U.S. patent application number 14/427195 was filed with the patent office on 2015-07-30 for fast dissolving ocular insert.
This patent application is currently assigned to UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG. The applicant listed for this patent is UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG. Invention is credited to Yahya Essop Choonara, Lisa Claire du Toit, Rafeeq Jhetam, Pradeep Kumar, Raeesa M. Moosa, Viness Pillay.
Application Number | 20150209275 14/427195 |
Document ID | / |
Family ID | 49585457 |
Filed Date | 2015-07-30 |
United States Patent
Application |
20150209275 |
Kind Code |
A1 |
Choonara; Yahya Essop ; et
al. |
July 30, 2015 |
Fast Dissolving Ocular Insert
Abstract
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.
Inventors: |
Choonara; Yahya Essop;
(Johannesburg, ZA) ; du Toit; Lisa Claire;
(Roodepoort, ZA) ; Kumar; Pradeep; (Johannesburg,
ZA) ; Pillay; Viness; (Sandton, ZA) ; Moosa;
Raeesa M.; (Johannesburg, ZA) ; Jhetam; Rafeeq;
(Johannesburg, ZA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG |
Johannesburg |
|
ZA |
|
|
Assignee: |
UNIVERSITY OF THE WITWATERSRAND,
JOHANNESBURG
Johannesburg
ZA
|
Family ID: |
49585457 |
Appl. No.: |
14/427195 |
Filed: |
September 11, 2013 |
PCT Filed: |
September 11, 2013 |
PCT NO: |
PCT/IB2013/058453 |
371 Date: |
March 10, 2015 |
Current U.S.
Class: |
424/428 ; 264/28;
514/236.8; 514/567; 514/772.1; 514/773; 514/778; 514/781 |
Current CPC
Class: |
A61K 31/196 20130101;
A61K 31/5377 20130101; A61K 9/2059 20130101; A61K 9/0051 20130101;
A61K 9/2077 20130101; A61K 31/138 20130101; A61K 9/2031 20130101;
A61K 9/2072 20130101; A61K 31/4196 20130101; C07H 15/02 20130101;
A61K 9/2013 20130101; A61K 9/2054 20130101; A61K 45/06 20130101;
A61K 9/2095 20130101; A61K 9/2027 20130101 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 31/5377 20060101 A61K031/5377; A61K 31/196
20060101 A61K031/196; A61K 9/20 20060101 A61K009/20 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 11, 2012 |
ZA |
2012/06792 |
Sep 11, 2012 |
ZA |
2012/06803 |
Claims
1-15. (canceled)
16. 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, hydroxpropyl cellulose (HPC), and an anti-collapsing
agent.
17. The pharmaceutical dosage form according to claim 16, wherein
the polyethylene oxide block copolymer is a
polyoxyethylene-polyoxypropylene block copolymer, preferably a
pluronic polymer, further preferably Pluronic F-68 (PF-68).
18. The pharmaceutical dosage form according to claim 16, wherein
the anti-collapsing agent is an amino acid chain.
19. The pharmaceutical dosage form according to claim 18, wherein
the amino acid chain has 1 amino acid residue.
20. The pharmaceutical dosage form according to claim 18, wherein
the amino acid chain has 2 amino acid residues.
21. The pharmaceutical dosage form according to claim 18, wherein
the anti-collapsing agent is diglycine.
22. The pharmaceutical dosage form according to claim 16, further
comprising a lyoprotectant.
23. The pharmaceutical dosage form according to claim 22, wherein
the lyoprotectant is maltodextrin.
24. The pharmaceutical dosage form according to claim 16, further
comprising a superabsorbent polymer.
25. The pharmaceutical dosage form according to claim 24, wherein
the superabsorbent is polyacrylic acid sodium salt (PAA-Na
salt).
26. The pharmaceutical dosage form according to claim 16, further
comprising at least one active pharmaceutical ingredient (API)
homogenously dispersed therein selected from the group:
prostaglandin analogs, beta blockers, alpha agonists and carbonic
anhydrous inhibitors, or combinations of thereof.
27. 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 Pluronic F-68 and hydroxpropyl
cellulose (HPC); an anti-collapsing agent in the form of diglycine;
a lyoprotectant in the form of maltodextrin; and a superabsorbent
polymer in the form of polyacrylic acid sodium salt (PAA-Na
salt).
28. The pharmaceutical dosage form according to claim 27, further
comprising an active pharmaceutical ingredient (API).
29. The pharmaceutical dosage form according to claim 16, wherein
the pharmaceutical dosage form is 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.
30. The pharmaceutical dosage form according to claim 29, wherein
the solid ocular pharmaceutical dosage form is formed as a
tablet.
31. The pharmaceutical dosage form according to claim 30, wherein
the tablet is a mini-tablet having circular and/or discoid shaped
dimensions wherein the thickness is 2 mm and the diameter is 3
mm.
32. A method of manufacturing a pharmaceutical dosage form
comprising the steps of: (a). dissolving polyethylene oxide block
copolymer and hydroxpropyl cellulose (HPC) in a liquid medium,
preferably deionized water to produce Solution 1; (b). adding to
Solution 1 a lyoprotectant, preferably maltodextrin; a
superabsorbent polymer, preferably polyacrylic acid sodium salt
(PAA-Na salt); and an anti-collapsing agent, preferably diglycine,
to produce Solution 2; (c). freezing Solution 2; and (d).
lyophilizing the frozen Solution 2 to form the solid ocular
pharmaceutical dosage form.
33. The method according to claim 32, wherein Step (b) further
comprises adding an active pharmaceutical ingredient (API) to
Solution 1.
34. The method according to claim 32, wherein Step (c) takes place
for 24 hours at -82.degree. C.
35. The method according to claim 32, wherein Step (d) takes place
at -42.degree. C. for between 24 to 48 hours.
36. The method according to claim 32, wherein Step (c) takes place
in polyvinyl chloride (PVA) blister packs of predetermined size in
order to produce dosage forms having circular and/or discoid
dimensions of about 2 mm in thickness and 3 mm in diameter.
Description
FIELD OF INVENTION
[0001] 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.
BACKGROUND TO THE INVENTION
[0002] 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.
[0003] 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.
[0004] 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 (Jarvinen and
Jarvinen 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).
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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 (Suverkrup 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).
[0011] 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.
[0012] However, there exists a need for novel, easy to manufacture,
rapid pharmaceutically active ingredient (API) releasing solid
pharmaceutical dosage forms for topical ocular use.
Definitions
[0013] The following terms used through the course of this patent
specification shall have the following meanings: [0014] Instantly
soluble--implies rapid ingress of fluid into a dosage form and
immediate onset of dissolution and/or disintegration of the dosage
form; [0015] Biocompatible--not displaying toxic effects on
biological tissue; and [0016] Biodegradable--capable of being
decomposed through biological processes.
SUMMARY OF THE INVENTION
[0017] 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.
[0018] 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).
[0019] 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).
[0020] 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.
[0021] In a preferred embodiment of the invention, the
anti-collapsing agent comprises an amino acid chain having two
amino acid residues, preferably diglycine.
[0022] The pharmaceutical dosage form may further comprise a
lyoprotectant, preferably maltodextrin.
[0023] The pharmaceutical dosage form may further comprise a
superabsorbent polymer, preferably polyacrylic acid sodium salt
(PAA-Na salt).
[0024] 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.
[0025] In a preferred embodiment of the pharmaceutical dosage form,
the dosage form comprises: [0026] a homogenous polymeric matrix
consisting of a Pluronic F-68 and hydroxpropyl cellulose (HPC);
[0027] an anti-collapsing agent, preferably diglycine; [0028] a
lyoprotectant, preferably maltodextrin; [0029] a superabsorbent
polymer, preferably polyacrylic acid sodium salt (PAA-Na salt); and
[0030] an active pharmaceutical ingredient (API).
[0031] 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.
[0032] 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.
[0033] 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: [0034] (a). dissolving polyethylene oxide block copolymer and
hydroxpropyl cellulose (HPC) in a liquid medium, preferably
deionized water to produce Solution 1; [0035] (b). adding to
Solution 1 a lyoprotectant, preferably maltodextrin; a
superabsorbent polymer, preferably polyacrylic acid sodium salt
(PAA-Na salt); and an anti-collapsing agent, preferably diglycine,
to produce Solution 2; [0036] (c). freezing Solution 2; and [0037]
(d). lyophilizing the frozen Solution 2 to form the solid ocular
pharmaceutical dosage form.
[0038] Step (b) may further comprise adding an active
pharmaceutical ingredient (API) to Solution 1.
[0039] Step (c) may typically take place for about 24 hours at
about -82.degree. C. and Step (d) may take place at about
-42.degree. C. for about 24 to about 48 hours.
[0040] 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.
[0041] 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.
DESCRIPTION OF THE DRAWINGS
[0042] The invention is now described by way of example only, with
reference to the accompanying diagrammatic drawings, in which
[0043] FIG. 1 shows a) a flow diagram showing simplified
methodology employed for the fabrication of the pharmaceutical
dosage form, b) a schematic of the pharmaceutical dosage form, and
c) a digital image of a typical pharmaceutical dosage form in
accordance with this invention;
[0044] FIG. 2 shows a typical a) force-time profile for matrix
resilience calculation, b) force-distance profile for the
determination of matrix tolerance, yield value and energy of
absorption and c) force-distance profile for computation of Brinell
Hardness Number (BHN);
[0045] FIG. 3 a calibration curve of the drug or API, Timolol (TM)
in simulated aqueous humour (SAH) (pH7.4, 35.degree. C.) at 295 nm
N=3 in all cases (SD.ltoreq.0.0543);
[0046] FIG. 4 shows a typical distance-time graph for
disintegration profiling;
[0047] FIG. 5 shows scanning electron microscopy (SEM) images
depicting the porous lyophilized matrix surface of the
pharmaceutical dosage form according to the invention
(magnification 615.times.);
[0048] FIG. 6 shows stereomicroscopy images for visualization of
the rapid dissolution process (magnification 10.times.) of the
pharmaceutical dosage form according to the invention;
[0049] FIG. 7 shows a bar graph depicting drug entrapment
efficiency (DEE) of design formulations for the pharmaceutical
dosage form according to the invention;
[0050] FIG. 8 shows graphs indicating release of active
pharmaceutical ingredient (API) of a) formulations 1-5, b)
formulations 6-10 and c) formulations 11-13;
[0051] FIG. 9 shows digital images of: a) incubation process, b)
eggs placed in an equatorial position after albumin removal, c)
observation of embryo formation on day 3 and d) preparation of egg
for sample testing;
[0052] FIG. 10 shows digital images outlining the step-wise in vivo
studies: a) left eye of New Zealand Albino rabbit, b) insertion of
the OISED into the cul-de-sac, c) aqueous humour aspiration, d)
removal of surrounding tissue, e) enucleation of the eyeball, f)
complete removal of the eye;
[0053] FIG. 11 shows a digital image of the OISED in comparison to
a US one Dime coin;
[0054] FIG. 12 shows FTIR spectra of components of the OISED;
[0055] FIG. 13 shows DSC thermograms of a) pure drug timolol (TM)
maleate, b) native PF68, c) native maltodextin (MD), d) physical
mixture, e) DL final OISED formulation f) ADSC graph drug-loaded
OISED formulation, g) TGA graph of pure TM, h) DF OISED formulation
and i) DL OISED formulation (N=3 in all cases);
[0056] FIG. 14 Porosity analysis plots of drug-loaded optimized
OISED formulation: a) linear isotherm, aa) with corresponding SEM
images of optimized OISED formulation, b) BET surface area of
optimized OISED formulation, c) BJH adsorption cumulative pore
volumes of optimized OISED formulation, d) BJH desorption
cumulative dA/dD pore areas of optimized OISED formulation, e)
typical t-plot generated for optimized OISED formulation (N=3 in
all cases);
[0057] FIG. 15 shows a graphical representation of drug/API flux of
the optimized OISED formulation and TM drug dispersion (error bars
indicate standard deviation);
[0058] FIG. 16 shows a 2D chromatograph of TM and DS;
[0059] FIG. 17 shows graphs depicting a) in vitro OISED drug
release results and b) in vivo OISED and commercial eye drop
concentrations;
[0060] FIG. 18 shows graphs depicting: a) residual plots, b) in
vitro release, c) in vivo absorption and d) point-to-point Level
AIVIVC model plot; and
[0061] FIG. 19 shows digital images depicting corneal layer with a)
DL OISED at 30 minutes and b) with DF OISED at 30 minutes, c) DL
OISED at 240 minutes and d) DF OISED at 240 minutes.
DETAILED DESCRIPTION OF THE DRAWINGS
[0062] 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.
[0063] 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.
[0064] The solid ocular pharmaceutical dosage form comprises at
least one active pharmaceutical ingredient (API) homogenously
dispersed therein.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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: [0071] (a). dissolving polyethylene oxide block copolymer and
hydroxpropyl cellulose (HPC) in a liquid medium, preferably
deionized water to produce Solution 1; [0072] (b). adding to
Solution 1 a lyoprotectant, preferably maltodextrin; a
superabsorbent polymer, preferably polyacrylic acid sodium salt
(PAA-Na salt); and an anti-collapsing agent, preferably diglycine,
to produce Solution 2; [0073] (c). freezing Solution 2; and [0074]
(d). lyophilizing the frozen Solution 2 to form the solid ocular
pharmaceutical dosage form.
[0075] 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.degree. C. Lyophilization may
take place at about -42.degree. C. for about 24 to about 48
hours.
[0076] 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.
[0077] 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.
[0078] 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).
EXAMPLE 1
Manufacturing & Analyses
Materials
[0079] Hydroxypropylcellulose (HPC) (M.sub.w=80 000 g/mol)
(Klucel.RTM., Hercules Incorporated, Willington, Del., USA),
glycyl-glycine (diglycine, DG) (M.sub.w=132.12 g/mol) (Fluka
BioChemika, Belgium), Interfix 1001 Polycaprolactone CAPA.RTM. 6400
(Interfix CC, Johannesburg, South Africa), poly(acylic acid sodium
salt) (PAA-Na salt) (M.sub.w=5100 g/mol), Maltodextrin (MD)
(dextrose equivalent 4.0-7.0) and Pluronic.RTM. F-68 (M.sub.w=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) (M.sub.w=432.49 g/mol)
purchased from Sigma-Aldrich (St. Louis, Mo., USA).
Development of an Experimental Design
[0080] 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).
TABLE-US-00001 TABLE 1 Thirteen formulations of pharmaceutical
dosage forms according to the invention generated from the
Face-Centered Central Composite Design with variations of MD and
PF68. Each Formulation contained 5 mg (0.5% before lyophilization)
of the active pharmaceutical ingredient (API) in the form of
Timolol maleate salt (TM) in order to produce a drug-loaded (DL)
dosage form. All % w/v is prior to lyophilization. PF68 (% w/v)
(Pluronic Maltodextrin HPC DG (% w/v) PAA-Na API (TM) Formulation
F68) (MD) (% w/v) (% w/v) (diglycine) (% w/v) (% w/v) 1 1 5 0.5
0.25 0.25 0.5 2 5 3 0.5 0.25 0.25 0.5 3 3 5 0.5 0.25 0.25 0.5 4 1 3
0.5 0.25 0.25 0.5 5 5 1 0.5 0.25 0.25 0.5 6 3 3 0.5 0.25 0.25 0.5 7
3 3 0.5 0.25 0.25 0.5 8 3 3 0.5 0.25 0.25 0.5 9 5 5 0.5 0.25 0.25
0.5 10 3 3 0.5 0.25 0.25 0.5 11 1 1 0.5 0.25 0.25 0.5 12 3 3 0.5
0.25 0.25 0.5 13 3 1 0.5 0.25 0.25 0.5
Preparation of Polymeric Solutions and Synthesis of Pharmaceutical
Dosage Forms According to FCCCD
[0081] 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
.mu.L 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.degree. 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. FIG. 1
depicts the methodology and schematic of the proposed
formulation.
[0082] 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.
[0083] 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.
Determination of the Physicomechanical Properties of the
Pharmaceutical Dosage Forms According to the Invention
[0084] 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.degree.
C.). The following textural properties were determined:
[0085] 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.
[0086] The MR (%) was determined by finding the ratio between
anchors 2 and 3 and between anchors 1 and 2 (FIG. 2a).
[0087] 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 FIG. 2b.
[0088] 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 (FIG. 2b).
[0089] 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 (FIG. 2b).
[0090] 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 (FIG. 2c). The indentation hardness was
represented by a conversion to the Brinell Hardness Number (BHN)
(N/mm.sup.2) using the following equation:
BHN = 2 F .pi. D ( D - D 2 - d 2 ) Equation 1 ##EQU00001##
[0091] Where:
[0092] F=force generated from indentation (N) obtained from maximum
peak of curve,
[0093] D=diameter of ball probe indenter (3.125 mm) and
[0094] d=indentation depth which is half the probe diameter (1.5625
mm)
[0095] Textural profiling was determined as per the settings in
Table 2.
TABLE-US-00002 TABLE 2 Parameters employed for textural analysis
Parameters MR.sup.1 BHN.sup.2 Test mode Compression Force Pre test
speed 1.0 mm/sec 1.0 mm/sec Test speed 0.5 mm/sec 0.5 mm/sec Post
test speed 1.0 mm/sec 1.0 mm/sec Compression force -- 40N Trigger
force 0.05N 0.05N Load cell 5 kg 5 kg Compression strain 50% --
Compressive distance 1.5625 mm .sup.1Matrix resilience
.sup.2Brinell hardness number
[0096] 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.degree. 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.
TABLE-US-00003 TABLE 3 Preparation of simulated lachrymal fluid
(SLF) Components Quantity NaHCO.sub.3 218 mg NaCl 678 mg
CaCl.sub.2.cndot.2H.sub.2O 8.4 mg KCl 138 mg Deionized water 100 mL
(Gonjari et al., 2009)
TABLE-US-00004 TABLE 4 Parameters employed for disintegration
profiling Parameters Settings Test mode Compression Hold until
reset Pre test speed 2.0 mm/sec Test speed 3.0 mm/sec Post test
speed 10.0 mm/sec Force 0.098N Trigger force 0.029N Data
Acquisition rate 500 points per second Maximum tracking speed 5
mm/sec
Surface Morphology by Scanning Electron Microscopy (SEM)
[0097] The in-depth surface morphology and internal structure of
the dosage form was visualized by the use of scanning electron
microscopy (SEM) (FEI Phenom.TM., Hillsboro, Oreg., USA). Samples
were mounted on a spud and gold plated by the sputter-coater (SPI
modul.TM. sputter-coater and control unit, West Chester, Pa.
USA).Samples were then viewed under the SEM at different
magnifications.
Visualization of Dissolution Process Employing Stereomicroscopy
[0098] 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.RTM. 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.
Computation of Drug Entrapment Efficiency (DEE)
[0099] 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) (FIG. 3) in Simulated Aqueous Humour
(SAH, Ph 7.4, 37.degree. C.) (Table 5) for determination of drug or
API content. All tests were conducted in triplicate. The following
equation was employed for the DEE calculation:
DEE = D a D t .times. 100 Equation 2 ##EQU00002##
[0100] Where:
[0101] DEE=Drug entrapment efficiency,
[0102] D.sub.a=the actual quantity of drug (mg) measured by UV
spectroscopy and
[0103] D.sub.t=the theoretical quantity of drug (mg) added in the
formulation.
[0104] FIG. 3 shows calibration curves of TM in SAH (pH7.4,
35.degree. C.) at 295 nm (SD.ltoreq.0.0543).
TABLE-US-00005 TABLE 5 Preparation of simulated aqueous humour
(SAH) Components Quantity NaCl 6400 mg KCl 750 mg CaCl.sub.2 480 mg
MgCl.sub.2 300 mg Sodium acetate 3900 mg Sodium Citrate 1700 mg
Deionized water 1000 mL (Giannola et al., 2008)
In Vitro Drug Release Studies
[0105] 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 (M.sub.w=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.degree. 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). MDT.sub.50% data point was
selected for the design formulations. The following equation was
used for the determination of MDT:
MDT = i = 1 n t i M t M .infin. Equation 3 ##EQU00003##
[0106] Where:
[0107] M.sub.t=the fraction of dose released in time t.sub.t
[0108] t.sub.t=(t.sub.i+t.sub.i-1)/2 and
[0109] M.sub..infin.=loading dose
Results and Discussion
Textural Profile Analysis of Instantly Soluble Solid Eye Drops
(ISEDs)
[0110] 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/mm.sup.2). 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.
TABLE-US-00006 TABLE 6 Textural analysis results conducted on
design formulations Matrix Matrix yield Matrix absorption Matrix
value tolerance energy resilience BHN Formulation (N/mm) (N/mm) (J)
(%) (N/mm.sup.2) 1 2.918 17.402 0.026 8.469 2.062 2 2.121 11.057
0.019 4.589 2.042 3 2.874 15.592 0.023 6.519 2.055 4 1.395 15.878
0.020 5.439 2.058 5 1.307 16.935 0.018 4.712 2.061 6 1.035 16.164
0.018 5.274 2.032 7 2.261 19.531 0.018 5.257 2.057 8 1.932 13.814
0.022 4.698 2.059 9 0.947 11.941 0.022 4.137 2.054 10 1.096 14.892
0.018 4.934 2.050 11 1.289 11.513 0.016 3.814 2.055 12 2.171 15.849
0.016 6.519 2.048 13 1.462 12.454 0.022 5.895 2.054
[0111] Results are expressed as the mean of at least three
measurements, SDs (standard deviation) obtained were within: yield
value .ltoreq.0.010, tolerance .ltoreq.0.059, absorption energy
.ltoreq.0.002, resilience .ltoreq.0.081, BHN.ltoreq.0.011.
Disintegration Profiling
[0112] 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 FIG. 4 shows a typical profile.
TABLE-US-00007 TABLE 7 Disintegration results obtained End Point
disintegration Disintegration time rate (EPDT) (DR) Formulation (s)
(mm/sec) 1 0.3200 6.250 2 3.0800 0.649 3 0.4800 4.167 4 0.2700
7.407 5 3.0400 0.658 6 0.5200 3.846 7 0.5800 3.448 8 0.5400 3.704 9
3.3400 0.598 10 0.5600 3.571 11 0.2000 10.00 12 0.5500 3.636 13
0.6300 3.175
Surface Morphology
[0113] The lyophilized ISED appeared to have a sponge-like surface
and an interconnecting network with the presence of pores (FIG. 5).
FIG. 5 shows SEM images depicting the porous lyophilized ISED
(magnification 615.times.). The pores were generally circular or
asymmetrical and widespread across the matrix surface. Entrance of
aqueous media into these pores allowed for rapid hydration and
disintegration of the ISED. It was noted that a relationship
between pores and disintegration time existed. Formulations with a
spongy pore appearance had a quicker disintegration time. It is the
specific combination of compounds comprising the ISED that provide
for the forming of pores, and which impart the physico-chemical
and/or physico-mechanical properties described herein, particularly
the characteristic of rapid dissolution in use.
Stereomicroscopy
[0114] Microscopic imaging of the ISED was performed to aid with
visualization of the rapid dissolution process (FIG. 6). FIG. 6
shows stereomicroscopy images for visualization of the rapid
dissolution process (magnification 10.times.). This was apparent by
the images captured. Initially the unhydrated solid eye drop and
water front were distinctly separated. Upon addition of water,
rapid ingression was noted and the ISED dissolved in less than 1
second.
Drug Entrapment Efficiency (DEE)
[0115] 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 FIG. 7 is a graphical
representation of DEE. The API in these examples was timolol (TM)
as described above.
TABLE-US-00008 TABLE 8 Drug entrapment efficiency (DEE) of various
formulations Formulation DEE (%) 1 81.573 2 83.295 3 94.451 4
93.262 5 88.227 6 85.987 7 90.139 8 95.225 9 91.293 10 89.982 11
78.922 12 96.132 13 92.918 Results are expressed as the mean of
three measurements, SD obtained was .ltoreq.2.517.
In Vitro Drug Release
[0116] Drug release was determined and plots are depicted in FIG.
8. The mean dissolution time (MDT) was calculated in order to
determine the drug or API release characteristics of the
formulations (Table 9). With respect to drug release patterns, low
MDT values represent rapid release and high MDT values indicate a
prolonged release. All formulations showed initial burst release.
This can be attributed to the porous nature of the system since it
was instantaneously soluble upon contact with SLF due to the
presence of hydrophilic polymers (PF68 and HPC) and the drug or API
contained therein was released. This is favorable for this dosage
form since it allowed for rapid dissolution which results in
minimal irritation and with the added advantage of reduced loss due
to washout as the initial form is a solid. Formulations 2, 5 and 9
which contained a higher concentration of PF68 (5%) showed longer
MDT values due to the gel-like properties of the polymer with
increased concentration.
TABLE-US-00009 TABLE 9 Mean dissolution times (MDT) calculated for
drug or API release characteristics Formulation MDT.sub.50%
(minutes) 1 25.0000 2 54.5000 3 42.1000 4 27.2500 5 55.0000 6
49.5000 7 49.0000 8 48.7500 9 56.5000 10 48.2000 11 21.2000 12
48.1000 13 45.2500 Results are expressed as the mean of three
measurements, SD obtained was within .ltoreq.0.430.
CONCLUSIONS
[0117] 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.
References for Example 1
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EXAMPLE 2
Further Testing
[0160] 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
(R.sup.2=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.sup.-2.min.sup.-1) and permeability co-efficient
(1.7.times.10.sup.-4 cm.min.sup.-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 C.sub.max was obtained (1.97 ug/mL).
Level A point-to-point IVIVC plots a Wagner Nelson method indicated
a satisfactory R.sup.2 value of 0.84. The biodegradability and
biocompatibility by histological toxicity studies was
confirmed.
1. Introduction
[0161] 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).
[0162] 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 .mu.L) 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.
[0163] 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.
[0164] 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.
[0165] 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 (.gamma.)-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).
[0166] 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).
[0167] 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.
2. Materials and Methods
2.1 Materials
[0168] Hydroxypropylcellulose (HPC) (M.sub.w=80 000 g/mol)
(Klucel.RTM., Hercules Incorporated, Wilingtion, Del., USA),
glycyl-glycine (diglycine, DG) (M.sub.w=132.12 g/mol) (Fluka
BioChemika, Belgium), poly(acylic acid sodium salt) (PAA-Na salt)
(M.sub.w=5100 g/mol), Maltodextrin (MD) (dextrose equivalent
16.5-19.5), Pluronic.RTM. F-68 (PF68) (M.sub.w=8400 g/mol), Sodium
Dodecyl Sulphate (SDS) (M.sub.w=288.38 g/mol) and Timolol maleate
salt (TM) (an example of an active pharmaceutical ingredient (API)
or drug) (M.sub.w=432.49 g/mol) were all purchased from
Sigma-Aldrich (St. Louis, Mo., USA), diclofenac sodium (another
example of an API or drug) (M.sub.w=318.13 g/mol) were purchased
from Sigma Aldrich, ammonium acetate (M.sub.w=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.TM., Johannesburg), Glaucosan.RTM. 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).
2.2 Methods
2.2.1 Attainment of an Optimized Formulation of the ISED
[0169] 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.RTM..
2.2.2 Preparation of Polymeric Solutions and Synthesis of the
Optimized ISED (OISED)
[0170] 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 .mu.L 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.degree. 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.
[0171] 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.
[0172] All analytical experiments were conducted on the drug-loaded
(DL) OISED wherein TM is the API.
2.2.3 Determination of the Molecular Vibrational Transitions
[0173] 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.sup.-1
and a resolution of 4. The spectrum software (Spectrum 100) was
used for interpretation of the results.
2.2.4 Determination of Thermophysical Properties by Temperature
Modulated Differential Scanning Calorimetry (TMDSC)
[0174] 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, STAR.sup.eSystem, 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.degree. C. under constant N.sub.2 purge to reduce
the oxidation rate of 1.degree. C./min.
2.2.5 Determination of Supplementary Thermal Properties via Thermal
Gravimetric Analysis (TGA)
[0175] 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 N.sub.2 to prevent the occurrence of any undesirable
reactions. Samples were placed in a pan using tweezers followed by
heating between 50-550.degree. C. at a rate of 10.degree. C./min.
Weight vs. temperature plots were generated and analysed using
Pyris.TM. software.
2.2.6 Complementary Surface Morphology and Porositometric
Analysis
[0176] 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.TM., Hillsboro, Oreg., USA).
Samples were mounted on a spud and gold plated by the
sputter-coater (SPI module.TM. 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.RTM. 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.
TABLE-US-00010 TABLE 10 Parameters employed for degassing phase of
OISEDs Parameters Settings a) Degassing conditions (evacuation)
Temperature ramp rate 10.degree. C./min Target temperature
30.degree. C. Evacuation rate 50.0 mmHg/s Unrestricted evacuation
from 30.0 mmHg Vacuum set-point 500 mmHg Evacuation time 60 min b)
Degassing conditions (heating) Ramp rate 10.degree. C./min Hold
temperature 30.degree. C. Hold time 900 min Hold pressure 100 mmHg
c) Analysis conditions (adsorptive) Adsorptive Nitrogen gas
(N.sub.2) Maximum manifold pressure 925 kPa Nonideality factor
0.0000620 Density conversion factor 0.0015468 Hard sphere diameter
3.860 A.degree. Molecular cross-sectional area 0.162 nm.sup.2
2.2.8 Ocular Toxicity Evaluation Employing Modified Hen Egg
Chorioallontoic (HET-CAM) Test
2.2.8.1 Assay Preparation and Procedure
[0177] 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.times.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.degree. C. The
following features were used for observation of injury: hemorrhage,
vascular lysis and coagulation. FIG. 9 depicts the procedure for
the test.
TABLE-US-00011 TABLE 11 Scoring chart employed for the HET-CAM test
(10) IS Effects Score ranges Inference No visible haemorrhage 0
0-0.9 Non-irritant Visible membrane discoloration 1 1-4.9
Mildy-irritant Structures covered due to partial 2 5-8.9 Moderately
haemorrhage/discolouration irritant Structures covered fully due to
3 9-21 Severely irritant haemorrhage/discolouration IS = irritation
score
[0178] 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.
IS = 301 - secH .5 + 300 301 - secL .7 + 300 301 - secC .9 300
Equation 4 ##EQU00004##
[0179] Where:
[0180] IS=irritation score
[0181] Sec=onset of reaction in seconds
[0182] H=hemorrhage
[0183] L=lyses
[0184] C=coagulation
2.2.9 Treatment of Dissolution Data by Comparison of Release
Profiles of the OISED and a Commercial Product
[0185] 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.
f 1 = j = 1 n R j - T j j = 1 n R j j Equation 5 ##EQU00005##
[0186] 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:
f 2 = 50 .times. log { [ 1 + ( 1 n ) j = 1 n R j - T j 2 ] - 0.5
.times. 100 } Equation 6 ##EQU00006##
[0187] Where:
[0188] n=sampling number
[0189] R and T=reference and test products at each time point j
2.2.10 Ex Vivo Permeation Studies of the Optimized Formulation
(OISED) in Comparison to a Commercial Anti-Glaucoma Product
[0190] 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 mm.sup.2) 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.degree. 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.
J s = Q r A .times. t Equation 7 ##EQU00007##
[0191] Where:
[0192] J.sub.s=drug flux (mg cm.sup.-2 min.sup.-1)
[0193] Q.sub.r=amount of drug that passed through to the receptor
compartment (mg)
[0194] A=Active cross-sectional area for diffusion (cm.sup.2)
and
[0195] t=time of exposure (min)
[0196] The permeability co-efficient can be described as the
velocity of drug diffusion through the cornea based on its
mathematical unit using Equation 8.
kp = J s C d Equation 8 ##EQU00008##
[0197] Where:
[0198] Kp=permeability co-efficient (cm.min.sup.-1)
[0199] J.sub.s=Flux at steady state (mg cm.sup.-2 min.sup.-1)
[0200] C.sub.d=drug concentration in donor compartment
(mg.cm.sup.-3)
2.2.11 Ultra Performance Liquid Chromatography (UPLC)
Methodology
[0201] The equipment consisted of an ultra-performance liquid
chromatography system (Acquity.TM. 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
C.sub.18column (1.7 .mu.m; 1.times.100 mm) at 25.degree. 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.
2.2.12 Aqueous Humor Collection and Drug Extraction Via
Liquid-Liquid Procedure
[0202] 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.degree. 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 (FIG. 2).
The lowest standard concentration of the calibration curve was
regarded as the lowest limit of quantification (LOQ). Calibration
curve obtained for TM in blank aqueous humour samples was
constructed. R.sup.2 of 0.99 was obtained.
2.2.13 In Vivo Characterization of the OISED in the Rabbit
Model
[0203] 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.RTM., 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, .about.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.degree. C. FIG. 11 provides digital
images of the process.
2.2.14 Pharmacokinetic Data Analysis
[0204] 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.
[0205] The following pertinent pK parameters were determined:
[0206] 1. Apparent absorption rate constant (k.sub.a)
[0207] 2. Apparent elimination rate constant (k.sub.e)
[0208] 3. Peak TM concentration (C.sub.max)
[0209] 4. Area under the concentration vs. time curve (AUC)
[0210] 5. Area under the zero and first moment curves from the last
sampling time to infinity (AUC.sub.t-.infin.)
[0211] 6. Area under the moment curve (AUMC)
[0212] 7. Peak time (t.sub.max)
[0213] 8. Mean residence time (MRT)
[0214] The following diagnostic criteria were used to determine
suitability of the selected model:
[0215] 1. Co-efficient (R)
[0216] 2. Weighted sum squares of residuals (SS)
[0217] 3. Standard error of residuals (SE)
[0218] 4. Akaike's information criteria (AIC)
[0219] 5. Schwarz criteria (SC)
2.2.15 In Vitro In Vivo Correlation (IVIVC)
[0220] 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.
2.2.16 Histological Evaluation of OISED Ocular-Compatibility
[0221] 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 .mu.m 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.
3. Results and Discussion
3.1 Preparation and Sterilization of the OISED
[0222] The optimized OISED was prepared as outlined previously.
Drug loaded (DL) and drug-free (DF) OISEDs were prepared. FIG. 11
depicts the size of the OISED (diameter 3 mm, thickness 2 mm) in
comparison to a US one dime coin (diameter 17.91 mm, thickness 1.35
mm).
3.2 Molecular Vibration Transitions
[0223] 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 FIG. 12) and the drug-loaded final OISED formulation
(FF shown on FIG. 12) was analyzed for possible interactions (FIG.
12). From the spectrum of pure drug the major peaks were observed
thus confirming that the drug was timolol maleate (TM). A
quaternary ammonium peak (N--H) was found at 3280.98 cm.sup.-1.
Presence of an OH group was seen at 3037.96 cm.sup.-1. Also,
C--O--C stretching (1702.51 cm.sup.-1) and C.dbd.C aromatic ring
(1583.32 cm.sup.-1 and 1448.66 cm.sup.-1) were noted. Furthermore,
CH stretching and CH bending were observed at 1352.7 cm.sup.-1 and
2964.98 cm.sup.-1 respectively. The broad peak at 2879.95 cm.sup.-1
was attributed to the OH moeity present in PF68. Additionally, C--O
stretching occurred at 1145.98 cm.sup.-1. For MD, OH stretching
occurred around 3304.07 cm.sup.-1. C--O stretching of MD
corresponds to peaks at 1059.52 cm.sup.-1 and 961.79 cm.sup.-1. All
major peaks of pure drug appeared in the physical mixture
indicating that no interaction occurred (3294.94 cm.sup.-1, 1586.96
cm.sup.-1). For the final formulation, drug peaks were not clearly
defined and this could be attributed to the homogenous dispersion
within the matrix. In terms of the final formulation, the major
components were PF68 and MD. Since no major shifts in peaks were
observed, it can be proposed that the absence of any appreciable
variations or chemical interaction between polymers or drug was
noted and the components maintained their initial structure. The
peaks occurring in the region of >3000 cm.sup.-1 could be
attributed to the summation of peaks associated with polymer HPC
(3423.75 cm.sup.-1), excipients diglycine (DG) (3283.1 cm.sup.-1)
and sodium polyacrylate (PAANa) (3285.27 cm.sup.-1). Hence, this
assisted in confirming that the formulation remained stable during
the preparation procedure.
3.3 Thermal Analysis of OISED
[0224] 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.degree. C. confirming its crystalline nature (FIG. 13a). This
corresponded to the melting peak (T.sub.m) of the drug which is the
transition from a solid to liquid state with subsequent
degradation. In terms of the drug-loaded OISED formulation, PF68
and MD were the major components. Native PF68 produced a
distinguishable single step endothermic degradation at 53.9.degree.
C. (FIG. 13b). For pure MD, peaks that occur before 220.degree. C.
(106.51.degree. C.) (FIG. 13c) were possibly due to the elution of
water (Elnaggar et al., 2010). The T.sub.m of MD was observed as a
broad peak at 230.98.degree. C. In terms of the physical mixture, a
less defined broad TM peak in the region of 193.89.degree. C. was
seen confirming the presence of the drug (FIG. 13d). TM uniformly
dispersed within the final optimized OISED formulation as well as
increased polymer and excipient concentration compared to drug,
possibly contributed to the diminished broad T.sub.m of the drug
(FIG. 13e). Furthermore, Simon and co-workers (Simon et al., 2003),
reported that a lower endothermic peak of lyophilized samples when
compared to the individual components may be due to a decrease in
thermal conductivity. This may be as a result of the presence
voids/pores within the matrix. Additionally, the endothermic peak
was reduced in the final formulation, postulated due to MD
(16.5-19.5 dextrose equivalence). It has been investigated to be a
lyoprotectant (lyophilization stabilizer) by being amorphous and
displaying a diluting ability during the lyophilization process
(Corveleyn and Remon, 1996).
3.4 Thermal Gravimetric Analysis
[0225] 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.degree. C. and an endset of 350.degree.
C. which can be explained as the decomposition temperature (FIG.
13g). A sharp endothermic peak is observed in this area which is in
agreement with the findings of Joshi and co-workers (2009). The
derivative curve of TM shows weight loss corresponding to a three
step pattern at 231.14.degree. C., 260.degree. C. and
313.95.degree. C. respectively. In terms of the optimized OISED
formulations, similar weight loss patterns were noticed. For both
DF and DL OISED formulations, sharp weight loss occurred in the
region of 200.degree. C.-450.degree. C. (FIG. 13h and i).
Additionally, in the derivative curves for both formulations, 3
points of degradation were noted at approximately 230-240.degree.
C., 305-310.degree. C. and 411-413.degree. C. It is explicable that
incorporation of drug did not alter the thermal properties.
3.5 Porositometric and Surface Imaging Analysis Comparison
[0226] 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
FIGS. 14a and b respectively. The linear plot indicates that
degassing occurred since the adsorption and desorption isotherms
are noted. The presence of a hysteresis loop was noted since a
difference in the adsorption and desorption curves were seen as per
definition of the loop. The loop infers nitrogen evaporation from
pores (Guirguis and Moselhey, 2012). A type 3 hysteresis loop (H3)
can be possibly classified due to the isotherms sloping. This
implies the presence of slit-like pores. This sample can thus be
described as mesoporous (pore diameter between 2-50 A) and
macroporous (pore diameter >50 A) according to the IUPAC
definition (Condon, 2000).
[0227] 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 m.sup.2/g, R.sup.2=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 FIGS. 14c and d (Halsey, 1948). The average pore size
for BJH adsorption and desorption were 46.644 .ANG. and 53.949
.ANG. respectively. The large pore distribution can be explained on
account of the entrapped air spaces within the matrix as a result
of the freeze-drying methodology for production. FIG. 14e displays
t-plots based on Lippins, Linsen and de Boer Lippins et al., 1964).
This is used for the determination of micropore or mesopore
presence <20 .ANG.. Positive BET values enabled the computation
of the t-plot. The micropore volume is obtained by extrapolation of
the isotherm. A t-plot micropore volume of -0.010393 cm.sup.3/g was
obtained indicating the presence of micropores. Hence, these
findings emphasized that the lyophilization process significantly
contributed to the porous nature of the samples and ultimately
results in a light structure that rapidly disintegrates. This
explains the possible method of disintegration and drug release i.e
via fluid filling into the pores of the matrix with subsequent
dissolution. Table 12 computes results obtained for optimized
drug-loaded samples.
TABLE-US-00012 TABLE 12 Results obtained from porosity analysis
Parameter DLO BET total surface area (m.sup.2/g) 27.2052 BET-C
4.785274 Qm (cm.sup.3/g STP) 6.2495 BET slope(g/cm.sup.3 STP)
0.126575 .+-. 0.007612 BET y-intercept (g/cm.sup.3 STP) 0.033439
.+-. 0.001053 R.sup.2 (BET plot) 0.99 Micropore volume (cm.sup.3/g)
-0.010393 External surface area (m.sup.2/g) 38.5234 R.sup.2
(t-plot) 0.99 t-plot slope (cm.sup.3/g .ANG. STP) 2.490519 .+-.
0.099199 t-plot y-intercept (cm.sup.3/g STP) -6.718757 .+-.
0.403672 Pore diameter range (.ANG.) 7-3000 Pore thickness range
(.ANG.) 3.5-5 BJH adsorption pore size (.ANG.) 46.644 BJH
desorption pore size (.ANG.) 53.949 DLO = drug-loaded optimized
BET-C = intensity of the N.sub.2-surface interaction Qm = monolayer
surface capacity
3.7 Ocular Toxicity Evaluation
[0228] 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.
TABLE-US-00013 TABLE 13 Results obtained for the HET-CAM test
Substance Score Inference IS Negative control NaCl 0 Non-irritant 0
Positive control SDS 2.5 Severe 10.40 Samples DF 0 Non-irritant 0
DL 0 Non-irritant 0 Results expressed as a mean of 3 values NaCl =
saline, DF = Drug free, DL = drug loaded, SLS = sodium lauryl
sulphate
3.8 Comparison of Dissolution Data
[0229] 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.
3.9 Determination of Ex Vivo Permeation of OISED in Comparison to a
Marketed Product
[0230] 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.sup.-2.min.sup.-1)
compared to pure drug 2 (0.000378 mg.cm.sup.-2.min.sup.-1) and eye
drops 3 (0.00039 mg.cm.sup.-2.min.sup.-1) (FIG. 15). This can be
attributed to the polymers employed in the formulation. PF68 forms
a slight gel-like substance due increased temperature of SLF which
assists in increases the corneal contact time. Also, HPC is a
non-ionic surface-active viscosity adjusting polymer that favours
pre-corneal retention thus influencing the drug permeation. A low
concentration of the polymers was incorporated to promote rapid
disintegration of the device, avoidance of a highly viscous
gel-like residue yet allowance of sufficient corneal contact. The
influx of fluid in the pores influenced the matrix disentanglement
flowed by rapid dissolution and drug liberation. Pure drug solution
is diluted and remains in the SLF or on the corneal surface as it
is dispersed within a liquid (saline) and thus drug takes longer
period to diffuse across the corneal epithelium as opposed to a
polymeric device that favours adhesion and corneal interaction. The
kp calculated for the samples differed, and this disparity is
likely to be due to the difference in composition. As an oral drug,
timolol has been classified as a class 1 drug according to
Biopharmaceutics Classification system (BCS) (Kasim et al., 2004;
Dahan et al., 2009). This infers that it is a highly permeable and
soluble drug across the intestinal tissue. On the eye surface,
across the corneal epithelium, it also displays favorable
permeability due to the advantageous hydrophilic and lipophilic
property. The polymers and excipients employed in the optimized
OISED formulation are all hydrophilic thus rapid drug liberation
and permeation was attained and thus this was suitable for
immediate drug release. Table 14 shows differences in kp
values.
TABLE-US-00014 TABLE 14 Results obtained for permeability
co-efficients Sample Kp (cm min.sup.-1) Optimized formulation 1.7
.times. 10.sup.-4 Pure drug dispersion 1.2 .times. 10.sup.-4
Marketed eye drops 1.3 .times. 10.sup.-4 Results expressed as a
mean of 3 values
3.12 In Vivo Results from the OISED in Comparison to Marketed Eye
Drop Preparation
[0231] FIG. 16 depicts the chromatograph of TM and IS DS obtained
from UPLC 1 hour after analysis. TM was eluted at 0.594 minutes and
DS at 1.595 minutes. Intra- and inter-day precision and accuracy
and drug recovery were seen as satisfactory (R.sup.2=0.98). The
developed method used perchloric acid and methanol in order to
de-proteinate samples. The mobile phase maintained at a pH of 4.5
was acceptable to allow for drug retention and peak separation. No
difference in retention times of TM and DS in blank samples was
noted.
[0232] 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.
TABLE-US-00015 TABLE 15 Score for clinical assessment of OISED on
ocular tissue Effect Score DL Score DF Conjunctival rednesss 0 0
Conjunctival chemosis 0 0 Discharge 0 0
[0233] 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 C.sub.max 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). FIG. 17a-b displays the in vivo
and in vitro drug release curves for the OISED as well as the eye
drops.
TABLE-US-00016 TABLE 16 PK parameters and diagnostic criteria for
"goodness of fit" of TM in the aqueous humour following topical
application Parameters ISED Eye drops Pk parameters K.sub.a
(min.sup.-1) 0.012 0.011 K.sub.e (min.sup.-1) 0.013 0.01 T.sub.max
(min) 100.09 92.2 C.sub.max (ug/mL) 3.14 1.97 AUC.sub.0-t (ng/ml)
628.39 447.03 AUC.sub.t-.infin. 664.77 496.78 AUMC
(ug/ml/min.sup.-2) 103272.52 91924.99 MRT(min) 155.34 185.04
Diagnostic criteria R obs pred 0.92 0.86 SS 0.94 1.50 SC 6.17 6.86
SE 1.007 0.866 AIC 7.74 8.032
3.13 In Vitro In Vivo Correlation (IVIVC)
[0234] FIG. 18 (a-d) provides a graphical representation of the
data obtained. A Wagner Nelson method was employed to calculate the
in vivo drug release. Level A point-to-point IVIVC plots indicated
a R.sup.2 value of 0.84. This serves to imply that the in vitro
dissolution data can be compared to and may serve as a surrogate to
that of in vivo pK data.
3.14 Histological Assessment
[0235] 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. FIG. 19 provides images of the
corneal layer at different time points.
4. Conclusions
[0236] 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 R.sup.2 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.
Ethical Approval
[0237] Ethics clearance was obtained from the Animal Ethics
Screening Committee (AESC) of the University of the Witwatersrand.
Ethics clearance number: Dec. 5, 2012.
[0238] 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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* * * * *
References