAK concentrations (50 and 80 ppm).

[0089] Similarly, the combination of P188 0.01% with TW 20 0.02% and 0.1%, and P188 0.01% with EL 0.01%, caused a decrease in the apparent permeability of BMTP. The values obtained were lower for all the blends tested, with respect to the commercial product and the references containing up to 80 ppm of BAK (FIG. 4).

Corneal Permeation Study Ex Vivo

[0090] Experimental design: BMTP permeation through isolated rabbit corneas was performed using a plexiglass perfusion apparatus where rabbit corneas (New Zealand) were mounted immediately after animal sacrifice, to separate a donor compartment with volume of 1.0 ml and an acceptor compartment with a volume of 5.0 ml. Both compartments were oxygenated with O.sub.2:CO.sub.2 mixture (95:5) and the experiments were conducted in Ringer's glutathione bicarbonate solution (GBR) at pH 7.2 and 32° C. to maintain corneal viability up to at 5 hours. The formulations were added to the donor compartment, replacing 500 μL of donor solution. At different times (approximately every hour) samples of the receiving solution were collected and analysed by HPLC. The quantitative analysis of the BMTP in the reception phase at different times of sampling and in the donor phase at the end of the experiments was calculated using an appropriate calibration curve.

TABLE-US-00011 TABLE 2 P.sub.app values of formulations used for ex vivo corneal permeability study BAK BAK P.sub.app (ppm) (ppm) (cm/s) 1 Lumigan 0.01% 200 9.10 10.sup.−6 2 P188 (0.1%)-TW20 (0.02%) 100 5.06 10.sup.−6 3 P188 (0.1%)-TW20 (0.02%) 50 0.032 10.sup.−6  4 P188 (0.1%)-RH40 (0.5%) 100 3.04 10.sup.−6 5 P188 (0.1%)-TW80 (1.0%) 100 4.03 10.sup.−6

[0091] The formulations No. 2, 4 and 5 contained P188 (0.1%) plus TW20 (0.02%) or RH40 (0.5%) or TW80 (1.0%) and the BAK concentration was 100 ppm. Formulation No. 3 had the same composition as formulation No. 2 but contained 50 ppm of BAK.

[0092] In all cases the formulations tested did not determine the apparent permeability of Lumigan 0.01%, and a reduction in P.sub.app was detected by reducing the concentration of BAK from 100 ppm to 50 ppm respectively for formulations No. 2 and No. 3.

[0093] The present invention has been described with reference to some specific embodiments thereof, but it is to be understood that variations or modifications may be made to it by those skilled in the art without departing from the scope of protection as defined in the appended claims.