Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1841—Transforming growth factor [TGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
  • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/10—Ophthalmic agents for accommodation disorders, e.g. myopia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/495—Transforming growth factor [TGF]

Definitions

• the present disclosure relates to compositions and methods useful for the treatment and/or prevention of conditions of the eye.
• the disclosure relates to compositions and methods that can be used in augmenting and regenerating the cornea, and in correcting refractive errors of the eye.
• Myopia results from excessive curvature of the cornea so that light entering the eye focuses in front of the retina. It is the most prevalent vision impairment worldwide affecting the vision of 70 to 90% of people in some Asian countries and 30 to 40% in Europe and the United States (Frederick 2002). In most cases, myopia first occurs in school-age children and progresses until about the age of 20. It is also associated with increased prevalence of macular degeneration, retinal detachment, and glaucoma in adulthood (Ebenstein & Pruitt 2006).
• changes associated with keratoconus include a decrease in the number of lamellae and keratocytes (Ku, Niederer, Patel, Sherwin, & McGhee 2008; Sherwin & Brookes 2004), and changes in organisation of the lamellae and distribution of collagen fibrillary mass (Meek et al. 2005).
• Keratoconus corneas are known to have decreased levels of enzyme inhibitors and an increased level of degradative enzymes (Kenney & Brown 2003).
• compositions and methods for modulating corneal cells to alter collagen expression and extracellular matrix formation in corneal tissue. These compositions and methods are useful for regenerating and/or augmenting the cornea, and thereby treating and/or preventing various conditions of the cornea and refractive errors of the eye.
• composition is formulated as an eye drop.
• composition is administered once daily or twice daily.
• the composition is administered in conjunction with use of a contact lens, corneal insert, corneal implant, or intrastromal ring.
• the contact lens, corneal insert, corneal implant, or intrastromal ring is adapted for moulding or holding corneal shape during and/or following treatment with the composition.
• the condition is selected from the group consisting of: keratoconus, myopia, and astigmatism.
• composition is formulated with gellan gum.
• stromal cells from adult human and rat corneas can be reprogrammed to produce neuron specific proteins when treated with neuronal lineage specifying growth factors (Greene et al. 2013). This data demonstrates that an adult cell population can be reprogrammed simply by the modulation of the growth factor environment both in vitro and in vivo.
• the composition may include, for example, 0.04 ng/ml to 4 ng/ml; or 0.04 ng/ml to 0.4 ng/ml; or 0.4 ng/ml to 4 ng/ml; or 4 to 40 ng/ml; or 40 ng/ml to 400 ng/ml, or 40 ng/ml to 4000 ng/ml dexamethasone, or derivative thereof or related steroidal agent; or about 0.04 ng/ml, about 0.08 ng/ml, about 0.12 ng/ml, about 0.4 ng/ml, about 0.8 ng/ml, about 1.2 ng/ml, about 4 ng/ml, about 12 ng/ml, about 24 ng/ml, about 40 ng/ml, about 80 ng/ml, about 120 ng/ml, about 240 ng/ml, about 400 ng/ml, about 800 ng/ml, about 1000 ng/ml, about 1600
• the composition may include, for example, 1 ng/ml to 1 ⁇ g/ml; or 1 ng/ml to 10 ng/ml; or 10 ng/ml to 100 ng/ml; or 100 ng/ml to 1 ⁇ g ml TGFp3 polypeptide or variants or fragments thereof, or about 1 ng/ml, about 5 ng/ml, about 10 ng/ml, about 20 ng/ml, about 50 ng/ml, about 100 ng/ml, about 200 ng/ml, about 500 ng/ml, about 800 ng/ml, or about 1 ⁇ g/ml TGFP3 polypeptide or variants or fragments thereof.
• the composition may include at least 40 ng/ml dexamethasone, or derivative thereof or related steroidal agent, along with at least 4 ng/ml TGFP3 polypeptide, or variants or fragments thereof.
• the compositions may be formulated with a pH range of 5.0 to 8.0. This pH range may be achieved by the addition of buffers to the solution. It is preferred that the formulations are stable in buffered solutions. That is, there is no adverse interaction between the buffer and the active agents that would cause the composition to be unstable, e.g., by precipitation or aggregation.
• the dosage range may be 1.5 ⁇ g to 150 ⁇ g; 2.6 ⁇ g to 260 ⁇ g; or 6.5 ⁇ g to 650 ⁇ g of dexamethasone, or derivative thereof or related steroidal agent; or about 1.5 ⁇ g, about 2 ⁇ g, about 3 ⁇ g, about 4.5 ⁇ g, about 6 ⁇ g, about 6.5 ⁇ g, about 7.5 ⁇ g, about 10 ⁇ g, about 15 ⁇ g, about 22.5 ⁇ , about 32.5 ⁇ g, about 20 ⁇ g, about 26 ⁇ g, about 30 ⁇ g, about 40 g, about 45 ⁇ g, about 60 ⁇ g, about 65 ⁇ g, about 75 ⁇ g, about 80 ⁇ g, about 90 ⁇ g, about 100 ⁇ g, about 120 ⁇ g, about 130 ⁇ g, about 150 ⁇ g, about 180 ⁇ g, about 225 ⁇ g, about 240 ⁇ g, about 260 ⁇ g, about 200 ⁇ g, about 300
• Dosage for one eye may be about one drop of the disclosed composition.
• One drop of composition may be 10 ⁇ to 200 ⁇ , 20 ⁇ and 120 ⁇ , or 50 ⁇ to 80 ⁇ or any values in between.
• dispensers such as pipettors can dispense drops from 1 ⁇ to 300 ⁇ and any value in between.
• the dispenser metes out about 15 ⁇ , about 20 ⁇ , about 30 ⁇ , about 45 ⁇ , about 60 ⁇ , or about 65 ⁇ per drop of the disclosed composition.
• the contact lens or insert/implant may include, for example, 0.01 mg to 10 mg of dexamethasone, or derivative thereof or related steroidal agent; or about 0.01 mg, about 0.1 mg, about 0.5 mg, about 0.7 mg, about 1 mg, about 5 mg, or about 10 mg of dexamethasone, or derivative thereof or related steroidal agent.
• the disclosed compositions can be used to avoid or counter the deleterious effects of crosslinking procedures, such as stromal haze and cell loss (described in more detail, below).
• corneal regeneration with the disclosed compositions can allow crosslinking to be performed on subjects who were previously ineligible for such procedures, e.g., those with corneal thickness less than 400 ⁇ .
• the disclosed compositions can be used to slow or halt progressive corneal thinning, which would not be addressed by the use of crosslinking on its own.
• Non-limiting examples of anaesthetics include: benzocaine, bupivacaine, cocaine, etidocaine, lidocaine, mepivacaine, pramoxine, prilocalne, chloroprocaine, procaine, proparacaine, ropicaine, and tetracaine.
• Non-limiting examples of antiinflammatory agents include: aspirin, acetaminophen, indomethacin, sulfasalazine, olsalazine, sodium salicylate, choline magnesium trisalicylate, salsalate, diflunisal, salicylsalicylic acid, sulindac, etodolac, tolmetin, diclofenac, ketorolac, ibuprofen, naproxen, flurbiprofen, ketoprofen, fenoprofen, suprofen, oxaproxin, mefenamic acid, meclofenamic acid, oxicams, piroxicam, tenoxicam, pyrazolidinediones, phenylbutazone, oxyphenthatrazone, pheniramine, antazoline, nabumetone, COX-2 inhibitors (Celebrex®), apazone, nimesulide, and zileuton
• compositions may be used in conjunction with contact lenses, corneal inserts, corneal implants, or intrastromal rings, to assist in supporting or reshaping the subject's cornea.
• corneal inserts include corneal inlay and corneal onlay devices.
• contact lenses, intrastromal rings, or other inserts/implants may be used for moulding or holding corneal shape preceding, during, and/or following treatment with the composition.
• a corneal 'insert' typically refers to a temporary device inserted into the cornea
• a corneal 'implant' typically refers to a more permanent device.
• many well known devices are described interchangeably in the art as implants/inserts. Therefore, the terms 'insert/implant' as used herein are not to be deemed as strictly limiting based on time of usage.
• the contact lens, corneal insert, corneal implant, or intrastromal ring may be used with the disclosed composition for treatment of corneal defects, diseases, damage, injury, and/or degeneration, as well as refractive errors of the eye.
• the contact lens, corneal insert, corneal implant, or intrastromal ring may act as a carrier for the composition or as a composition eluting device.
• the contact lens, intrastromal ring, or other corneal insert/implant may be utilised with the composition that is suitable for administration to the eye, e.g., eye drops, as described in detail herein.
• the contact lenses may be gas permeable. Moulding contact lenses may be obtained from commercial sources. Examples of commercially available lenses include, at least, DreamLite, OK Lens, EyeDream, MiracLens, DreamLens, i-GO OVC, GOV, Wake and See, CRT, Fargo/iSee, Emerald and Wave Contact Lens System lenses.
• the composition described herein e.g., eye drops
• the flattest keratometry is taken.
• One of skill in this art could also use the steeper keratometry or an average of both and based on this corneal curvature make the necessary calculations to flatten or steepen the radius of curvature of the anterior surface of the cornea and thus correct the refractive defect of the eye.
• the base curve of the moulding contact lens may be calculated based on the change in the refractive power for each eye separately.
• the base curve of the moulding contact lens may be calculated starting with one to four flatter or steeper diopters, more preferably one to three flatter or steeper diopters, even more preferably one to two flatter or steeper diopters, depending on the refractive error that is required.
• the peripheral base curve depends on the adaptation of the moulding contact lens and is calculated to be 0.5 mm of radius greater than the central zone, but can vary depending on the design.
• the diameter of the moulding contact lens used in accordance with the invention may be from 8.0 mm to 18.0 mm. Commercially available lenses are produced with such diameters.
• the moulding contact lens may be a hard contact lens with a diameter ranging from 8.0 mm to 12.0 mm.
• the moulding contact lens may be a soft contact lens with a diameter ranging from 13.0 mm to 15.0 mm. Soft contact lenses may cover the entire cornea and go from sclera to sclera.
• the moulding contact lens may be comprised of hard and soft materials.
• the power of the moulding contact lenses can be determined to the nearest possible refractive power that the subject requires to see comfortably.
• the subject is prescribed eyeglasses while the subject is undergoing treatment.
• various optometric measurements may be repeated to confirm that the treatment is progressing as planned and is adequate.
• Such measurements may include assessment of visual acuity for near and far vision, orthotypes, keratometry measurements, objective and subjective retinoscopy, diagrams of the adaptation of the moulding contact lens, movement of the moulding contact lens, and comfort of the moulding contact lens.
• exemplary enzymes include but are not limited to hyaluronidase, chondroitinase ABC, chondroitinase AC, keratanse, and stromelysin, which have been shown to work on various proteoglycan components of the cornea. Included also are the enzyme collagenase, matrix metalloproteinase 1 (interstitial collagenase), and matrix metalloproteinase 2 (gelatinase).
• composition is coadministered with any such enzymes
• a vehicle such as a polymer (e.g., methylcellulose, polyvinyl alcohol, cellulose, etc.) in the composition to enhance the working of such enzymes.
• Additional agents may be included to activate metalloproteinase enzymes, e.g., interleukin-la, tumour necrosis factor ⁇ / ⁇ and any subtypes thereof, monosodium urate monohydrate, 4-amino phenylmercuric acetate, human serum amyloid A, human B 2 microglobin, and copper chloride.
• carbamide urea
• the composition may also be co-administered with one or more enzymes that degrade other sugars or proteins found in the cornea.
• the composition may be coadministered with one or more anaesthetics used to reduce the irritation of the moulding contact lens or any corneal insert/implant to the cornea.
• the composition may be coadministered with one or more lubricants to improve the comfort of the subject during the treatment.
• the composition may be co-administered with one or more anti-microbial agents such as anti-bacterial, anti-viral, and/or anti-fungal agents.
• the composition may also be co-administered with one or more vasoconstrictors. The person of skill in the art can determine the appropriate agents for co-administration to the subject based on the condition being treated.
• Isolated keratocytes were cultured in either 12 or 24 well cluster plates (Falcon) on plastic or glass coverslips in 2-3 ml of cell culture media. Cells were kept in a humidified incubator at 37°C with 5% C0 2 . Culture media was changed after 24 hours then every two days subsequently or more frequently if required. Cultures were viewed daily with a Leica DM IL bench top inverted microscope. For cell pellet culture, freshly obtained cells after tissue were pelleted by centrifuging at 300 g for 7 minutes at 20°C in a plastic conical tube. Appropriate culture media was added to the tubes.
• the cells After 24 hours of incubation at 37°C, the cells had contracted and formed a pellet which did not adhere to the walls of the tube.
• the pellets were cultured in 2 ml of media in a humidified atmosphere of 5% C0 2 at 37°C for three weeks. Media was changed every other day.
• Human and rat corneal and cartilage tissue was thin-sliced (1-2 mm) in an anteroposterior plane with a blade and the slices were placed in an organotypic air-liquid interphase culture system (Figure 2). Briefly, the explants of healthy tissue were cultured on 0.4 ⁇ pore size cell culture inserts (Millicell, France) at the interface between culture medium and a C0 2 rich environment. Corneal sections were placed epithelium side up on cell culture plate inserts with 3 ml of culture medium. The culture media was changed every other day.
• Control medium Dulbecco's Modified Eagle 1% GlutaMAXTM (100X stock), 1% Anti- Medium (DMEM) Anti (100X stock)
• Tissue slices were cultured in the chondrogenic differentiation medium for varying time intervals to determine the optimum time required for the growth factor treatment. Samples were collected for each time point (Table 2). For obtaining a monolayer of cells, keratocytes were seeded on glass coverslips at a density of 15 x 10 4 per cm 2 . The cells were allowed to attach to the coverslips for 24 hours and culture media was changed every other day. Cultures were maintained for up to 3 weeks.
• Eye drops were formulated using gellan gum which is a water soluble polysaccharide produced by the bacterium, Pseudomonas elodea.
• gel base formulation allows a prolonged corneal residence time and increased ocular bioavailability of the therapeutic agent.
• polymeric gellan gum is an anionic polymer it undergoes in situ gelling in the presence of mono- and divalent cations such as Ca 2+ , Mg 2+ , K + , and Na + (Bakliwal, & Pawar 2010) .
• the corneas were immersed in 20% sucrose solution for 5 hours at 4°C and then moved to a 30% sucrose solution and kept at 4°C until the tissue sinks (usually overnight).
• the corneas were then embedded in OCT compound and immersed in liquid nitrogen to bring about rapid freezing.
• the frozen blocks of tissue were stored at -80°C until further use.
• Approximately 10-15 ⁇ thick cryostat sections were mounted on SuperFrostTM Plus slides and the slides were stored at -80°C until needed.
• the cells cultured on coverslips were rinsed with PBS and fixed with 4% PFA for 15 minutes. Coverslips were immersed in PBS until further use.
• RNA extraction from samples was carried out using the PureLink® RNA MicroKit (Invitrogen).
• tissue samples were mixed with 0.75 ml TRIzol® and carrier RNA and homogenised using a hand held homogeniser (PRO Scientific, Inc.).
• the samples where then incubated with 0.2 ml of chloroform followed by centrifugation at 12000 rpm and 4°C for 15 minutes.
• the upper phase was separated and was mixed with ethanol and then transferred to the collection column tube.
• Example 7 Testing of biomechanical and optical properties of corneas following in situ stromal ECM protein deposition
• Nanoindentation provides mechanical measurements of materials of interest through the application of ultra-small forces perpendicular to the sample plane of interest and measurement of the resultant sample indentation (Dias & Ziebarth 2013). Nanoindentation has recently emerged as a powerful tool for measuring nano- and microscale mechanical properties in tissues and other biomaterials (Ebenstein & Pruitt 2006). The more recent advancement of in situ scanning probe microscopy (SPM) imaging, where the nanoindenter tip is simultaneously used as a 3D imaging device combined with nanoindentation has enabled a new wave of novel materials research (Dickinson & Schirer 2009). Force, displacement, and time are recorded simultaneously while a nanoindentation tip is pushed into the corneal tissue under a controlled load.
• SPM in situ scanning probe microscopy
• Nanoindentation tests are output as a load-displacement curve which can be analysed using well defined equations to calculate the mechanical properties relating to rigidity, integrity, and elasticity of the cornea.
• Nanoindentation testing was carried out at the Chemical and Materials Engineering lab at the University of Auckland. In order to test the cornea in its natural position a mould was required for nanoindentation. Previous studies have used polystyrene and blue tack to hold the corneas in place. The effect of the mould deforming under the load was a potential source of error so a hard mould was decided on for testing.
• the first material that was used to create a mould was conventional play dough. This was formed to the exact shape and curvature of human cornea samples (Figure 6(A)). The play dough was then left to harden over the next two days before being used in testing. The testing of the rat eyes was slightly different as the entire globe was used. To hold the globes in place a petri dish filled with a resin and having small indent to hold the globe was used (Figure 6(B)). PBS was used to keep the samples from drying out.
• the samples are very soft biological samples a conospherical fluid tip was used for all nanoindentation testing.
• the indent load used for the human samples was 50 ⁇ .
• For the rat globes a range of loads between 3 and 5 uN were used.
• the fibre optic light was switched on and the sample placed directly under the stream of light from the microscope.
• the central section of the cornea was placed directly in the stream of light as accurately as possible ( Figure 6(C)).
• the sample was focused by adjusting the Z slider until the surface of the cornea could be observed in good resolution. To ensure that the focus was on the central highest point of the cornea sample, the view was moved in the x and y directions to observe how the focus changed.
• dexamethasone a synthetic steroid drug has been used to treat inflammatory eye conditions. Therefore a combination of TGFP3 and dexamethasone was used in the chondrogenic differentiation medium to drive the differentiation of keratocytes towards a chondrocyte phenotype.
• type I and type II collagen was specifically noted. It is known that fibrillar types of collagen such as types I and II self- assemble and crosslink to form highly crystalline fibres exhibit a very high stiffness, low extensibility and a remarkable elastic energy storage capacity (Wells 2003). It is the crosslinking which contributes towards the stiffness and tensile strength of the fibres.
• the corneal stromal extracellular matrix is composed of tightly packed heterotypic collagen fibrils made up mostly of collagen types I and V. Similar to corneal fibrils, cartilage fibrils are heterotypic (made up of types II and XI) and have a uniform diameter of 25 nm (slightly smaller than corneal fibrils) (Mendler, Eich-Bender, Vaughan, Winterhalter, & Bruckner 1989). Collagen II is the major fibril component of cartilage and is similar to collagen I in that the molecule essentially consists of a single uninterrupted helical domain 300 nm in length. Owing to their similarities, collagens II and XI are considered to be the cartilage analogues of collagens I and V (corneal stroma collagens) in other tissues.
• corneal keratocytes from adult corneas were seeded in either the chondrogenic differentiation medium containing TGFP3 and dexamethasone or a standard fibroblast proliferation medium.
• the keratocytes seeded in the chondrogenic differentiation media formed cell aggregations/spheres (Figure 7(A)) approximately 50-100 ⁇ in diameter.
• Figure 7(C) the fibroblast proliferation media cells from the spheres started spreading outwards (Figure 7(C)) to populate the culture dish thereby forming a cell monolayer.
• the regions where the cells had once been aggregated labelled for collagen type II whereas the cells in monolayer did not ( Figure 7(D)).
• Keratocytes seeded in the fibroblast proliferating medium formed an even monolayer of fibroblast-like cells ( Figure 8(A)) which did not label for either nestin or collagen type II ( Figure 8(B)).
• Figure 8(A) Keratocytes seeded into fibroblast proliferation medium failed to form the necessary cell aggregations. Therefore, in order to form fibroblast clusters, the confluent fibroblasts were dissociated from the culture dish, pelleted, and grown as a pellet culture in chondrogenic differentiation medium for a further three weeks.
• Example 11 TGFP3 and dexamethasone treatment does not induce deposition of fibrotic proteins or cause corneal opacity
• Example 12 Change in mRNA expression of collagen type II and type I upon treatment in vivo
• Rat corneas which were treated in vivo for 1 week, 7 weeks, and 3 weeks followed by a non-treatment period of 4 weeks were subjected to quantitative gene expression analysis. The aim was to determine whether type II collagen expression decreases again and/or permanently ceases after growth factor treatment is withdrawn. The effect of the treatment on native corneal collagen type II was also investigated.
• the explants of healthy tissue were cultured on 0.4 ⁇ pore size cell culture inserts (Millicell, France) at the interface between culture medium and a C0 2 rich environment. Corneal sections were placed epithelium side up on cell culture plate inserts with 3 ml of culture medium. The culture media was changed every other day.
• the basal medium used was Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% Anti-Anti (antibiotic-antimycotic solution) and 1% GIutaMAXTM (GIBCO®).
• DMEM Dulbecco's Modified Eagle Medium
• Anti-Anti antibiotic-antimycotic solution
• GIutaMAXTM GIutaMAXTM
• Example 15 Comparative dosages for TGFP3 and dexamethasone
• human scleral cells have been shown to retain their chondrogenic potential in vivo after being transplanted into a rat cartilage defect (Seko et al. 2008). It is known that the fibroblastic cells of the sclera and the corneal stroma share a common embryological origin.
• keratocytes seeded in culture medium containing TGFp3 and dexamethasone and in the absence of serum spontaneously formed cell spheroids within 2-3 days by cell aggregation and by three weeks these cell clusters labelled positive for cartilage specific type II collagen.
• type I collagen expression was also increased.
• the medium was changed to a control medium containing fetal calf serum the cell clusters dispersed into a monolayer of cells. Cells growing in the monolayer no longer expressed type II collagen.
• keratocytes which were first proliferated as fibroblasts in serum containing medium did not secrete collagen type II when the medium was changed to the TGF 3 and dexamethasone containing chondrogenic differentiation medium. This suggests that once proliferated as fibroblasts the cells lose the ability to differentiate along a chondrogenic pathway. Further to this, fibroblasts grown in three-dimensional culture in chondrogenic differentiation medium as a pellet also failed to express cartilage specific collagen type II. These results suggest that the quiescent keratocyte phenotype and cell aggregation are important to chondrogenic differentiation.
• the results here indicate a marked decrease in keratocyte density in the posterior part of the stroma of the untreated keratoconic cornea also.
• keratoconus there is a general thinning of the cornea. It is not known, however, whether this is due to the apoptosis of keratocytes and subsequent decreased production of ECM or whether keratocyte apoptosis is secondary to the process of corneal thinning.
• Collagen crosslinking one of the current treatments for keratoconus, results in an initial period of keratocyte apoptosis in the anterior part of the stroma. This is then followed by a period of repopulation of the stroma by the keratocytes. Keratocyte cell death is generally seen in response to an injury and in the case of crosslinking is understood to be as a result of UVA-induced cellular damage. This apoptotic response is thought to have evolved in order to protect the cornea from further inflammation (Wilson, Netto, & Ambrosio 2003).
• Nanoindentation has been employed in the assessment of postoperative therapeutic methods such as crosslinking for keratoconus (a corneal dystrophy) and post- LASIK ectasia in the eye.
• collagen crosslinking caused a two-fold increase in the elastic modulus in the anterior corneal stroma while the posterior stroma was unaffected by the treatment (Dias, Diakonis, Kankariya, Yoo, & Ziebarth 2013).
• anterior corneal elasticity was measured.
• the results in this study do indicate that posterior stroma keratocyte density was altered in the TGFP3 and dexamethasone treated corneas.
• nanoindentation does not measure the properties of the individual collagen fibrils it can measure the changes in the inherent elastic property of the cornea which will be altered on collagen II deposition with a subsequent increase in collagen crosslinking. Structural differences within the stroma are reflected in the corresponding differences in biomechanical properties. The results here show that there was almost a three-fold increase in elastic modulus and hardness in the growth factor treated rat corneas. These results indicate that the treatment results in a stiffer cornea with higher elasticity.
• the elastic modulus is a measure of a substance's resistance to being deformed elastically and therefore a higher elastic modulus indicates that a material is more difficult to deform.
• the immunohistochemical labelling results coupled with the gene expression studies and biomechanical testing show that keratocytes within an intact cornea are amenable to reprogramming along a chondrogenic pathway by treatment with TGFP3 and dexamethasone.
• the reprogramming by combined TGFP3 and dexamethasone treatment is stochastic and may be controlled via the modulation of the growth factor treatment period to result in stiffer, more elastic corneas.
• administration of both agents is required; when TGF 3 and dexamethasone are tested separately, no collagen type II production in keratocytes is observed.
• a novel treatment is therefore proposed for keratoconus and other eye conditions using in vivo tissue engineering, by administration of TGFP3 and dexamethasone, as described herein.
• Sheep are sedated in accordance with standard operating procedure in the housing facility.
• the eye drop formulation with optimal TGFP3 and dexamethasone concentrations (volume scaled) based upon the rodent dose optimisation studies are instilled in the right eye followed by the placing of corneal INTACS® (or similar scleral rings) to hold the desired curvature of the cornea during collagen deposition (Figure 18). Eye drops are continued to be administered either once or twice daily (as determined in rodent optimisation studies) for a period of three weeks.
• the INTACS® are then removed and the animals are continued to be housed for a further three weeks or six months.
• corneal thickness and curvature measurements are taken before treatment and at the end of the treatment (when the INTACS® are removed.
• the portable corneal pachymeter is used to detect changes in corneal thickness of treated versus control contralateral corneas in vivo.
• a portable Pentacam® is used to measure corneal curvature as well as corneal thickness of the sheep eyes before and after treatment ( Figures 19(E) and (F)). Corneal measurements are repeated again at three weeks after lenses removal with the final (most accurate) Pentacam® measurements. These are taken after killing the animal but prior to eye removal for immunohistological and biomechanical analysis as described above for rodent corneas. In the unlikely event that the sheep are unable to tolerate a hard contact lens (signs of infection, inflammation or irritability), the study is continued without lenses, which allows completion of key parameters such as type II collagen deposition and distribution, and biomechanical properties.

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