Patent Application
20150209275
This invention relates to a pharmaceutical dosage form, particularly to a topical ocular pharmaceutical dosage form comprising a polymeric matrix of polyethylene oxide block copolymer, preferably polyoxyethylene-polyoxypropylene block copolymer and hydroxpropyl cellulose, and a pharmaceutically active ingredient incorporated within the matrix. The invention extends to a method of manufacturing the pharmaceutical dosage form.
Ocular disorders, conditions or diseases of the anterior segment of eye require an effective topical, locally acting, drug delivery system that ensures penetration of the drug through the cornea, effective therapeutic drug levels and patient compliance. Topical methods for drug delivery to the anterior eye remain one of the most convenient and common means of drug delivery. In particular, the use of eye drops still dominates as the drug delivery system of choice by patients. Thus, focus has been directed to topical systems such as eye drops and gels which currently are the most popular pharmaceutical dosage forms for topical ocular use available on the market.
Despite the convenience and compliance using eye drops, limitations are evident. Most eye drops remain on the eye for between about 5 to about 25 minutes and only about 1 to about 10% of drug or active pharmaceutical ingredient (API) is absorbed (Anumolu et al., 2009). This results in low bioavailability of ophthalmic drugs when administered using topical dosage forms such as eye drops (du Toit et al., 2011). Patients often have difficulty administering conventional eye drops due to the following reasons: Firstly, compliance is a problem since eye drops typically have to be administered several times during the course of the day. Secondly, certain patients with conditions such as arthritis have difficulty administering the eye drops since handling of the eye drop bottle poses a problem. A certain level of pressure has to be applied to the bottle to expel the eye drop and a dispensing end of the bottle has to be specially directed such that an expelled eye drop engages and/or enters the eye. These patients are then disadvantaged due to lack of control and synchronization of the bottle together with the eye-lid closure reflex.
Over the years, various attempts have been made to optimize the bioavailability of topically administered ocular drugs. Some of these approaches include: viscous vehicles and hydrogels (Rajas et al., 2011), facilitated transport via prodrugs (Järvinen and Järvinen 1996), nanoparticles (Diebold and Colonge, 2010; Zimmer and Kreuter, 1995), contact lens delivery systems (Ali et al., 2007) and penetration enhancers (Kikuchi et al., 2005). These systems have been developed and used to increase corneal contact time and allow for improved corneal drug penetration to the anterior segment of the eye (Weyenberg et al., 2004). However, their use is associated with several disadvantages. For example, gels and ointments can blur vision and cause reflex blinking. Contact lenses may prove difficult to insert and requires removal (Saettone and Salimen, 1995).
Limitations with respect to the anatomical and physiological aspects of the eye itself are also evident. When drug is administered topically to the eye, reflex closure of the eye lid occurs due to a foreign-body sensation (Urtti, 2006). This results in the liquid preparations being forced out or even prevents entry into the anterior eye altogether. Also, the flow of lacrimal fluid removes administered compounds from the surface of the eye and this fluid, which may entrain the administered drug or API, is then drained by the nasolacrimal duct (Barar et al., 2009). The cornea serves as a protective layer of the eye and comprises several layers (Patel et al., 2004). The corneal epithelium is the most anterior layer of the eye and is approximately 0.1 mm in thickness (Mannermaa et al., 2006). On the outside is the multicellular epithelium which consists of 56 layers of cells and these tight junctions and hydrophobic regions pose a barrier to drug penetration (Chang, 2010; Rathore and Nema, 2009). Therefore, the corneal epithelium limits drug absorption to the anterior segment of the eye.
Apart from improving bioavailability of topically administered ocular drugs, an improved administration system is required to ensure patient compliance and that the drug reaches the target site. As mentioned, eye drops may be difficult to administer and are easily flushed out thus resulting in poor bioavailability especially in elderly patients (Davies, 2000). Thus, there has been effort directed in developing novel drug delivery systems for ocular use. Solid drug delivery systems such as mini-tablets have been investigated as potential vehicles to overcome the challenges associated with the use of conventional eye drops. Using the concepts of improving contact time and bioavailability of ocular preparations, mini-tablets for ocular drug delivery have been formulated. Mini-tablets can be defined as tablets with a diameter of about 2 to about 3 mm (Lennartz and Mielck, 1998). Some advantages that these systems offer over conventional eye drops include: avoiding flushing out of the drug by lachrymation and drainage once administered; increased corneal contact time due to the use of bioadhesive polymers and the fact that the outer layers of mini-tablets swell upon hydration and as the hydration front penetrates into the tablet core providing a gradual release of drug or API over time; low cost and ease of manufacture; and such systems have been reported not to induce mucosal irritation (Ceulemans et al., 2001; Weyenberg et al., 2003; Weyenberg, 2005). Disadvantages of these systems are that they dissolve in the range of hours and may be displaced from their original position within the eye after administration causing some degree of irritation.
Rapid disintegrating systems are defined as systems that have rapid disintegration in less than about 1 minute without water or a small amount of water (about 1 to about 2 mL) (Fu et al., 2004). Advantages of rapid disintegrating solid drug delivery systems include: good stability, accurate dosing, ease of manufacture and ease of use (Dobetti, 2001, Chandrasekhar et al., 2009). Common uses of such rapid disintegrating drug delivery systems include use in oral methods of administration for geriatric or paediatric patients. In terms of ocular delivery, the concept of fast disintegrating systems has not been thoroughly explored. The advantages of solid drug delivery systems to ocular surfaces include: comfortable and non-irritant use due to small and light structure; convenient form of administration and accurate dosing compared to liquid formulations (Virely and Yarwood, 1990); rapid hydration of system due to porous structure thus reducing foreign body sensation in the eye (Refai and Tag, 2011); and preservatives are not required as in the case of conventional liquid formulations. Once in contact with the lachrymal fluid on the eye surface, disintegration of the system occurs with rapid release of the pharmaceutically active compound.
Research has been conducted on several rapid disintegrating solid delivery systems for ocular use typically comprising polymeric matrices that gel on insertion into the lower cul-de-sac of the eye for controlled release of pharmaceutically active compound.
Using solid hydrophilic matrices in the form of mini-tablets for ocular use has gained popularity over the years. Further, the use of freeze-dried mini-tablets may provide a quicker disintegration time compared to conventional tablets. The lyophilization or freeze drying method is defined as: sublimation of frozen water in the sample from a solid phase to a gas phase under reduced pressure (Tsinontides et al., 2004). The process involves selection of polymer-excipient concentrations and subjecting the solution to freezing followed by lyophilization of the sample. This results in the formation of the porous product. On exposure to fluid, ingress of fluid into the matrix occurs with resultant dissolution and release of the pharmaceutically active compound.
The concept of employing lyophilized systems in topical ocular drug delivery has been investigated. Freeze drying results in a solid dosage form which further results in reduced degradation reactions. In addition, easier handling during transportation and storage is noted (Virely and Yarwood, 1990). In terms of solid rapidly disintegrating delivery systems, they offer the following advantages: i) comfort of use due to small and light structure, ii) convenient form of administration and accurate dosing compared to liquid and/or gel formulations (Carpenter et al., 1997), iii) benefit of liquid formulation in a solid form, iv) preservatives are not required since water content is below 5% and this does not favour microbial growth (Süverkrüp et al., 2004) and v) rapid hydration of system due to porous structure thus reducing foreign body sensation in the eye (Refai and Tag, 2011).
Ocular tolerability of solid dry drops comprising hydroxypropylmethylcellulose (HPMC) was demonstrated in healthy human eyes in phase 1 studies (Diestelhorst et al., 1998). Lux and co-workers (2003) concluded that a HPMC lyophisilate displayed higher sodium florescein levels in the anterior eye compared to liquid eye drops in human volunteers. A superior intraocular bioavailability of sodium florescein was seen with a 1% hypromellose lyophisilate compared to eye drops and thus a pharmacokinetic advantage was demonstrated (Abduljalil et al., 2008). A study by Refai and Tag (2011), investigated freeze-dried sponge-like ocular mini-tablets for ocular keratitis treatment comprising sodium carboxymethyl cellulose, HPMC, xanthan gum, chitosan and Carbopol 943P. These mini-tablets showed significant sustained release of acyclovir and good bioadhesive properties and permeation across the cornea in rabbits.
However, there exists a need for novel, easy to manufacture, rapid pharmaceutically active ingredient (API) releasing solid pharmaceutical dosage forms for topical ocular use.
The following terms used through the course of this patent specification shall have the following meanings:
In broad terms this invention relates to a pharmaceutical dosage form for the delivery of at least one active pharmaceutical ingredient (API) to a desired target site in a human or animal, the pharmaceutical dosage form comprising at least one matrix forming polymer, preferably two matrix forming polymers, the two matrix forming polymers preferably being from the class of polymers comprising polyethylene oxide block copolymers as well as cellulosic polymers such as hydroxpropyl cellulose (HPC), such that the at least one matrix forming polymer is formed into a solid pharmaceutical dosage form.
According to a first aspect of this invention there is provided a pharmaceutical dosage form for the delivery of at least one active pharmaceutical ingredient (API) to a target site in a human or animal, the pharmaceutical dosage form comprising a homogenous polymeric matrix consisting of a polyethylene oxide block copolymer and hydroxpropyl cellulose (HPC).
In a preferred embodiment of the invention the polyethylene oxide block copolymer may be polyoxyethylene-polyoxypropylene block copolymer. The polyethylene oxide block copolymer may be a Pluronic polymer, preferably Pluronic F-68 (PF-68).
The pharmaceutical dosage form may further comprise an anti-collapsing agent. The anti-collapsing agent may comprise an amino acid chain, preferably the amino acid chain having 1 amino acid residue, more preferably 2 amino acid residues.
In a preferred embodiment of the invention, the anti-collapsing agent comprises an amino acid chain having two amino acid residues, preferably diglycine.
The pharmaceutical dosage form may further comprise a lyoprotectant, preferably maltodextrin.
The pharmaceutical dosage form may further comprise a superabsorbent polymer, preferably polyacrylic acid sodium salt (PAA-Na salt).
The pharmaceutical dosage form may further comprise at least one active pharmaceutical ingredient (API) homogenously dispersed therein selected from the group: prostaglandin analogs such as latanoprost, beta blockers such as timolol, alpha agonists such as brimonidine, carbonic anhydrous inhibitors such as dorzolamide hydrochloride, or combinations of these such as timolol and dorzolamide hydrochloride or timolol and brimonidine.
In a preferred embodiment of the pharmaceutical dosage form, the dosage form comprises:
The pharmaceutical dosage form may be formed into a solid ocular pharmaceutical dosage form for the delivery of the at least one active pharmaceutical ingredient (API) to a region of the eye.
In a preferred embodiment of the invention the solid ocular pharmaceutical dosage form is formed as a tablet, particularly a mini-tablet having substantially circular and/or discoid shaped dimensions wherein the thickness is about 2 mm and the diameter is about 3 mm.
According to a second aspect of this invention there is provided a method of manufacturing a pharmaceutical dosage form of the first aspect of this invention, the method comprising the steps of:
Step (b) may further comprise adding an active pharmaceutical ingredient (API) to Solution 1.
Step (c) may typically take place for about 24 hours at about −82° C. and Step (d) may take place at about −42° C. for about 24 to about 48 hours.
The method may include freezing Solution 2 in polyvinyl chloride (PVA) blister packs of predetermined size in order to produce dosage forms having substantially circular and/or discoid dimensions of about 2 mm in thickness and 3 mm in diameter.
The composition of dry components dissolved in the deionized water prior to freezing in % w/v may be as follows: the polyethylene oxide block copolymer, preferably PF-68 may be in the range of about 1 to about 5% w/v; the hydroxpropyl cellulose (HPC) may be about 0.5% w/v; the lyoprotectant, preferably maltodextrin, may be in the range of about 1 to about 5% w/v; the superabsorbent polymer, preferably polyacrylic acid sodium salt (PAA-Na salt), may be about 0.25% w/v; and an anti-collapsing agent, preferably diglycine, may be about 0.25% w/v.
The invention is now described by way of example only, with reference to the accompanying diagrammatic drawings, in which
In broad terms this invention relates to a pharmaceutical dosage form for the delivery of at least one active pharmaceutical ingredient (API) to a desired target site in a human or animal, the pharmaceutical dosage form comprising at least one matrix forming polymer, preferably two matrix forming polymers, the two matrix forming polymers preferably being from the class of polymers comprising polyethylene oxide block copolymers as well as cellulosic polymers such as hydroxpropyl cellulose (HPC), such that the at least one matrix forming polymer is formed into a solid pharmaceutical dosage form. It is to be understood that the dosage form may be substantially homogenous or may be formed to be layered, either like an onion or like a sandwich. The matrix forming polymers facilitate providing structural integrity to the dosage form.
According to a first aspect of this invention there is provided a pharmaceutical dosage form for the delivery of at least one active pharmaceutical ingredient (API) to a target site in a human or animal, the pharmaceutical dosage form comprising a homogenous or mono-layered polymeric matrix consisting of a polyethylene oxide block copolymer and hydroxpropyl cellulose (HPC). The pharmaceutical dosage form may be a solid ocular pharmaceutical dosage form in the form of a solid eye drop for the delivery of API to a region of the eye. The HPC typically acts as a matrix forming polymer to facilitate providing structural integrity to the solid eye drop.
The solid ocular pharmaceutical dosage form comprises at least one active pharmaceutical ingredient (API) homogenously dispersed therein.
The solid eye drop is light weight and porous in nature. Typically, the solid eye drop is inserted into the eye, preferably administered to the cul-de-sac of the eye. The solid eye drop is soluble upon contact with the mucosal surface of the eye such that dissolution and/or disintegration initiates substantially instantaneously upon contact with the mucosal surface. The rapid dissolution and/or disintegration causes release of API to a region of the eye. Typically, the released API moves through the cornea to posterior regions of the eye.
The particular chemical composition of the solid eye drop causes certain mechanical properties, particularly its porosity facilitates the ingress of liquid media which causes dissolution and/or disintegration and associated API release. The applicant found that the specific combination of a polyethylene oxide block copolymer and HPC, preferably Pluronic F68 and HPC, produced a solid dosage form having a inter-connecting network of pores. These pores facilitated in providing the dosage forms rapid disintegration characteristics in use, and contributed to its rigidity prior to use. The API can be at least one selected from the group, but not limited to: prostaglandin analogs such as latanoprost, beta blockers such as timolol, alpha agonists such as brimonidine, carbonic anhydrous inhibitors such as dorzolamide hydrochloride, or combinations of these such as timolol and dorzolamide hydrochloride or timolol and brimonidine.
The chemical composition of the solid eye drop is biocompatible and biodegradable in nature. Although the preferred embodiment later herein described comprises Pluronic F-68 this should not be seen as limiting. The invention may very well employ the use of other known polyethylene oxide block copolymers in the stead of Pluronic F-68, or other polymers from classes of polymers which the aforementioned can be categorized under.
The solid ocular pharmaceutical dosage form may further comprise an anti-collapsing agent, preferably diglycine; a lyoprotectant, preferably maltodextrin; and a superabsorbent polymer, preferably polyacrylic acid sodium salt (PAA-Na salt). The dosage form is typically formed into a tablet, particularly a mini-tablet, having substantially circular and/or discoid shaped dimensions wherein the thickness is about 2 mm and the diameter is about 3 mm.
It is important to understand that the pharmaceutical dosage form according to this invention may be formulated as an oral wafer matrix, a graft lubricant, a chromatography gel, a wound dressing, a mesh, a degradable bone fixation glue, a degradable ligament glue and sealant, a tendon implant, a dental implant, a reconstituted nerve injectable, a disposable article, a disposable contact lens, an ocular device, a rupture net, a rupture mesh, an instant blood bag additive, an instant haemodialysis additive, an instant peritoneal dialysis additive, an instant plasmapheresis additive, an inhalation drug delivery device, a cardiac assist device, a tissue replacing implant, a drug delivery device, an endotracheal tube lubricant, a drain additive, and a dispersible suspension system.
According to a second aspect of this invention there is provided a method to manufacture the pharmaceutical dosage form of the first aspect of this invention, the method comprising the steps of:
Step (b) typically further includes adding an active pharmaceutical ingredient (API) to Solution 1. The method may include freezing Solution 2 in polyvinyl chloride (PVA) blister packs of predetermined size in order to produce dosage forms having substantially circular and/or discoid dimensions of about 2 mm in thickness and about 3 mm in diameter. The freezing typically takes place for about 24 hours at about −82° C. Lyophilization may take place at about −42° C. for about 24 to about 48 hours.
The method employed in the manufacture of pharmaceutical dosage forms according to this invention was the lyophilization process which will be described below in detail. This method offers the advantages of simplicity, reproducibility, cost-effectiveness and generation of a stable yet rapidly soluble system.
Design of Experiments (DOE) is defined as a mathematical strategy for setting up experiments in such a manner that the information required is obtained as efficiently and precisely as possible (Lewis et al., 1999; Anderson and Whitcomb, 2007). This statistically based methodology was employed in order to obtain formulations for the purpose of characterization. Tests carried out on the manufactured dosage forms included: textural analysis, disintegration profiling, moisture content studies, scanning electron microscopy (SEM), stereomicroscopy, drug entrapment efficiency (DEE) and in vitro drug release studies.
Representative examples of the invention are described in detail hereunder. The representative examples of the dosage form were formulated as solid topical ocular pharmaceutical dosage forms also termed solid eye drops or instantly soluble solid eye drops (ISEDs).
Hydroxypropylcellulose (HPC) (Mw=80 000 g/mol) (Klucel®, Hercules Incorporated, Willington, Del., USA), glycyl-glycine (diglycine, DG) (Mw=132.12 g/mol) (Fluka BioChemika, Belgium), Interfix 1001 Polycaprolactone CAPA® 6400 (Interfix CC, Johannesburg, South Africa), poly(acylic acid sodium salt) (PAA-Na salt) (Mw=5100 g/mol), Maltodextrin (MD) (dextrose equivalent 4.0-7.0) and Pluronic® F-68 (Mw=8400 g/mol) all purchased from Sigma-Aldrich (St. Louis, Mo., USA). All other reagents were of analytical grade and used as supplied. The drug or API is Timolol maleate salt (TM) (Mw=432.49 g/mol) purchased from Sigma-Aldrich (St. Louis, Mo., USA).
A two-factor, three-level Face Centered Central Composite Design (FCCCD) was applied for the construction of a second order polynomial model describing the effect of formulation constituents on the characteristics of the system. Pharmaceutical dosage forms according to the invention, in the form of instantly soluble solid eye drops ISEDs, of various combinations were prepared in accordance with the FCCCD (Table 1). The term “instantly soluble” was assigned to the dosage form since it dissolved rapidly (see Definitions section).
Aqueous solutions of polymer were prepared in various concentrations in accordance with the FCCCD. Table 1 depicts formulations generated from the design based on influential components as determined from preformulation studies. Components were dissolved in 100 mL deionized water and agitated for 30 minutes until complete dissolution had occurred. Samples of 150 μL were injected into each mould of the polyvinyl chloride (PVC) blister packs employing a 1 mL syringe. Samples were then frozen (Sanyo Ultralow Temperature freezer, MDF-U73V, Sanyo Electric, Japan) for 24 hours at −82° C. to solidify the product. The product was placed in a lyophilizer (Labconco Freeze-Dry Systems, Labconco Corp., Kansas City, Mo., USA) for 48 hours to extract excess water. On attainment of the samples they were stored in glass vials in the presence of 2 g desiccant sachets.
The method above was utilized to prepare a drug-loaded (DL) ISEDs. When manufacturing a drug-free (DF) ISED the API or drug was omitted from the remaining components which were dissolved in the deionized water prior to freezing.
All analytical experiments outlined below as part of Example 1 were conducted on drug-loaded (DL) ISEDs where Timolol maleate salt (TM) was the API.
Textural analysis was used to characterize the compressibility of the dosage form using a Texture Analyzer (TA.XTPlus, Stable Microsystems, UK). The following tests were conducted for characterization at room conditions (about 25° C.). The following textural properties were determined:
1. Matrix Resilience (MR): Resilience can be defined as the ability of a material to return to its original position or state after stress has been applied to it. Resilience of the dosage form provides an elucidation of the ability of the dosage form to withstand an applied stress. An analyzer (TA.XTPlus, Stable Microsystems, UK) was fitted with a suitable 10 mm diameter delcin probe for resilience measurement. Force-time profiles were generated and analyzed.
The MR (%) was determined by finding the ratio between anchors 2 and 3 and between anchors 1 and 2 (
2. Energy of Absorption (EA): The energy of absorption is an indirect indication of the porosity of the dosage form. A highly porous dosage form will exhibit a greater value for the energy of absorption. The energy of absorption is calculated by determining the area under the curve (AUC) of a profile illustrating force (N) and distance (m) as depicted in
3. Matrix Yield Value (MYV): Matrix yield value assists in the determination of the strength of the surface structure of the dosage form. This is determined by obtaining a gradient between anchors 1 and 2 of a force-distance profile (
4. Matrix Tolerance (MT): Matrix tolerance indicates the overall strength of the dosage form. The gradient between anchors 1 and 3 provides the matrix tolerance value. This is the point of total collapse of the dosage form (
5. Brinell Hardness Number (BHN): Hardness is described as the resistance of an object to permanent shape change when a force is applied. Brinell hardness is an indication of the force required to indent the surface of the dosage form. Brinell hardness was assessed using a ball point probe. Force-distance profiles were generated and assessed (
Where:
F=force generated from indentation (N) obtained from maximum peak of curve,
D=diameter of ball probe indenter (3.125 mm) and
d=indentation depth which is half the probe diameter (1.5625 mm)
Textural profiling was determined as per the settings in Table 2.
1Matrix resilience
2Brinell hardness number
Disintegration profiling of the pharmaceutical dosage form according to the invention Disintegration testing of rapidly dissolving dosage forms or delivery systems is an important test to be carried out for the evaluation of the time taken for such a system to dissolve in use. The Texture Analyzer method has been employed for the evaluation of disintegration of fast dissolving oral wafers (Dor et al., 2000; El-Arini and Class, 2002; Fu et al., 2004). Advanced testing utilizing improved methods were conducted employing a Texture Analyzer (TA) (Stable Micro Systems, Surrey, UK) with a flat, cylindrical probe. The probe head was magnetically attached to the shaft which was screwed onto the load cell carrier. This allowed for preparation of 5 samples at a time and quick removal and interchanging of probe heads was done. The dry ISED was attached to the probe head by means of a thin strip of double sided adhesive tape across its diameter. This was lowered into a Perspex test vessel containing 5 mL Simulated Lachrymal Fluid (SLF, pH 7.4, 35° C.) (Table 3). Upon entering the medium and quickly reaching an immersed perforated platform the Texture Analyzer applied a minimal force (0.098N) for a chosen period of time. The clear vessel allowed for observation of the process. Typical distance-time profiles were generated according to set parameters (Table 4) and disintegration rate (mm/sec) and end point disintegration time (s) were determined.
The in-depth surface morphology and internal structure of the dosage form was visualized by the use of scanning electron microscopy (SEM) (FEI Phenom™, Hillsboro, Oreg., USA). Samples were mounted on a spud and gold plated by the sputter-coater (SPI modul™ sputter-coater and control unit, West Chester, Pa. USA).Samples were then viewed under the SEM at different magnifications.
The visualization of the solubilization of the ISED was determined by microscopy using light illumination for images in a 3-dimensional level. A stereomicroscope (Olympus SZX7 stereomicroscope, Olympus, Japan) connected to a digital camera (CC 12, Olympus, Japan) and image analysis system (AnalySIS® Soft Imaging System, GmbH, Germany) was employed. The unhydrated and hydrated samples were imaged to observe the entrance of fluid into the sample on a microscopic level.
DEE was conducted by dissolving the ISEDs in SLF and spectrophometrically analyzing the solution at 295 nm employing a standard calibration curve for timolol maleate (the drug or API in this representative example) (
Where:
DEE=Drug entrapment efficiency,
Da=the actual quantity of drug (mg) measured by UV spectroscopy and
Dt=the theoretical quantity of drug (mg) added in the formulation.
The Franz diffusion cell is a method that can be used for studying the diffusion of drug or API from semisolid ocular dosage forms (Gorle and Gattani, 2009; Gilhotr et al., 2010; Gilhotra et al., 2011). A Franz diffusion cell consists of a donor chamber and a receptor chamber with a membrane clamped in between. Commercial cellophane membranes (Mw=12000, Sigma-Aldrich Corp. St. Louis, Mo., USA) were used to simulate the corneal epithelial barrier for ocular penetration of drug. Membranes were presoaked in dissolution media overnight. The diffusion cell donor chamber contained 2 mL SLF which was maintained to simulate tear volume and solid eye drops were placed in the donor compartment in contact with the membrane. The receptor solution of 12 mL Simulated Aqueous Humour (SAH, pH 7.4, 35° C.) (Table 6) was contained in the receptor chamber. The membrane was placed such that the surface was in contact with the receptor solution which was continuously stirred by a magnetic stirrer at 20 rpm to simulate blinking. Aliquots of 2 mL were withdrawn from the receptor compartment at regular intervals (30, 60, 120, 240, 360 minutes after insertion) and replaced an equal volume of dissolution medium. The samples were analyzed spectrophotometrically at 295 nm (Hewlett Packard 8453 Spectrophotometer, Germany) to determine the drug release. Tests were conducted in triplicate. The dissolution data was analyzed by calculation of the Mean Dissolution time (MDT). This is determined as the sum of individual period of time during which a specific fraction of the total drug or API is released (Pillay and Fassihi, 1999; Govender et al., 2005). MDT50% data point was selected for the design formulations. The following equation was used for the determination of MDT:
Where:
Mt=the fraction of dose released in time tt
tt=(ti+ti-1)/2 and
M∞=loading dose
The physical characteristics of the ISEDs as a dosage form are essential for determination of the stability during storage and use of the product in the eye. It provides an indication of the integrity of the formulations. The numerical values obtained from the analysis at room conditions are listed in Table 6. Low MD and PF68 concentrations resulted in ISEDs that were fragile. Higher MD and PF68 concentrations produced well formed and easily removable ISEDs. Higher MD imparted rigidity and improved hardness of the formulations. Formulation 6, 7, 8, 10 and 12 comprised the same formulation combinations. An increase in matrix resilience is attributed to higher strength and this is an important parameter to help understand the characteristics of the dosage form. Resilience of these formulations was noted to be higher than other formulations ranging from 4.698 to 6.519%. Elevated BHN values are due to higher stability characteristics. The BHN values did not vary much among the formulations. Highest BHN value was seen for formulation 1 (2.062 N/mm2). Energy of absorption was used as an indication of indirect quantification of matrix porosity (Patel et al., 2007). Formulation 1 displayed the highest energy of absorption (0.026 J) which indicates a superior stability between the surface and polymeric network and thus a more robust sample. More pores within a dosage form cause a higher energy of absorption, and was observed as such for formulation 1. Highest tolerance was noted for formulation 7 (19.531 N/mm) which indicates a high overall matrix strength. In terms of yield value, the highest value was seen for formulation 1 (2.9180 N/mm). The correlation between yield and tolerance values is such that initially a low yield value is observed and subsequent to fracturing of the dosage form this energy is dissipated resulting in complete collapse.
Results are expressed as the mean of at least three measurements, SDs (standard deviation) obtained were within: yield value ≦0.010, tolerance ≦0.059, absorption energy ≦0.002, resilience ≦0.081, BHN≦0.011.
Disintegration end point times (EPDTs) were determined by noting the end point on graphs. The EPDT was taken as the time taken for complete disintegration to occur as the solid eye drop was immersed in SLF. Instantaneous disintegration was noted for all the formulations due to the porous nature of the ISED and quick ingress of the fluid. All formulations dissolved to form a liquid with the exception of formulations 2, 5 and 9. This is due to the higher PF68 concentration (5%). The polymer displayed a tendency to form a gel-like residue with increasing concentration. This can be attributed to PF68 being a solid hydrophilic block co-polymer which upon heating and increased concentrations displays a tendency to form a low viscosity thermoreversible gel-like substance (Maghraby and Alomrani, 2009). Formulation 11 displayed the fastest EPDT of 0.200 s while formulation 9 had the slowest time of 3.340 s. Disintegration rate was taken as the first gradient of the descending region from the onset of disintegration. The highest disintegration rate was noted for formulation 11 (10 mm/sec). This correlated with the highest disintegration time. This can be explained in terms of the low PF68 and MD concentration which produced a fragile matrix of formulation 11 and thus resulted in rapid breakdown of the matrix. Table 7 indicates values obtained from the study while
The lyophilized ISED appeared to have a sponge-like surface and an interconnecting network with the presence of pores (
Microscopic imaging of the ISED was performed to aid with visualization of the rapid dissolution process (
The purpose of calculating the DEE was to determine the percentage of API or drug loaded into the formulations. A homogenous dispersion of API is required. This would ultimately affect the API release profile and a high DEE is desirable with a maximum of 100%. DEE for the dosage form ranged from 79-96%. Table 8 outlines DEE values calculated and
Drug release was determined and plots are depicted in
Herein described is a rapidly disintegrating pharmaceutical dosage form formulated for specific topical ocular delivery of an API, and preferably termed an instantly soluble solid eye drop (ISED). The dosage form having sufficient strength. Textural analysis revealed a robust dosage form was manufactured. Disintegration testing demonstrated that instant dissolution was achieved and thus rapid liberation of API or drug was possible. Surface morphology allowed for visualization of the porous surface of the formulations. The pharmaceutical dosage form formulated may be suitable for ocular use due to the biocompatibility of the polymers and excipients employed. Furthermore, the rapid disintegration would allow minimal irritation to the ocular surface due to reduced foreign body sensation. In addition, the method of production was simple and relatively time effective thus possible cost-effectiveness in terms of manufacturing may be advantageous. The applicant found that the specific combination of a polyethylene oxide block copolymer and HPC, preferably Pluronic F68 and HPC, produced a solid dosage form having a inter-connecting network of pores. These pores facilitated in providing the dosage forms rapid disintegration characteristics in use, and contributed to its rigidity prior to use.
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The focus of further testing was to gauge the in depth pertinent pharmaceutical properties and in vivo behavior of a pharmaceutical dosage form, particularly a solid ocular pharmaceutical dosage form, namely, an optimized instantly soluble solid eye drop (OISED) in accordance with the invention. In brief, thermal and molecular transition analysis showed congruent findings with no incompatibility between components of the OISED. Porositometric studies confirmed the presence of interconnecting pores across the matrix surface. Drug release kinetic evaluation predicted that best model fit was first-order release (R2=0.98). The HET-CAM test indicated an irritation score of 0 with the inference of good tolerability. Ex vivo permeation across excised rabbit cornea showed an improved steady state drug flux (0.00052 mg.cm−2.min−1) and permeability co-efficient (1.7×10−4 cm.min−1) for the OISED device compared to pure drug and a marketed eye drop preparation. Ultra-performance liquid chromatography (UPLC) analysis indicated drug (timolol maleate, TM) and internal standard (diclofenac sodium) elution at 0.5 and 1.5 s respectively. Gamma irradiation served as an effective terminal sterilization procedure. In vivo results revealed a peak concentration was reached at 104.9 minutes. In the case of eye drops a lower Cmax was obtained (1.97 ug/mL). Level A point-to-point IVIVC plots a Wagner Nelson method indicated a satisfactory R2 value of 0.84. The biodegradability and biocompatibility by histological toxicity studies was confirmed.
Formulation of an ocular pharmaceutical dosage form requires special attention due to the intricacy and sensitive nature of the eye. Solid ocular pharmaceutical dosage forms for use in the eye have been studied due to the advantages noted. These include lack of flushing, more accurate dosing and better stability. One such example is the application of fast dissolving systems, conventionally for oral application, to the eye surface. However, the process of lyophilization may have an impact on the physical properties, thermal behavior and chemical stability of the polymers involved (Guo et al., 2000). Additionally, the porosity of a freeze-dried system is significant since the voids within a matrix often display a relationship with the appearance and in vitro behavior of the dosage form (Sznitowska et al., 2005). As pointed out by Vargas and co-workers (2007), in order to evaluate novel drug delivery systems, the use of animal models is required but has the disadvantage of being costly, difficult and time-consuming. Thus, alternative means have to be delved into. The Hens Egg Choriollointic Membrane Test (HET-CAM) is an alternative to the Draize rabbit eye test for the assessment of potential ocular irritants and has been applied in various ocular studies (Gorle and Gattani, 2005; Gilhotra and Mishra, 2012). This test has been accepted in Europe and documented in the current EU guidance to The UN Globally Harmonized System of Classification and Labeling of Chemicals (UN GHS) (EU, 2009; Scheel, 2011).
Topical ocular drug delivery using a conventional system such as liquid eye drops is associated with several drawbacks. The following specific points are of paramount importance when considering the use of such formulations: i) a very small volume of lachrymal fluid (7-9 μL) is found on the eye surface while a dropper bottle dispenses more than this present volume of liquid. Majority of this is then flushed out upon instillation which consequently results in a loss of contained drug; ii) instillation of the liquid, due to foreign body sensation, may trigger a flow of tears which forces more of the liquid to be lost and iii) content of substances for preservation of the solution which may not be well tolerated with triggering of blinking and further loss of drug.[1-3] Furthermore, these dosage forms are convenient and widely available, but have poor bioavailability due to dilution in lachrymal fluid upon eye drop instillation and subsequent removal by the naso-lacrimal drainage system.[4] Thus, continual administration of liquid drops is needed in order to achieve therapeutic effects. This can result in poor patient compliance and in turn hinders the overall treatment process.
In order to improve the bioavailability of topical ocular drugs, several other systems have been proposed and investigated. These include formulations with an increased viscosity and thus increased corneal contact time such as inserts and viscous polymers.[5,6] However, these systems may suffer from the disadvantage of transient blurring of vision and foreign body sensation, which in turn may negatively affect patients willingness to use such systems.
Drug delivery systems research is making strides as investigators gain better insight to the factors that affect their mechanism of action. Rapid disintegrating systems (also called ‘fast-melt’ or ‘fast-dissolving’) as novel replacements for conventional solid tablets have been investigated. These systems are solid dosage forms which disintegrate rapidly, usually in the time frame of minutes or seconds. Most commonly applied for oral use, these systems quickly dissolve once exposed to salivary fluid and this minimizes the need for external water or fluid medium.[7-9] To help improve the administration, compliance and bioavailability of topically administered drugs the concept of fast disintegrating systems can be implemented to ocular drug delivery systems.
Acquiring in vivo data from any pharmaceutical dosage form in the animal model is essential to corroborate the feasibility of the novel system in human patients. Prior to that, it is important to ensure that the dosage form is rendered sterile before proceeding to in vivo analysis. This is also a requirement of Good Manufacturing Practice (GMP) for pharmaceuticals. Of note in the selection of a method is that the procedure should ideally be validated and not impinge on the quality and properties of the product intended for sterilization. For purposes of this further testing, gamma (γ)-irradiation was employed. Commonly, this method has been used for ocular systems such as mini-tablets, implants and gels (Petrich and Rosen 1995, Weyenberg et al., 2006; Choonara, 2010). The purpose of selecting this method was due to the apparent advantages such as: safety, simplicity, controlled conditions and importantly, no residual activity after the process. The use of this method is recognized by the American National Standards Institute (ANSI), the American Association of Medical Instrumentation (AAMI) and the International Standards Organization (ISO) (Martin, 2012).
In terms of data analysis, pharmacokinetics studies the relationship of the active pharmaceutical ingredient (API) or drug in the body. It allows for a mathematical means to interpret data obtained from in vivo analysis. However, from an ocular perspective, this is the study of absorption, distribution and elimination of drug after topical instillation on the eye surface (Mayers et al., 1991). In vitro in vivo correlation (IVIVC) is a concept can had been described by the Food and Drug administration FDA as model that shows the relationship between the in vitro drug dissolution and in vivo absorption results. In vivo absorption can be determined by means of model independent Wagner Nelson method (one compartment model) or Loo-Riegelmin (multi-compartment model). There are mainly 4 levels of IVIVC i.e. A, B, C and multiple C. These levels are based on the ability to reflect the complete plasma time profile of which Level A provides a point-point to point relationship and is often preferred. IVIVC serves to support a dissolution method and assists in quality control during manufacturing (Leeson, 1995). To determine the pK parameters complicated calculations have to be carried out, however software packages can be used for time effective results. Some programs such as WinNonlin may be expensive and have a steep learning curve, thus alternatives are required. One such example is the Microsoft Excel (MSE) 2003 add in PKSolver. This is a freely available add-in programme that has demonstrated to be simple to install and operate, compatible with MSE 2007 and 2010, has various calculation options and most importantly values for parameters generated have been compared to that of WinNonlin with acceptable results (Zhang, 2010).
Therefore, the aim of this further testing was to evaluate the OISED in New Zealand Albino rabbit eye. A novel UPLC method for the detection of timolol maleate (TM) in aqueous humour was developed and the pharmacokinetic parameters of in vivo release were determined in comparison to a marketed eye drop preparation in the New Zealand Albino rabbit eye. Finally, the compatibility of the OISED with the ocular tissue via histology was assessed.
Hydroxypropylcellulose (HPC) (Mw=80 000 g/mol) (Klucel®, Hercules Incorporated, Wilingtion, Del., USA), glycyl-glycine (diglycine, DG) (Mw=132.12 g/mol) (Fluka BioChemika, Belgium), poly(acylic acid sodium salt) (PAA-Na salt) (Mw=5100 g/mol), Maltodextrin (MD) (dextrose equivalent 16.5-19.5), Pluronic® F-68 (PF68) (Mw=8400 g/mol), Sodium Dodecyl Sulphate (SDS) (Mw=288.38 g/mol) and Timolol maleate salt (TM) (an example of an active pharmaceutical ingredient (API) or drug) (Mw=432.49 g/mol) were all purchased from Sigma-Aldrich (St. Louis, Mo., USA), diclofenac sodium (another example of an API or drug) (Mw=318.13 g/mol) were purchased from Sigma Aldrich, ammonium acetate (Mw=77.09 g/mol, Saarchem, Muldersdrift, South Africa), glacial acetic acid (Associate Chemical Enterprises, Southdale, Johannesburg), acetonitrile 200, perchloric acid and methanol (UPLC grade, ROMIL™, Johannesburg), Glaucosan® 0.5% eye drops (Hexal Pharma (SA)(PTY)(LTD) and double deionized water was obtained by use of a Millipore filter (Millipore water purification system, Millipore, Molsheim, France).
A two-factor, three-level Face Centred Central Composite Design (FCCCD) as shown in Example 1 (Table 1) was applied for the construction of a second order polynomial model describing the effect of formulation constituents on the characteristics of the system. ISEDs of various combinations were prepared in accordance with the FCCCD. In order to determine the optimal formulation, several responses were tested: Textural characteristics, disintegration testing and in vitro drug release. Constraint optimization predicted an optimized formulation of MD and PF68 concentrations with sufficient strength and rapid disintegration as carried out in previous studies using Minitab®.
Aqueous solutions of polymer were prepared in accordance with the optimized formulation components obtained using: HPC 1% w/v, PAANa 0.25% w/v, DG 0.25% w/v, MD 5% w/v, and PF68 2.94% w/v. (The optimized formulation comprised 0.5% timolol maleate as the API). Components were dissolved in 100 mL deionized water and agitated for 30 minutes until complete dissolution had occurred. Samples of 150 μL were injected into each mould of the polyvinyl chloride (PVC) blister packs. Samples were then frozen (Sanyo Ultralow Temperature freezer, MDF-U73V, Sanyo Electric company, Japan) for 24 hours at −82° C. to solidify the product. The product was placed in a lyophilizer (Labconco Freeze-Dry Systems, Labconco Corp., Kansas City, Mo., USA) for 48 hours to extract excess water. Lyophilized drug-loaded OISED samples were stored in glass vials in the presence of 2 g desiccant sachets.
The method above was utilized to prepare a drug-loaded (DL) OISED. When manufacturing a drug-free (DF) OISED the API or drug is omitted from the remaining components which are dissolved in the deionized water prior to freezing.
All analytical experiments were conducted on the drug-loaded (DL) OISED wherein TM is the API.
Fourier transform infrared (FTIR) spectroscopy is used for the detection of interactions between native polymers and blends as well as to detect whether a specific functionality is present. FTIR was carried out to detect vibration characteristics of chemical functional groups in response to infrared light interactions. A Perkin Elmer Spectrum 2000 FTIR spectrometer with a MIRTGS detector, (PerkinElmer Spectrum 100, Llantrisant, Wales, UK) was used. Samples were be prepared as pellets against a blank ZnCn pellet background at a wave number ranging from 4000-650 cm−1 and a resolution of 4. The spectrum software (Spectrum 100) was used for interpretation of the results.
Thermal analysis was carried out to determine the thermal properties of the formulations as per the FCCD shown in Table 1. A Temperature Modulated Differential Scanning Calorimeter (TMDSC) (Mettler Toledo, DSC1, STAReSystem, Swchwerzenback, ZH, Switzerland) equipped with software for computation evaluation of numerical values was used. The glass transition temperature (Tg), melting temperature (Tm) and temperature of crystallization (Tc) were determined. Temperature calibration was attained by a melting transition of 6.7 mg indium. Samples (10 mg) were weighed out on aluminium crucibles, sealed and tested within a temperature gradient of 0-300° C. under constant N2 purge to reduce the oxidation rate of 1° C./min.
Supplementary thermal characterization was conducted using a thermogravimetric analyser (TGA) (PerkinElmer, TG-IR 8000, Llantrisant, Wales, UK) in order to determine weight variations of samples as a function of temperature. The instrument was purged with N2 to prevent the occurrence of any undesirable reactions. Samples were placed in a pan using tweezers followed by heating between 50-550° C. at a rate of 10° C./min. Weight vs. temperature plots were generated and analysed using Pyris™ software.
The in-depth surface morphology and internal structure of the matrix was visualized. This was done by the use of scanning electron microscopy (FEI Phenom™, Hillsboro, Oreg., USA). Samples were mounted on a spud and gold plated by the sputter-coater (SPI module™ sputter-coater and control unit, West Chester, Pa. USA). Samples were then viewed by the SEM at different magnifications. In addition, the visualization of the surface of the OISED was determined by microscopy using light illumination for images on a 3-dimensional level. A stereomicroscope (Olympus SZX7 stereomicroscope, Olympus, Japan) connected to a digital camera (CC 12, Olympus, Japan) and image analysis system (AnalySIS® Soft Imaging System, GmbH, Germany) was employed. The porosity of the OISED was determined by employing a porositometer (Micromeritics ASAP 2020, GA) with the use of Brunauer-Emmett-Teller (BET) isotherm of adsorption/desorption of nitrogen. The process involved: i) degassing of samples was carried out which involves an evacuation and heating phase, parameters of which are outlined in Table 10. Samples were inserted into tubes and underwent this phase and ii) asdorbtive properties were then determined. Data analysis by means of the following were obtained: i) BET calculation which was obtained from determination of the monolayer volume of adsorbed gas from isotherm results, ii) t-plot calculation for analysis of area and total volume due to micropores in the matrix and iii) Barrett-Joiner-Halenda (BJH) for the determination of the mesopore volume/area distribution which accounts for both the change in adsorbate layer thickness and the liquid condensed in the matrix pore cores.
The methodology involved using hen's eggs (50-60 g fertile) that were purchased from a local hatchery (Bruce Hatcheries, South Africa) and less than a week old. The eggs were placed in commercial incubators (Surehatch, Brakenfell, South Africa) and rotated every 12 hours. On day 3, a hole was made and the albumin was removed. The eggs were sealed by sterile heated parafilm and left in an equatorial position. On day 5 and everyday thereafter eggs were candled for viability and non-viable ones were discarded. On day 10, a window (2×2 cm) was made and 2-3 mL saline (0.9% NaCl) was added and eggs were returned to the incubator. The following samples were prepared for testing: Standard solution: 1% sodium dodecyl sulphate (SDS), test sample: test sample drug-loaded (DL) OISED dissolved in 0.9% NaCl, Placebo sample: drug-free (DF) OISED dissolved in 0.9% NaCl, Control: 0.9% NaCl. The eggs were then dosed with 0.3 mL of samples and observed for 5 minutes for any effects of injury in accordance with Table 11 and this was noted. Saline was used as a control for comparison purposes. Eggs were scored for severity of any reaction and time taken to occur (Table 11). At the end of the assay the embryos were discarded immediately by placing the eggs into a freezer −80° C. The following features were used for observation of injury: hemorrhage, vascular lysis and coagulation.
The period taken for any untoward reaction to occur was noted and the irritation threshold was determined (highest concentration of samples required for a minimal reaction to occur). The irritation score was calculated by means of Equation 4.
Where:
IS=irritation score
Sec=onset of reaction in seconds
H=hemorrhage
L=lyses
C=coagulation
A model independent approach was used for mathematical analysis of dissolution data. According to the US FDA, the similarity and difference factors are significant to identify for this purpose. Two factors were determined with the time point set at 360 minutes. The difference factor (f1) is a measurement of percentage error of two curves at each time point. Curves are similar if f1 is close to 0. This was calculated by Equation 5.
The similarity factor (f2) is the logarithmic transformation of the sum-squared error of differences between test and reference products over all time points. Curves are considered similar when f2 is between 50 -100. Equation 6 was employed to determine f2:
Where:
n=sampling number
R and T=reference and test products at each time point j
Franz diffusion cell apparatus was used to conduct ex vivo permeation studies. Excised rabbit corneas (11.83±0.04 mm in diameter and area 3.0±0.01 mm2) were mounted on the Franz diffusion cell apparatus (Permagear, Arnie Systems, USA) connected to a heating bath system for temperature control. The diffusion cell donor chamber was filled with 2 mL Simulated tear (lacrimal) fluid (SLF) (Gonjari et al., 2009) (which was maintained to simulate tear volume). 12 mL Simulated Aqueous Humor (SAH, pH 7.4, 37° C.) (Giannola et al., 2007) solution was contained in the receptor chamber. The cornea was placed such that the surface was in contact with the receptor solution which was continuously stirred by a magnetic stirrer at 20 rpm to simulate blinking. Samples of one optimized formulation (OISED), pure drug dispersion and one drop of marketed product (0.5% timolol maleate) were tested in triplicate for comparison purposes. Aliquots of sample were withdrawn from the receptor compartment at regular intervals (30, 60, 120, 180, 240 minutes and 24 hours after insertion) and replaced with an equal volume of dissolution medium. Samples were then subjected to the quantification of drug through spectrophotometric analysis at 295 nm (Hewlett Packard 8453 Spectrophotometer, Germany) to determine the drug release and flux (rate of drug permeation per unit area). Drug flux values were calculated at the steady state per unit area by regression analysis of permeation. Drug flux values were calculated employing Equation 7.
Where:
Js=drug flux (mg cm−2 min−1)
Qr=amount of drug that passed through to the receptor compartment (mg)
A=Active cross-sectional area for diffusion (cm2) and
t=time of exposure (min)
The permeability co-efficient can be described as the velocity of drug diffusion through the cornea based on its mathematical unit using Equation 8.
Where:
Kp=permeability co-efficient (cm.min−1)
Js=Flux at steady state (mg cm−2 min−1)
Cd=drug concentration in donor compartment (mg.cm−3)
The equipment consisted of an ultra-performance liquid chromatography system (Acquity™ Ultra Performance LC, UPLC, Waters, coupled with PDA detector and ELC detector). Data acquisition and interpretation was conducted using Waters Empower software. Analysis was performed with ammonium acetate (AA): acetonitrile (ACN) (49:51 v/v) as a mobile phase which was pumped at flow rate of 0.25 ml/min in isocratic mode employing a C18column (1.7 μm; 1×100 mm) at 25° C. The wavelength was kept at 295 nm for detection of TM and 2 ul of sample was injected for analysis. The run time was set at 3 minutes. AA buffer was prepared by dissolving 7.7 g in 100 ml water as solvent A. pH was adjusted to 4.5 with glacial acetic acid in a drop-wise manner. ACN 100% was employed as solvent B. TM and internal standard (IS) were prepared and solutions were kept in amber glass vials until use. Standard solutions of drug and IS were prepared and calibration curves were constructed. A mixture in a 1:1 ratio of analyte and IS were prepared. Samples were filtered (0.22 um) and injected into 2 mL vials for analysis.
Enucleated rabbit eye balls were collected and punctured for blank aqueous humour sample aspiration (200 uL). Samples were stored in plastic eppendorf tubes at −80° C. until used for analysis. Samples were thawed out (200 uL) and spiked with standard solutions of analytes. An equal volume of IS was added and samples were vortexed for 2 minutes. An equal volume of 6% v/v perchloric acid and methanol were added for deproteination. Samples were centrifuged for 10 min at 1500 rpm (MSE minor laboratory centrifuge, Scientific instruments, West Palm Beach, Fla., USA) and the clear supernatant was removed. This was made up to a volume of 2 mL with mobile phase. Samples were prepared and analyzed as explained above and a calibration curve was constructed (
Ethics clearance was obtained from the Animal Ethics Screening Committee (AESC) of the University of the Witwatersrand. All animals and biological tissue were handled according to Standard Operating Procedures (SOP) of the Central Animal Services (CAS). Furthermore, guidelines of Association for Research in Vision and Ophthalmology (ARVO) Resolution on the Use of Animals in Ophthalmic Research and Vision Research (Rockville, Md., USA) were followed. Cage activity by means of observation for 1 hour periods daily were used to assess state of well-being. Assessment of wellbeing was determined with the use of a score sheet. Score sheets can be used for routine monitoring and thus contribute to the welfare of the animals. Samples were prepared and packaged into labeled glass poly-tops sealed with a plastic lid. Samples were transported to Isotron (Pty) Ltd. (Isando, South Africa) and irradiated at a dose of 25 KGy (sufficient for sterilization without biological validation according to the European Guideline, 1992). A total of 66 New Zealand Albino rabbits of weight approximately 2.25 kg were used. An initial pilot study was conducted on (6 rabbits). The remaining 60 rabbits were randomly assigned to 2 groups with 30 rabbits each for group 1 and 2. Rabbits were divided into the respective groups (test/placebo group 1 and comparison group 2). A pilot study was conducted on 6 rabbits (1 rabbit for each sampling point) in order to collect samples after OISED insertion. The following groups were assigned: Pilot study (6 rabbits): To determine ease of administration at the defined site and presence of any untoward reactions in the rabbit eye following visual assessment after insertion. Test and Placebo group 1 (30 rabbits): Rabbits in this group received a drug-loaded OISED containing in the cul-de-sac of the left eye. Drug-free OISEDs were inserted into the right eye for the placebo effect. Comparison group 2 (30 rabbits): Rabbits in this group received eye drops of a commercial product (Glaucosan®, 0.5%) in the cul-de-sac of the left eye. Rabbits were anaesthetized with an intramuscular combination of ketamine hydrochloride (40 mg/kg) and xylazine (10 mg/kg) for ocular delivery of the drug-loaded OISED in the left cul-de-sac at time 0. Placebo OISED was inserted into the opposing eye (right). Rabbit eyes were assessed using thorough observation on a macroscopic level. This involved observing any possible effects of the OISED on the eye with reference to Table 13. Evaluation of irritation was conducted according to a scoring system of 0 (absence) to 3 (highest). The untreated eye served as a control. Overall irritation was calculated by addition of the total clinical evaluation scores (A, B, C) (Mishra and Gilholtra, 2008). Samples were withdrawn at 30, 60, 120, 240, 360 minutes and 24 hours after insertion. At each sampling point each animal was euthanised with an overdose of IV sodium pentobarbitone (>50 mg/kg, ˜2 mL) with consequent enucleation (surgical removal of the eyeball). The aqueous humour approximately 200 ul) was aspirated (paracentesis/tapping) employing an insulin syringe fitted with a 26 gauge needle inserted parallel to the iris (Lee and Robinson, 1979). The aqueous humor samples was frozen immediately and stored at —80° C.
A one-compartmental pharmacokinetic model (PK) was used for the assessment of ocular results (Mishma, 1981; Lee et al., 1991). This proposes that the drug distributes to a central compartment (aqueous humor) initially followed by a peripheral compartment. PKSolver (Microsoft Excel add-ins) was used for analysis.
The following pertinent pK parameters were determined:
1. Apparent absorption rate constant (ka)
2. Apparent elimination rate constant (ke)
3. Peak TM concentration (Cmax)
4. Area under the concentration vs. time curve (AUC)
5. Area under the zero and first moment curves from the last sampling time to infinity (AUCt-∞)
6. Area under the moment curve (AUMC)
7. Peak time (tmax)
8. Mean residence time (MRT)
The following diagnostic criteria were used to determine suitability of the selected model:
1. Co-efficient (R)
2. Weighted sum squares of residuals (SS)
3. Standard error of residuals (SE)
4. Akaike's information criteria (AIC)
5. Schwarz criteria (SC)
A mathematical model for the relationship between an in vitro property and in vivo response was developed using R-console (version 2.15.3, R foundation for statistical computing) with ivivc package (0.1.6). Input data was the drug release data from the optimized OISED as well as in vivo pK data obtained from UPLC analysis. Level A correlation was selected for IVIVC.
Each eye was cut in a sagittal section and each select block of the eye was placed in a histological cassette and processed in an automated tissue processor according to procedures. Following automated tissue processing, wax blocks were prepared and sections of 6 μm were cut on a microtome. The prepared sections were stained in an automated Haematoxylin and Eosin stainer. After staining, the specimen was mounted and the slides examined.
The optimized OISED was prepared as outlined previously. Drug loaded (DL) and drug-free (DF) OISEDs were prepared.
FTIR spectroscopy is employed for the detection of interaction of polymers or drugs as well as observation of important groups present in constituents. The vibrational bands highlight changes on a molecular level. FTIR spectra and corresponding structures of the pure drug TM, individual maltodextrin (MD) and Pluronic-F68 (PF68), a physical mixture (PM shown on the
Thermal analysis of OISEDs is of key importance in order to gather the significant characteristics of the sample. Specifically, the ‘collapse temperature’ which is the temperature over-which the sample displays loss of its structure and collapses during the lyophilization process (Pikal, 1990). This is critical in terms of manufacturing and storage. Testing provides information regarding the possibility of drug-polymer interactions. Analysis was conducted via DSC and thermal curves of the pure drug TM and a drug-loaded final formulation were investigated. In terms of the pure drug a single defined endothermic peak was observed at 203.2° C. confirming its crystalline nature (
TGA was utilized in order to assist in interpretation of thermal properties of the samples using heat and stoichiometric ratios in determination of the percent by mass of solute. Pure TM displayed a definate weight loss in the region comprising an extrapolated onset of 200° C. and an endset of 350° C. which can be explained as the decomposition temperature (
Porosity can be described as the measurement of the spaces within a sample network. With regards to lyophilized matrices, the resultant product is often porous due to the sublimation of water post-freezing. This can be quantified in terms of porosity analysis and visualization by SEM. The basic principle behind this method is the adsorption of nitrogen gas on a material's surface or on the pores, if present. A degassing linear isotherm and the Brunauer-Emmet-Teller (BET) surface area plots are depicted in
Corresponding SEM and stereomicroscope images for qualitative analysis enumerated the findings of porosity analysis and allowed for visualization of the surface and pore distribution. Pores were demarcated, interconnecting and circular to assymetrical over the matrix surface. BET plots indicated that a positive value was obtained (27.2052 m2/g, R2=0.99). Explanation of the BET concept is of the assumption that there is a uniform surface exposure and that nitrogen is more attracted to the surface then other nitrogen molecules. BET-C ranges between 5-100. Values <5 implied that the gas-gas affinity was competing with the gas-solid affinity as a result of the significantly reduced surface area and minimization of pores (Everett, 1972; Siminiceanu et al., 2008). A value of 4.785274 was obtained for the formulation tested. A method for determination of pore volume or surface area as a function of pore diameter was proposed by Barret, Joyner and Halender (Barret et al., 1951) BJH adsorption plots, BJH Desorption dV/dlog(D) Pore volume plots with implementation of the Halsey-Faas correction and are seen in
The purpose of employing this test was due to the CAM being tissue that is vascularized (arteries, veins and capillary plexus). The occurrence of any injury with inflammation due to a test substance is an indication of the damage that can occur and be compared to that of the eye surface in vivo and served as the rationale for this test (Spielman, 1991). The HET-CAM test acts as an alternative to the use of mammalian tissue and application of the highly controversial and harmful Draize-irritancy test performed on live animals. In addition, the HET-CAM test is rapid and cost-effective for the evaluation of novel drug-delivery systems. Testing was carried out according to Globally Harmonized System of classification and labeling of chemicals (GHS) guidelines for testing of chemicals (UN, 2009). Results indicated that after exposing the CAM to samples (300 uL) for 5 minutes, drug-free (DF) and drug-loaded (DL) samples of OISEDs were practically non-irritant and therefore well-tolerated with a mean score of 0. This can be attributed to the constituents of the formulation. They are ocular-compatible, biodegradable polymers (HPC and PF68) showed no adverse effects. Furthermore, the drug itself TM, is a standard treatment for glaucoma and its presence displayed no untoward reaction to the CAM. Saline, employed as a control also displayed a score of 0 implying no irritation potential as expected. SDS used as a positive control, showed a score of 2.5 inferring considerable toxicity with disruption of the CAM. It is an organic compound and non-ionic surfactant known to cause cell lysis and is often used for comparison purposes in studies. Equation 1 was used to determine the IS of formulations (0-21). An IS of 0 for NaCl, drug-free (DF) and drug-loaded (DL) formulations were obtained while in contrast, 10.4 for SDS. Notably, NaCl and optimized formulations were virtually non-irritant even at higher concentrations while SLS displayed toxicity at low concentrations. Table 13 outlines results obtained. The CAM remains unaffected when tested with the optimized formulation while disruption is clear with SDS. Thus, this study revealed that both drug-loaded and drug-free solid eye drops were non-irritant to the membrane and can be considered safe for use on the eye surface. It is a pre-requisite for ocular systems to have no adverse reaction on the eye, thus the selected polymers and excipients were suitable for the intended application.
Both drug-free (DF) and drug-loaded (DL) OISED formulations, since immediate release dosage forms, displayed initial burst release. However, the OISEDs displayed a superior result compared to the commercially available eye drops. The level of similarity was evaluated by means of the similarity and difference factors. A f1 value of 13.69 and a f2 of 61.61 were obtained. A f1 of 0-15 indicates that there are minor differences between the samples tested and f2 between 50-100 indicates the sameness of the samples. This can be used as a surrogate before conducting in vivo studies.
Permeation capabilities of the optimized formulation compared to the pure drug dispersion of TM and marketed eye drops were tested across excised rabbit cornea samples. Results had a positive outcome indicating that an improved steady state flux was noted for the optimized OISED. (0.00052 mg.cm−2.min−1) compared to pure drug 2 (0.000378 mg.cm−2.min−1) and eye drops 3 (0.00039 mg.cm−2.min−1) (
During in vivo studies, observations were made to determine the effect of the OISED on the ocular surface following administration. No negative effects were noticed and this served to confirm the results from the HET-CAM test. Table 15 provides the results obtained from scoring.
Results, as confirmed in previous studies, indicated that peak TM concentrations were achieved around 60-100 minutes in the aqueous humour (Phillips et al., 1985). For the OISED a peak concentration was reached at around 104.9 minutes. In the case of the eye drops a lower Cmax was obtained (1.97 ug/mL). As expected, the optimized OISED displayed a better drug release profile compared to the eye drops (Table 16). Diagnostic criteria for ‘goodness of fit’ of the selected model revealed that low AIC and SC values showed a good model fit. Reasons for the improved OISED behaviour can be attributed to the following: the eye surface contains hydrophilic glycoproteins termed ocular mucins. As explained by Mantelli and Argueso (2008), these mucins have the function of stabilizing the tear layer to postpone the break-up of this layer. Mucins are found on the cornea as well as the cul-de-sac and polymers have the tendency to bind non-covalently to these mucins. This allows for localization of drug to a specific area and reduces the possibility of drainage as in the case of the drug within a liquid vehicle. HPC is a surface-active polymer that assisted in alteration of the elimination of the drug instilled by means of the OISED. Similarly, the inclusion of PF68 allowed for pre-corneal interaction when exposed to the increased eye temperature (Wahg et al., 2008).
Histological analysis was performed to assess the safety of the OISED in the eye surface. The corneal topographical findings from the drug-loaded (DL) and placebo (DF) specimens of the OISEDs from the eyes of the rabbits were recorded. The presence of heterophils as well as the heterophil exocytosis into the conjunctival epithelium appeared minimal. Heterophils are usually indicative of sub-acute or chronic inflammation (Gervais et al., 2011). Absence of defects or inflammation was noticed. Based on the findings, there was minimal pathology or any marked changes and irritation due to the insertion of a drug-loaded device as well as from the placebo administration.
This study aimed to provide an extensive pharmaceutical analysis of an optimized instantly soluble solid eye drop device (OISED). Molecular transition studies and thermal analysis revealed that no incompatibility between drug, polymers and excipients existed. Porosity and morphological examination confirmed the porous-nature of the formulation. First-order release was determined to be the predominant release mechanism as indicated by kinetic analysis. Ocular toxicity studies via the HET-CAM test proved to be efficient for evaluation of samples and determined that the device was non-noxious and thus considered safe for topical ophthalmic application. Comparison with a pure drug dispersion and marketed eye drops for trans-corneal permeation revealed that the optimized formulation had an improved drug flux and permeability co-efficient attributed to the rapid disintegration and presence of hydrophilic adhesive polymers. The application of UPLC for drug detection allowed for an advantageous development of a novel and sensitive method. In vivo assessment indicated that the ISED had improved drug levels in the aqueous humor in comparison to the eye drops. The inclusion of polymers in the OISED allowed for improved corneal adherence and absence of drug drainage as in the case of liquid eye drops. Level A IVIVC correlation indicated an R2 value of 0.84. Histological assessment revealed the safety of the device as seen from observational studies. Thus, results from this study showed that the ISED was concluded to display advantageous behavior and considered safe for the eye surface. The applicant found that the specific combination of a polyethylene oxide block copolymer and HPC, preferably Pluronic F68 and HPC, produced a solid dosage form having a inter-connecting network of pores. These pores facilitated in providing the dosage forms rapid disintegration characteristics in use, and contributed to its rigidity prior to use. The specific combination of components comprising the OISED provided for a non-irritant solid ocular pharmaceutical dosage form in use.
Ethics clearance was obtained from the Animal Ethics Screening Committee (AESC) of the University of the Witwatersrand. Ethics clearance number: Dec. 5, 2012.
While the invention has been described in detail with respect to specific embodiments and/or examples thereof, it will be appreciated that those skilled in the art, upon attaining an understanding of the foregoing may readily conceive of alterations to, variations of and equivalents to these embodiments. Accordingly, the scope of the present invention should be assessed as that of the claims and any equivalents thereto, which claims appended hereto.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 2012/06792 | Sep 2012 | ZA | national |
| 2012/06803 | Sep 2012 | ZA | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2013/058453 | 9/11/2013 | WO | 00 |
