AU2011303580A1 - Method for performing a reversible refractive procedure - Google Patents

Abstract

The present invention is directed to a method for performing a reversible refractive procedure by reimplanting a refractive lenticule that has been obtained by a refractive procedure on the eye, the method comprising the steps of preparing the refractive lenticule for reimplantation, creating a corneal flap in an eye of an acceptor, transferring the refractive lenticule under the corneal flap, and repositioning and covering the acceptor's corneal flap; thereby reimplanting the refractive lenticule. The present invention is also directed to a refractive lenticule obtained by a refractive procedure on the eye of a donor for use in a method for subsequent reimplantation in a reversible refractive procedure on an acceptor, wherein the refractive lenticule is stored at a temperature below 0°C.

Description

WO 2012/035403 PCT/IB2011/002125 Method for Performing a Reversible Refractive Procedure Cross-Reference To Related Application [0001] This application makes reference to and claims the benefit of priority of an application for "Storage of Lenticules Following Femtosecond Laser Refractive Lenticule Extraction Procedures from Cornea for Subsequent Reimplantation" filed on September 13, 2010 with the United States Patent and Trademark Office, and there duly assigned serial number 61/382,037. The content of said application filed on September 13, 2010 is incorporated herein by reference for all purposes, including an incorporation of any element or part of the description, claims or drawings not contained herein and referred to in Rule 20.5(a) of the PCT, pursuant to Rule 4.18 of the PCT. Technical Field 10002] Various embodiments relate to the field of refractive procedures or surgeries, in particular, reversible refractive procedures on refractive lenticules. Background 100031 Surgical correction for refractive errors may be performed by a number of procedures. Laser correction involves procedures such as EpiLASIK, LASIK or conductive keratoplasty, while lens based corrections involve the use of phakic intra ocular lenses. Despite the emergence of other procedures, due to its fast visual rehabilitation and painless postoperative course, LASIK is still the procedure of choice for both patients and refractive surgeons. [00041 LASIK consists of two steps: the first step involves the formation of a corneal flap and the second step is when the excimer ablation is performed in the comeal stromal bed. Femtosecond lasers (FL) have been widely used in LASIK surgery to fashion the corneal

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WO 2012/035403 PCT/IB2011/002125 flap. The laser will cut a lamellar flap followed by a vertical corneal incision. A vertical cut may be produced since the femtosecond laser can deliver laser pulses of 1 micron diameter at a preselected depth in the cornea, which then expands to 20/30 microns with the formation of a cavitation bubble. These pulses create micro-photodisruption of the corneal tissue by the formation of an expanding bubble of carbon dioxide and water, which in turn cleaves the tissue and creates a plane of separation. The flap is then removed to expose the stroma and the refractive treatment is done by the excimer laser over the denuded stroma. The advantages of femtosecond laser over microkeratome LASIK flaps have been reduced postoperative dry eye symptoms, less chance of dislocation from trauma and reduced incidence of button holes or free caps. [0005] Even though Femto-LASIK has demonstrated good efficacy, it can still be associated with side effects found in standard LASIK, for example, glare, haloes, dry eye and still requires the use of two separate laser machines, one for the flap the other for the excimer ablation. This increases the length of time of the overall procedure and the costs. [00061 Recently, a "one laser" refractive surgery known as Refractive Lenticular Extraction (ReLEx) has been proposed. It consists of performing the entire refractive procedure without an excimer laser. ReLEx surgery involves 2 different surgical approaches, Femtosecond Lenticule Extraction (FLEx), and Small Incision Lenticule Extraction (SMILE). [0007] FLEx mimics conventional LASIK surgery by performing a LASIK type hinged flap to extract the lenticule. The femtosecond laser creates four stromal incisions to create a corneal lenticule and a corneal flap. The size and shape of the lenticule is calculated based on the patient refractive error. The lenticule is separated from the flap above and the rest of the cornea below using a small spatula. Following this, the lenticule is grasped with a forceps and extracted from the eye. Finally, the flap is repositioned and the interface washed. At this time the refractive error is already corrected without the use of a second laser. [0008] The SMILE procedure is a flap-less form of small incision surgery, in which the lenticule is extracted through a small superior pocket incision, without the formation or lifting of a full flap, which has the advantages of minimal incision or disturbance to the corneal surface, greater wound strength as a full flap is not raised, the possibility of less 2 WO 2012/035403 PCT/IB2011/002125 flap-related optical aberrations, with enhanced visual quality and less loss of contrast sensitivity, and less neurotrophic keratopathy or dry eye, as less comeal nerves may be disrupted at the corneal periphery when a full flap is used. Both procedures come under the umbrella term of Refractive Lenticule Extraction (ReLEx). 100091 The advantages of FLEx and SMILE treatments are obviation of the need for a separate excimer laser, shortened length of the refractive procedure, reduced patient inconvenience from moving from one laser to another, and more accurate ablation especially in high myopia as it appears that no algorithm adjustment is needed in ReLEx unlike that of excimer based LASIK surgery. The advantages of a pocket incision in SMILE as apposed to a full LASIK type flap have also been described above. [00101 There are few publications on FLEx or SMILE since as yet it is not commercially released and the procedure is only carried out by a few centres. Currently, following the creation of the refractive lenticule, the lenticule is removed from the eye and then disposed of. [00111 Presently, there are no laser based reversible refractive procedures. The only reversible refractive procedures currently available are artificial lens based systems that require intraocular surgery and considerable risks involved in reversal. [00121 Thus it is an object of the present invention to provide a method for storing the refractive lenticule upon its creation and subsequently performing a reversible refractive procedure thereon. Summary of the Invention [0013] In a first aspect, the present invention relates to a method for performing a reversible refractive procedure by reimplanting a refractive lenticule that has been obtained by a refractive procedure on the eye, the method comprising the steps of preparing the refractive lenticule for reimplantation, creating a corneal flap in an eye of an acceptor, transferring the refractive lenticule under the corneal flap, and repositioning and covering the acceptor's corneal flap; thereby reimplanting the refractive lenticule. [00141 According to a second aspect, the present invention relates to a refractive lenticule obtained by a refractive procedure on the eye of a donor for use in a method for 3 WO 2012/035403 PCT/IB2011/002125 subsequent reimplantation in a reversible refractive procedure on an acceptor, wherein the refractive lenticule is stored at a temperature below 0 0 C. Brief Description of the Drawings 100151 In the drawings, like reference characters generally refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention. In the following description, various embodiments of the invention are described with reference to the following drawings, in which: 100161 Figure 1 shows a flow diagram illustrating the processes performed for short term study of FLEx vs LASIK, in accordance to various embodiments; [00171 Figure 2 shows in vivo confocal microscopy of keratocyte activation, according to various embodiments; [00181 Figure 3a shows immunohistochemistry images of fibronectin expression of the central cornea, according to various embodiments; [00191 Figure 3b shows immunohistochemistry images of fibronectin expression of the periphery of flap, according to various embodiments; [00201 Figure 4a shows immunohistochemistry images of inflammation of the central cornea, according to various embodiments; [00211 Figure 4b shows immunohistochemistry images of inflammation of the periphery of flap, according to various embodiments; [00221 Figure 5 shows light microscopy of an extracted lenticule at (a) 100x magnification; (b) 400x magnification of the anterior edge; (c) 400x magnification of the posterior edge; and (d) 5600x magnification of a keratocyte, according to various embodiments; [0023] Figure 6a shows slit lamp (SL) images of the lenticule following re-implantation, in accordance to various embodiments; [00241 Figure 6b shows confocal microscopic images of the lenticule following re implantation in accordance to various embodiments; [0025] Figure 6c shows a plot of corneal thickness, in accordance to various 4 WO 2012/035403 PCT/IB2011/002125 embodiments; [00261 Figure 7 shows in vivo confocal microscopy of the lenticule, according to various embodiments; 100271 Figure 8 shows topography results 28 days after re-implantation of the lenticule, in accordance to various embodiments; [0028] Figure 9 shows immunohistochemistry images of inflammation of the eye 28 days after re-implantation of the lenticule compared against a control (in the rightmost column), according to various embodiments; [0029] Figure 10 shows confocal microscopic images of inflammation similar to Figure 9 using various labels and stains, in accordance to various embodiments; [00301 Figure I Ia shows a light microscopic image of extracted stromal lenticules, according to various embodiments; [00311 Figure 11 b shows a magnified light microscopic image of the extracted stromal lenticules, according to various embodiments; [0032] Figure 1 c shows a light microscopic image of a cell culture of the extracted stromal lenticules, according to various embodiments; [00331 Figure Ild shows a light microscopic image of the cell culture of Figure 1 ic after adding the stock freezing solution, according to various embodiments; 10034] Figure Ile shows a light microscopic image of the cell culture following a culture of a fresh lenticule, according to various embodiments; [0035] Figure 1 If shows a light microscopic image of the cell culture after 1 month of cryopreservation, according to various embodiments; and 100361 Figure 1 Ig shows gel electrophoresis for real-time PCR, in accordance to various embodiments. Detailed Description 100371 The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and 5 WO 2012/035403 PCT/IB2011/002125 structural, and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments. [00381 In one aspect, the present invention relates to a method for performing a reversible refractive procedure by reimplanting a refractive lenticule that has been obtained by a refractive procedure on the eye. The method comprising the steps of preparing the refractive lenticule for reimplantation, creating a corneal flap in an eye of an acceptor, transferring the refractive lenticule under the corneal flap, and repositioning and covering the acceptor's corneal flap; thereby reimplanting the refractive lenticule. [00391 In the context of various embodiments, the term "refractive procedure" may generally refer to any eye surgery used to improve the refractive state of the eye. This may include methods of surgical remodeling of the cornea or cataract surgery. For example, use of excimer lasers to reshape curvature of the cornea is a refractive procedure. The term "reversible refractive procedure" may refer to a procedure to invert or undo the refractive procedure. 10040] The term "refractive lenticule", as used herein, relates to a piece of stromal corneal tissue that is removed to correct refractive error of the eye. For example, the refractive lenticule may be peeled off the corneal bed. The refractive lenticule may have any shape and can, for example, be lense-shaped. 100411 In various embodiments, the refractive lenticule has been stored at a temperature below 0 'C. For example, the refractive lenticule may have been stored at about -80 'C. 10042] For storage, the refractive lenticule may be freezed and the frozen refractive lenticule may be stored in a freezer or a cryopreserving environment. During storage, the refractive lenticule may be held by a support to maintain a curved surface comparable to a corneal surface. For example, the support may be a rigid gas permeable contact lens or a mounting structure comprising a curved surface. As used herein, the term "mounting structure" may refer to any structure or mechanism to hold the refractive lenticule in place. The mounting structure may include a right and left mount to hold the refractive lenticule at each end such that the refractive lenticule is spreaded like a flat sheet. The mounting structure may also allow for easy removal of the refractive lenticule without 6 WO 2012/035403 PCT/IB2011/002125 damage. [00431 In various embodiments, the step of preparing the refractive lenticule for reimplantation may comprise thawing the refractive lenticule in a water bath of about 37 'C. Upon thawing, the refractive lenticule may be washed in a buffer solution. For example, the buffer solution may be selected from the group consisting of morpholinepropanesulfonic acid, a Phosphate Buffered Solution (PBS), a trehalose buffer, and a sucrose buffer. [0044] In various embodiments, the step of preparing the refractive lenticule for reimplantation may further comprise enzymatically digesting the thawed refractive lenticule for culture of corneal stromal fibroblast. The step of preparing the refractive lenticule for reimplantation may also further comprise adjusting refractive errors of the refractive lenticule to restore near vision, or to correct presbyopia, or to treat complications of keratectasia. [0045] According to various embodiments, the step of repositioning and covering the acceptor's corneal flap may comprise placing a bandage contact lens over the repositioned corneal flap. [0046] In various embodiments, the refractive lenticule for reimplantation may have been obtained by a method comprising the steps of creating an incision in an eye of a donor, and extracting the refractive lenticule through the incision. The incision may be made using a one-laser refractive surgery. The one-laser refractive surgery may be Refractive Laser Extraction (ReLEx). [0047] In one embodiment, the ReLEx may comprise Small Incision Lenticule Extraction (SMILE). [0048] In other embodiments, the incision may comprise one or two or three or four stromal incisions. For example, the four stromal incisions may further create a corneal flap in the donor's eye. The ReLEx in this embodiment may be Femtosecond Lenticule Extraction (FLEx). 10049] In various embodiments, the donor and the acceptor may be the same subject. In other embodiments, the donor and the acceptor may be different subjects. [00501 In another aspect, the present invention relates to a refractive lenticule obtained by a refractive procedure on the eye of a donor for use in a method for subsequent 7 WO 2012/035403 PCT/IB2011/002125 reimplantation in a reversible refractive procedure on an acceptor, wherein the refractive lenticule is stored at a temperature below 0 0 C. 100511 In various embodiments for storage, the refractive lenticule may be snap-freezed in liquid nitrogen for about 10 minutes. The snap-freezing process may be carried out for about 5 minutes to about 15 minutes, or about 5 minutes to about 8 minutes, or about 12 minutes to about 15 minutes. The refractive lenticule may be stored at about -80 'C. [00521 In various embodiments, the refractive lenticule may be cryopreserved. For the cryopreservation, the refractive lenticule may be suspended in a serum-containing medium in a vessel, a freezing solution of about 10% serum and about 20% organic solvent is added in a dropwise manner into the vessel to form a mixture of about 10% serum and about 10% organic solvent, the vessel containing the mixture is placed in a -80 'C freezer for at least 4 hours; and the frozen mixture is transferred into liquid nitrogen for storage. 100531 The organic solvent may be selected from the group consisting of organic solvent is selected from the group consisting of hexane, heptane, cyclohexane, benzene, pyridine, dichloromethane, chloroform, carbon tetrachloride, carbon disulfide, tetrahydrofuran, dioxane, diethyl ether, diisopropylether, ethylene glycol monobutyl ether, methyl ethyl ketone, methyl isobutyl ketone, acetone, cyclohexanone, ethyl acetate, isobutyl isobutyrate, ethylene glycol diacetate, dimethylformamide, acetonitrile, N,N-dimethyl acetamide, nitromethane, acetonitrile, N-methylpyrrolidone, dimethylsulfoxide water, methanol, ethanol, butyl alcohol and formic acid. The serum may be a fetal bovine serum. 100541 In various embodiments, the refractive lenticule may be obtained by creating an incision in an eye of the donor; and extracting the refractive lenticule through the incision. The incision may be created using a one-laser refractive surgery. For example, the one-laser refractive surgery is Refractive Laser Extraction (ReLEx). The ReLEx may comprise Small Incision Lenticule Extraction (SMILE) or Femtosecond Lenticule Extraction (FLEx). [00551 In one embodiment, the donor and the acceptor may be the same subject. In another embodiment, the donor and the acceptor may be different subjects. [00561 Prior to the subsequent reimplantation, the stored refractive lenticule may be 8 WO 2012/035403 PCT/IB2011/002125 processed to adjust its refractive errors to restore near vision, or to correct presbyopia, or to treat complications of keratectasia. [00571 In the context of various embodiments, the term "about" or "approximately" as applied to a value may encompass the exact value and a variance of +/- 5% of the value. [0058] The phrase "at least substantially" may include "exactly" and a variance of +/- 5% thereof. As an example and not limitations, "A is at least substantially same as B" may encompass embodiments where A is exactly the same as B, or where A may be within a variance of +/- 5%, for example of a value, of B, or vice versa. 100591 In order that the invention may be readily understood and put into practical effect, particular embodiments will now be described by way of examples and not limitations, and with reference to the figures. Examples 100601 Various embodiments provides the storage of refractive lenticules following the FLEx or SMILE ReLEx or other similar stromal lenticule extraction procedures to make these completely reversible or modifiable refractive procedures. [00611 The main advantage of FLEx/SMILE is that in theory it may be able to correct higher refractive powers with removal of less tissue and also it is a potentially reversible procedure. A FLEx model in an animal is firstly established. A rabbit model is chosen since this has been previously chosen to study refractive surgical procedure, for example, LASIK and PRK so as to provide basis for comparison. [00621 In an experimental model, the short term effect of the FLEx procedure compared to LASIK is assessed. Subsequently a model of re-implantation in an animal model is established and the viability of the lenticule following the cryopreservation method is assessed. 9 WO 2012/035403 PCT/IB2011/002125 Method [00631 Example A: Comparison to LASIK - Establishing ReLEx model [00641 Thirty six (36) New Zealand White rabbits (about 3-4 kg body weight) have been procured from animal holding unit of National University of Singapore, Singapore. Rabbits have been housed individually at room temperature on about 12 hours: 12 hours of light:darkness, with rabbit pellets and water available ad libitum. [00651 Eighteen rabbits (18) are used to study the short term effect of the surgery (i.e., to be sacrificed 1 day after surgery): [00661 - FLEx (mild correction: -3.OOD) on right eye and control left eye (naive)--+ 3 rabbits [00671 FLEx (moderate correction: -6.OOD) on right eye and control left eye (naive) -- 3 rabbits [0068] - FLEx (severe correction: -9.OOD) on right eye and control left eye (naive) - 3 rabbits [00691 - LASIK (mild correction: -3.OOD) on right eye and control left eye (naive) -> 3 rabbits 100701 LASIK (moderate correction: -6.OOD) on right eye and control left eye (naive) -> 3 rabbits [00711 - LASIK (severe correction: -9.OOD) on right eye and control left eye (naive) -> 3 rabbits [00721 Rabbits are anesthesized with a combination of xylazine hydrochloride (20 mg/kg) and kentamine hydrochloride (40 mg/kg) intramuscularly. In addition, preservative-free oxybuprocain hydrochloride eyedrops (Benoxinat SE 0.4%) are applied to each eye just before the surgery. After the surgery, the eyelids are closed with a temporary tarsorrhaphy placed with a 6-0 suture. [00731 The rabbits are then sacrificed under anesthesia by intracardiac injection of 5 ml embutramine 0.2 g/ml, mebezonium iodide 0.05 g/ml, and tetracaine hydrochloride 0.005 g/ml. All rabbits are treated in accordance with the ARVO Statement for the Use 10 WO 2012/035403 PCT/IB2011/002125 of Animals in Ophthalmic and Vision Research. The experimental protocol is approved by the Institutional Animal Care and Use Committee of SingHealth. 100741 Example B: Storage and re-implantation of stromal lenticule [0075] Eighteen different rabbits (18) are used to study the long term effect of the surgery (i.e., to be sacrificed 28 day after surgery). [00761 The extracted stromal lenticules are carefully transferred on the Rigid Gas Permeable (RGP) contact lens. A marking at 6 o'clock position is made on the lens to indicate the anatomical position of the lenticule on the cornea before extraction. A drop of 10% DMSO (Sigma, St. Louis, MI) is applied on the lenticule before a 10-minute snap freezing in the liquid nitrogen (N 2 ). The lenticule is stored in -80 OC until further use. [00771 The re-implantation of the lenticule is performed 28 days after the initial ReLEx procedure. A Siebel spatula is inserted under the flap near the hinge and the flap is then lifted. The lenticule is transferred onto the exposed stromal bed by a sliding motion from the RGP contact lens. After the flap is repositioned, a bandage contact lens (Bausch & Lomb, Rochester, NY) is placed over the flap and the eyelid is closed with a temporary tarsorraphy for 3 days. Post-operative follow up is done on day 3, 14, and 28 after the re-implantation. Gentamycin and Dexamethasone of 1 ml each are injected subconjunctivally following the re-implantation procedure and each post-operative follow up. 10078] Figure 1 shows a flow diagram illustrating the processes performed for short term study (sacrifice 1 day after surgery in Example A) and long term study (sacrifice 28 days after surgery in Example B) of FLEx vs LASIK. In the context of various embodiments, the term "OCT" for example in Figure 1 refers to optical coherence tomography. The term "immunofluorescent staining" may generally refer to fluorescent dyeing for detection and imaging of biological samples using light microscopy with a fluorescence microscope. The term "TUNEL" may generally refer to terminal deoxynucleotidyl transferase dUTP nick end labeling for detecting DNA fragmentation by labeling the terminal end of nucleic acids. The term "in vivo confocal" refers to in vivo confocal microscopy (IVCM), wherein confocal 11 WO 2012/035403 PCT/IB2011/002125 microscopy may generally refer to a microscopy for obtaining increased resolution by limiting the illumination and observation systems to a single point. The term "refractive states" may generally refers to a determining procedure for the purpose of obtaining glasses and includes specification of lens type (monofocal, bifocal, other), lens power, axis, prism, absorptive factor, impact resistance, and other factors. [00791 Example C: Lenticule Viability - Before and after Cryopreservation [00801 Extracted stromal lenticules from clinical patients and also from human cadaver eyes have undergone TUNEL assay, transmission electron microscopy (TEM), cell culture and real-time PCR. Extracted human stromal lenticule is washed twice (about 10 minutes each) in a phosphate buffered saline (PBS) buffered antibiotic/antimycotic solution. The lenticule is then transferred into a cryovial and resuspended in 500 pl medium containing 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA). A stock freezing solution containing 10% FBS and 20% dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA.) are added slowly in a dropwise manner to an equal volume ratio, to make a final volume of 1 ml freezing solution containing 10% FBS and 10% DMSO. [00811 Freezing of the cryovial containing the stromal lenticule is carried out in a cryo container ("Mr. Frosty", Nalgene, Denmark), within a -80 "C freezer overnight, and transferred into liquid nitrogen the following day for long-term storage. 10082] Thawing of frozen stromal lenticule is carried out after one month of cryopreservation where the vial containing the frozen stromal lenticule is rapidly thawed in water bath at 37 "C. Immediately after thawing, the stromal lenticule is rinsed twice in a PBS solution to remove the cryoprotectant agents. Thawed lenticules are either enzymatically digested for the culture of corneal stromal fibroblast, or immersed in a tissue-freezing compound and frozen for cryosection to be analyzed with TEM and TUNEL assay. 12 WO 2012/035403 PCT/IB2011/002125 Discussion [0083] Table 1 shows the data extracted from topography results for various refractive procedures as carried out in the short term study of Example A. [0084] Table 1 Control 44.81 ± 0.75 46.77 ± 0.32 LASIK (-3.OOD correction) 37.00 ± 1.44 39.15 ± 1.62 FLEx (-3.OOD correction) 37.16 ± 1.02 40.52 ± 0.08 LASIK (-6.OOD correction) 35.68 33.25 FLEx (-6.OOD correction) 38.34 ± 1.84 36.59 ± 1.35 FLEx (-9.OOD correction) 35.45 34.09 [0085] In Table 1, Kh and Kv represent horizontal Keratometry readings and vertical Keratometry readings, respectively, to determine the exact curvature (steepness) of an eye with respective standard deviations. That is, the larger the Keratometry reading, the steeper or the more curved is the eye. The control procedure is an unoperated eye. For the same myopic correction, less corneal flattening using the FLEx procedure as compared to using the LASIK procedure is observed. For example, with -3.001D correction, using the FLEx procedure Kh is about 37.16 diopter, while using the LASIK procedure Kh is 37.00 diopter. The Kh using the FLEx procedure is larger than that using the LASIK procedure; thus the eye using the FLEx procedure is steeper or more curved, therefore less flattening. [00861 In Figure 2, in vivo confocal microscopy shows that there is no increase in keratocyte activation following FLEx as compared to LASIK for the same myopic 13 WO 2012/035403 PCT/IB2011/002125 correction. For example, keratocyte activation at the direct anterior of the keratotomy site is represented by the solid small arrows. However there is evidence of more inflammation in the LASIK group at higher corrections (large arrows). [00871 Figures 3a and 3b show immunohistochemistry images of increasing fibronectin expression with increasing power of myopic correction with both ReLEx and LASIK. As used herein, the term "ReLEx" may be interchangeably referred to as "FLEx". In this regard, the myopic correction of -6.OOD is of a larger or increased power as compared to the myopic correction of -3.OOD. For example in Figure 3, increasing fibronectin expression in term of higher intensity shades are observed as indicated by the arrows. [0088] Figures 4a and 4b show immunohistochemistry images of inflammation. In Figure 4b, it is observed that there are similar levels of inflammation at low correction (i.e., at -3.OOD) between LASIK and ReLEx (as indicated by the area enclosed by the double-dashed rectangle). However, at higher corrections (i.e., at -6.OOD) there is more inflammation in the LASIK group (as indicated by the arrows in Figure 4a and the area enclosed by the single-dashed ellipse in Figure 4b). [0089] Light microscopy of an extracted lenticule shows evidence of a biconvex shaped lenticule (as seen in Figure 5a, magnified 100x), and high power TEM shows in Figure 5d that the keratocytes in the lenticule are viable indicating no evidence of degeneration or cell death. Figure 5b shows a 400x magnified image of the anterior edge and Figure 5c shows a 400x magnified image of the posterior edge. Figure 5d shows a 5600x magnified image of a keratocyte. [00901 Slit lamp (SL) images in Figure 6a and the confocal microscope images of Figures 6b show clearing of lenticule following re-implantation in accordance to various embodiments, for example, in Example B. Figure 6c shows an increase in corneal thickness back to preoperative stat on OCT. [00911 In vivo confocal microscopy of the lenticule in Figure 7 shows an increased hyper intense signal (as indicated by arrows) in the early postoperative period that reduces (in intensity) by 14 days and 28 days after re-implantation. 14 WO 2012/035403 PCT/IB2011/002125 [00921 The topography results of Figure 8 shows that 28 days after re-implantation of the lenticule, the keratometry readings are within -0.6 ± 0.8D of the preoperative keratometry. [00931 Figure 9 shows immunohistochemistry images of inflammation of the eye 28 days after re-implantation of the lenticule compared against a control (in the rightmost column). The leftmost column of Figure 9 shows 100x magnified immunohistochemistry images of the eye 28 days after re-implantation of the lenticule while the center column shows respective 200x magnified images of the enclosed rectangular areas correspondingly marked out in each of 100x magnified images in the leftmost column. It is shown from the images of the eye 28 days after re-implantation of the lenticule, more clearly in the enlarged images of the center column that new extracellular matrix formation and minimal inflammation are present, as indicated by the light shades within the area enclosed by the dashed ellipses. However, extracellular matrix formation and inflammation are minimal as the leftmost column is comparatively similar to the rightmost column of the control in terms of fluorescence intensity and distribution. Thus Figure 9 shows that there is still evidence of minimal extracellular matrix formation (e.g., Fibronectin, CD18, Tenascin-C) and tissue inflammation (e.g., a-SMA, Thy-1) deposition at 28 days of post-operation. [0094] Figure 10 shows confocal microscopic images of inflammation similar to Figure 9 using various labels (TUNEL) and stains (Ki-67 and Phalloidin). [00951 There is minimal evidence of proliferating cells and myofibroblasts at 28 days of post-operation, indicating minimal haze formation. [00961 Figure 11 a shows a light microscopic image of extracted stromal lenticules as described in Example C. Figure 11 b shows a magnified light microscopic image of the extracted stromal lenticules before cryopreservation following collagenase digestion as described in Example C. Figure 11 c shows a cell culture of the extracted stromal lenticules. After adding the stock freezing solution containing 10% FBS and 20% DMSO to the cell culture, longish cells (fibroblasts) are observed in Figure 11 d. Figure 11 e shows the cell culture following a culture of a fresh lenticule, and Figure 11 f shows cell culture after 1 month of cryopreservation. Figure 11 g shows the gel electrophoresis 15 WO 2012/035403 PCT/IB2011/002125 for real-time PCR of the extracted stromal lenticules after cryopreservation, wherein KERA refers to keratoctyes, ALDH31A refers to Aldehyde dehydrogenase 31A, GAPDH refers to glyceraldehyde-3-phosphate dehydrogenase, and the leftmost band of the gel electrophoresis being a marker. In Figure 11 g, the presence of keratocyte mRNA is observed. [00971 Using TEM and cell culture, presence of keratoctyes before and after cryopreservation are observed and that they were viable. Viability is shown both with 4',6-diamidino-2-phenylindole (DAPI)/TUNNEL assay and also cell culture. Cell phenotype is shown on real-time PCR to verify that they are keratocytes. [00981 The object of various embodiments is to store the lenticule following extraction for an indefinite period (or for at least the lifetime of a patient) in a freezing manner. [00991 Freezing may include all forms of cryobiology from fast freezing to vitrification. Following preservation, the lenticule is stored by freezing. Storage is required so that re-implantation of the lenticule at a later date can be carried out. The re-implantation is able to correct the original refractive error that has been removed by the lenticule; thereby reversing the procedure, or to enable further adjustment of the refractive errors, including restoration of near vision, This allows for solving presbyopia at a subsequent date, or to treat the potential complication of keratectasia due to, for example, inadequate stromal bed thickness and strength, which may be associated with collagen cross-linking of the lenticule and/or stromal recipient bed. This makes the ReLEx procedures reversible forms of corneal refractive surgery, which offers considerable advantages over current techniques. [00100] The reversible refractive procedure allows for (i) the reimplantation of the lenticule if needed to treat future presbyopia, or (ii) the correction of the refractive error if the patient wants the procedure reversed, or (iii) the treatment of keratectasia, or (iv) more accurate calculation of intraocular lens power for cataract surgery if the patient needs this as he/she gets older. Ultimately, it is desirable that the lenticules are allowed to be stored for the lifetime of the patient so that it may be replaced at anytime. [001011 The lenticules may also be modified by surgical or excimer laser techniques to alternative refractive powers. Various clinical scenarios are: 16 WO 2012/035403 PCT/IB2011/002125 - Scenario 1: Total reversal of refractive procedure in the same patient The unmodified lenticule is re-implanted back into the same patient either through the original flap or via a new femtosecond ablation procedure, potentially for treatment of keratectasia. - Scenario 2: Reimplantation into the same patient to treat subsequent alterations in refractive error The lenticule may be modified (e.g. by excimer ablation) to suit the required refractive error, e.g. hyperopia or astigmatism. - Scenario 3: Reimplantation to treat onset of presbyopia in the same patient The lenticule may be modified to a low hyperopic power (e.g. about 1.OOD or about 1.50D) and reimplanted in the non-dominant eye of a presbyopic patient to effect monovision correction. - Scenario 4: Allograft lenticule insertion The lenticule may be donated by the patient (with appropriate informed consent and donor serology testing) to be used as a lamellar comeal stromal allograft transplant to effect refractive treatments for other patients in the same situations as Scenarios 1 to 3 above. - Scenario 5: Allograft lenticule insertion The lenticules may be donated by the patient (with appropriate informed consent and donor serology testing) to be used as a lamellar corneal stromal allograft transplant for treating a variety of other corneal refractive, tectonic or therapeutic conditions, such as keratoconus, corneal thinning and irregularities, descemetoceles, and corneal perforations. - Scenario 6: Allograft lenticule insertion as a standardized presbyopic biological implant The lenticules may be donated by the patient (with appropriate informed consent and donor serology testing) to be used as a lamellar corneal stromal allograft transplant for treating presbyopia in other patients by standardized modification of the lenticules to presbyopic designs, such as multifocal designs, 17 WO 2012/035403 PCT/IB2011/002125 wavefront guided designs, diffractive designs, aspheric profile designs, and a small (e.g. about 3-5 mm) "centre near" presbyopic implant to effect a bifocal cornea effect. [001021 A lenticule banks linked to the ReLEx procedure with storing them on a RGP contact lens or similar devices or a mounting structure which has a curved surface replicating the corneal surface radius to ensure correct shape for storage. The patients' lenticules may be stored in such a bank underwritten costs by the patient for possible reimplantation at a future date. [00103] While the invention has been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is thus indicated by the appended claims and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced. 18

Claims (31)

1. A method for performing a reversible refractive procedure by reimplanting a refractive lenticule that has been obtained by a refractive procedure on the eye, the method comprising the steps of: preparing the refractive lenticule for reimplantation; creating a corneal flap in an eye of an acceptor; transferring the refractive lenticule under the corneal flap; and repositioning and covering the acceptor's corneal flap; thereby reimplanting the refractive lenticule.

2. The method as claimed in claim 1, wherein the refractive lenticule has been stored at a temperature below 0 'C.

3. The method as claimed in claim I or 2, wherein the step of preparing the refractive lenticule for reimplantation comprises thawing the refractive lenticule in a water bath of about 37 'C.

4. The method as claimed in claim 3, wherein upon thawing, the refractive lenticule is washed in a buffer solution.

5. The method as claimed in claim 4, wherein the buffer solution is selected from the group consisting of morpholinepropanesulfonic acid, a Phosphate Buffered Solution (PBS), a trehalose buffer, and a sucrose buffer.

6. The method as claimed in any one of claims 1 to 5, wherein the step of preparing the refractive lenticule for reimplantation further comprises enzymatically digesting the thawed refractive lenticule for culture of corneal stromal fibroblast.

7. The method as claimed in any one of claims I to 6, wherein the step of preparing the refractive lenticule for reimplantation further comprises adjusting refractive 19 WO 2012/035403 PCT/IB2011/002125 errors of the refractive lenticule to restore near vision, or to correct presbyopia, or to treat complications of keratectasia.

8. The method as claimed in any one of claims 1 to 7, wherein the step of repositioning and covering the acceptor's corneal flap comprises placing a bandage contact lens over the repositioned corneal flap.

9. The method as claimed in any one of claims 1 to 8, wherein the refractive lenticule for reimplantation has been obtained by a method comprising the steps of: creating an incision in an eye of a donor; and extracting the refractive lenticule through the incision.

10. The method as claimed in claim 9, wherein the incision is made using a one laser refractive surgery.

11. The method as claimed in claim 10, wherein the one-laser refractive surgery is Refractive Laser Extraction (ReLEx).

12. The method as claimed in claim 11, wherein the ReLEx comprises Small Incision Lenticule Extraction (SMILE).

13. The method as claimed in claim 10, wherein the incision comprises one or two or three or four stromal incisions.

14. The method as claimed in claim 13, wherein the four stromal incisions further create a corneal flap in the donor's eye.

15. The method as claimed in claim 11, wherein the ReLEx is Femtosecond Lenticule Extraction (FLEx).

16. The method as claimed in any one of claims 9 to 15, wherein the donor and 20 WO 2012/035403 PCT/IB2011/002125 the acceptor are the same subject.

17. The method as claimed in any one of claims 9 to 15, wherein the donor and the acceptor are different subjects.

18. A refractive lenticule obtained by a refractive procedure on the eye of a donor for use in a method for subsequent reimplantation in a reversible refractive procedure on an acceptor, wherein the refractive lenticule is stored at a temperature below 0 0 C.

19. The refractive lenticule for use as claimed in claim 18, wherein for storage, the refractive lenticule is snap-freezed in liquid nitrogen for about 10 minutes.

20. The refractive lenticule for use as claimed in claim 18 or 19, wherein the refractive lenticule is stored at about -80 'C.

21. The refractive lenticule for use as claimed in claim 18, wherein the refractive lenticule is cryopreserved.

22. The refractive lenticule for use as claimed in claim 21, wherein for the cryopreservation, the refractive lenticule is suspended in a serum-containing medium in a vessel; a freezing solution of about 10% serum and about 20% organic solvent is added in a dropwise manner into the vessel to form a mixture of about 10% serum and about 10% organic solvent; the vessel containing the mixture is placed in a -80 'C freezer for at least 4 hours; and the frozen mixture is transferred into liquid nitrogen for storage.

23. The refractive lenticule for use as claimed in claim 22, wherein the organic solvent is selected from the group consisting of organic solvent is selected from the group 21 WO 2012/035403 PCT/IB2011/002125 consisting of hexane, heptane, cyclohexane, benzene, pyridine, dichloromethane, chloroform, carbon tetrachloride, carbon disulfide, tetrahydrofuran, dioxane, diethyl ether, diisopropylether, ethylene glycol monobutyl ether, methyl ethyl ketone, methyl isobutyl ketone, acetone, cyclohexanone, ethyl acetate, isobutyl isobutyrate, ethylene glycol diacetate, dimethylformamide, acetonitrile, N,N-dimethyl acetamide, nitromethane, acetonitrile, N-methylpyrrolidone, dimethylsulfoxide water, methanol, ethanol, butyl alcohol and formic acid.

24. The refractive lenticule for use as claimed in claim 22 or 23, wherein the serum is a fetal bovine serum.

25. The refractive lenticule for use as claimed in any one of claims 18 to 24, wherein the refractive lenticule is obtained by creating an incision in an eye of the donor; and extracting the refractive lenticule through the incision.

26. The refractive lenticule for use as claimed in claim 25, wherein the incision is created using a one-laser refractive surgery.

27. The refractive lenticule for use as claimed in claim 26, wherein the one-laser refractive surgery is Refractive Laser Extraction (ReLEx).

28. The refractive lenticule for use as claimed in claim 27, wherein the ReLEx comprises Small Incision Lenticule Extraction (SMILE) or Femtosecond Lenticule Extraction (FLEx).

29. The refractive lenticule for use as claimed in any one of claims 18 to 28, wherein the donor and the acceptor are the same subject.

30. The refractive lenticule for use as claimed in any one of claims 18 to 28, wherein the donor and the acceptor are different subjects. 22 WO 2012/035403 PCT/IB2011/002125

31. The refractive lenticule for use as claimed in any one of claims 18 to 30, wherein prior to the subsequent reimplantation, the stored refractive lenticule is processed to adjust its refractive errors to restore near vision, or to correct presbyopia, or to treat complications of keratectasia. 23 WO 2012/035403 PCT/IB2011/002125 Method for Performing a Reversible Refractive Procedure ABSTRACT The present invention is directed to a method for performing a reversible refractive procedure by reimplanting a refractive lenticule that has been obtained by a refractive procedure on the eye, the method comprising the steps of preparing the refractive lenticule for reimplantation, creating a corneal flap in an eye of an acceptor, transferring the refractive lenticule under the corneal flap, and repositioning and covering the acceptor's corneal flap; thereby reimplanting the refractive lenticule. The present invention is also directed to a refractive lenticule obtained by a refractive procedure on the eye of a donor for use in a method for subsequent reimplantation in a reversible refractive procedure on an acceptor, wherein the refractive lenticule is stored at a temperature below 0 0 C. Figure 6a 24