JP4992071B2 - Composition and device for introduction of bioactive agents into ocular tissue - Google Patents

Description

  The present invention relates to a composition and apparatus for introducing a bioactive agent into ocular tissue using ultrasonic energy.

  Gene information related to diseases has been elucidated and attention has been focused on gene therapy to apply it to treatment. In gene therapy, a target gene must be taken into a target cell without causing cell damage, and the target gene must synthesize proteins. In addition to gene therapy, drug delivery that attempts to increase the efficiency of drug treatment and reduce side effects on normal cells by delivering the drug only to the target cell or target site (delivering) has also attracted a great deal of attention. ing.

  On the other hand, vision is an important sensation in daily life, and genetic eye diseases and refractory diseases continue to increase as the average lifespan is prolonged and has become a social problem.

  Since the eyeball tissue (FIG. 7) (particularly the eyeball internal tissue such as the retina, iris, and lens) is a tissue that does not communicate with the outside, it has been impossible to safely and efficiently administer bioactive agents.

  In the field of gene therapy in ophthalmology, it has been found that gene transfection with a viral vector using animals can improve symptoms of genetic diseases such as retinal degeneration, and there is great expectation for treatment. However, since the viral vector incorporates the gene of the vector at the same time as the target gene, it is currently searching for a new method with many problems in clinical application to humans in terms of safety.

  As for drug delivery in ophthalmology, clinical application has already begun, but it is simply in the form of supplying the administered drug close to the target cell and releasing it slowly.

  The sonoporation method is known as one of gene transfer methods. This method uses the phenomenon that when cells are irradiated with ultrasonic waves, small reversible pores appear in the cell membrane, and various substances are taken into the cells through the pores. (Patent Document 1). However, since the eyeball is a fragile tissue, the existing sonoporation method is too invasive, and clinical application to the eyeball tissue has not progressed.

JP 2004-182728 A

  The present invention is a composition suitable for introduction of genes, drugs, and other various substances (sometimes referred to herein as “bioactive agents” or simply “agents”) into ocular tissues using ultrasonic energy. The purpose is to provide.

  Another object of the present invention is to develop a device capable of safely irradiating ultrasonic waves in the eyeball in order to introduce a bioactive agent into cells by ultrasonic irradiation. So far, there are ultrasonic irradiation devices that can be used at the laboratory level to introduce bioactive agents by ultrasonic waves, but since the damage to the eyeball and surrounding tissues is large, it can be used practically. However, improvement is necessary.

  Here, the “apparatus capable of safely irradiating ultrasonic waves” preferably satisfies at least one of the following items. 1. An ultrasound irradiation device optimized for the shape and characteristics of the organ called the eyeball. 2. The cells that make up the eyeball (especially the retina and optic nerve) are sensitive cells like sensors called sensory cells. 3. It must have a structure that can efficiently supply not only the ultrasonic irradiation unit but also the bioactive agent to be administered to the target site. 4). Since the frequency and energy of the ultrasonic waves in the irradiation unit are not single and differ in each part of the eyeball, the device must be capable of adjusting the output of various factors.

The present invention includes the following inventions.
(1) A composition for introducing a bioactive agent into eyeball tissue, which contains the bioactive agent to be introduced and microspheres containing microbubbles, and introduces the bioactive agent into eyeball tissue Composition.

(2) The bioactive agent is at least one selected from the group consisting of oligonucleotides, antisense oligonucleotides, triple helix forming oligonucleotides, oligonucleotide probes, nucleotide vectors, viral vectors, and plasmids. The composition according to (1).

(3) An ultrasonic irradiation apparatus for introducing a bioactive agent into the internal tissue of the eyeball,
An ultrasonic irradiation unit including an ultrasonic generation unit, which is inserted into the internal tissue of the eyeball and irradiates the target site with ultrasonic waves;
The said apparatus provided with the cooling means for cooling the said ultrasonic irradiation part.
(4) The apparatus according to (3), wherein the cooling means is means for perfusing the perfusate so as to flow on the outer surface of the ultrasonic irradiation unit.

(5) The apparatus according to (3) or (4), wherein the ultrasonic wave irradiation section has a substantially rod-like shape having a thickness of 1.5 mm or less and a length of 30 mm or more.
(6) The apparatus according to any one of (3) to (5), further comprising supply means for supplying a bioactive agent to the target site.

(7) The apparatus according to any one of (3) to (6), further including observation means for observing the target site.
(8) It further comprises supply means for supplying the bioactive agent to the target site, and observation means for observing the target site,
The member included in the supply unit and directed to the target site, and the member included in the observation unit and directed to the target site are arranged such that the tip of each member is positioned at the tip of the ultrasonic irradiation unit The apparatus in any one of (3)-(5) arrange | positioned inside the said ultrasonic irradiation part.

(9) An ultrasonic irradiation device for introducing a bioactive agent into the ocular surface tissue,
An ultrasonic irradiation unit including an ultrasonic generation unit that irradiates ultrasonic waves to a target site of the ocular surface tissue; and
The said apparatus provided with the wrinkle contact prevention member arrange | positioned in the said ultrasonic irradiation part for preventing that a wrinkle contacts the front-end | tip part of the said ultrasonic irradiation part.

(10) The apparatus according to (9), wherein the wrinkle contact blocking member is a member that is formed so as to be widened toward the ultrasonic wave irradiation direction.
(11) All or a part of the heel contact blocking member is made of a substantially transparent material, or an opening is provided in a part of the heel contact blocking member (9) or ( 10) The apparatus described.

(12) The apparatus according to any one of (9) to (11), further including a cooling unit for cooling the ultrasonic irradiation unit.
(13) The apparatus according to any one of (9) to (12), wherein the cooling unit is a unit that perfuses the perfusate so as to flow on the outer surface of the ultrasonic irradiation unit.
(14) The device according to any one of (9) to (13), further comprising supply means for supplying a bioactive agent to the target site.

  By using the composition according to the present invention, it is possible to efficiently introduce a bioactive agent into eyeball tissue by the sonoporation method.

  When the sonoporation method is performed using the ultrasonic irradiation apparatus according to the present invention, the bioactive agent can be efficiently and safely introduced into each part (eyeball surface tissue or eyeball internal tissue) of the eyeball tissue. .

  In the present invention, the physiologically active agent to be introduced into the eyeball tissue is not particularly limited as long as it has physiological activity, and synthesis of nucleic acids, proteins (including peptides), sugars and other compounds having physiological activity or the like. Examples include natural physiologically active compounds. Among these, at least one bioactive agent selected from the group consisting of oligonucleotides, antisense oligonucleotides, triple helix forming oligonucleotides (TFO), oligonucleotide probes, nucleotide vectors, viral vectors and plasmids is preferred, Particularly preferred is a plasmid DNA encoding a gene having a bioactive agent. Other bioactive agents include anti-inflammatory agents (eg, corticosteroids), immunosuppressants (eg, cyclosporine, tacrolimus), various photosensitizers, and components that have an activity to promote or inhibit angiogenesis (eg, HGF, FGF, VEGF, oligotype siRNA against VEGF), IL-1 receptor antagonist, NFκB decoy and the like, but are not limited thereto.

  The physiological activity of the gene encoded by the plasmid DNA is not particularly limited. For example, there is a gene corresponding to a disease in the eye, that is, a gene that acts antagonistically to the disease in the eye or a gene that complements the lack in the disease. Used. Examples of such genes include cytokines, growth factors, antibodies or antibody fragments, receptors for cytokines or growth factors, proteins having antiproliferative or antiproliferative effects, enzymes, pulmonary inhibitors, thrombus inducers, and aggregation inhibitors. Group consisting of agents, fibrinolytic proteins, virus coat proteins, bacterial and parasitic antigens, tumor antigens, proteins having effects on blood circulation, peptide hormones, and ribonucleic acids such as ribozymes and antisense RNA Examples thereof include genes encoding pharmacologically active compounds selected more.

  Examples of the microbubble-containing microspheres used in the present invention include albumin-derived microbubble-containing microspheres containing perfluorocarbon which is an insoluble gas. Microbubble-containing microspheres are elastic and compressible, have a lower density than water, and cause an acoustic impedance imbalance between living tissue and body fluid. Therefore, ultrasonic waves are efficiently reflected by such properties. Microbubble-containing spherules also reduce the energy threshold for cavitation. In cavitation, ultrasonic energy is concentrated in the microscopic region, which causes small shock waves that do not affect safety and increases the cell permeability of the drug. Cavitation destroys the microbubble-containing microspheres, and the bioactive agent attached or enclosed in the microbubble-containing microspheres is released. Thus, the bioactive agent can be introduced into the cells by ultrasonic irradiation.

  As the microbubble-containing microspheres derived from albumin containing perfluorocarbon, Optison (trade name, Molecular Biosystems Inc.) is particularly preferably used. Optison has been conventionally used as a contrast agent for ultrasonic diagnosis, and its safety has been confirmed.

  The composition for introducing a bioactive agent of the present invention can be obtained by mixing the above-described bioactive agent and microbubble-containing microspheres. Although the mixing ratio is not particularly limited, for example, the microbubble-containing microspheres are 1 to 90% by weight, preferably 5 to 50% by weight, more preferably 7 to 30% by weight, and most preferably 10 to 10%, based on the bioactive agent. The ratio is 20% by weight. In the composition of the present invention, the bioactive agent is considered to be present in the vicinity of the bubbles of the microbubble-containing spherules, on the surface, or encapsulated inside the bubbles.

  More specifically, examples of the eyeball tissue that is a target for drug introduction in the present invention include eyeball internal tissues such as the retina, sclera, iris, lens, uvea, and optic nerve, and eyeball surface tissues such as the cornea and conjunctiva. It is done. In particular, the retina and sclera, which are deep eyeball tissues, have hitherto been considered particularly difficult to deliver drugs, but according to the present invention, drugs can be delivered noninvasively to these tissues. .

  Examples of the method for introducing a drug into the corneal tissue using the composition of the present invention include a method of performing ultrasonic irradiation after injecting the composition of the present invention into the corneal stroma. Examples of a method for introducing a drug into deep eyeball tissues such as the retina and sclera include a method in which the composition of the present invention is injected into the vitreous and then subjected to ultrasonic irradiation.

The frequency, intensity, irradiation time, and the like of the irradiated ultrasonic wave are not particularly limited because they can be appropriately selected according to various circumstances such as the type, amount, and disease state of the drug to be delivered. The frequency is 0.1 to 10 MHz, preferably 0.5 to 5 MHz, more preferably 0.7 to 3 MHz, most preferably 0.8 to 1.5 MHz (for example, 1 MHz), and the intensity is preferably 0.1. ~10w / cm ^2, more preferably 0.7~3w / cm ^2, most preferably 0.8~1.5w / cm ² (e.g. 1w / cm ^2), duty ratio (ultrasound per given time On / off ratio) is preferably 10-100%, more preferably 25-75%, most preferably 40-60% (eg 50%), and the irradiation time is preferably 1-999 seconds, more The Mashiku 30 to 300 seconds, most preferably 60 to 120 seconds. For example, when a luciferase plasmid is introduced into the retina, a frequency of 1 MHz, an intensity of 1 w / cm ² , an irradiation time of 120 to 240 seconds, and a duty ratio of 50% are the conditions that allow the most efficient gene introduction.

  Particularly preferred forms of ultrasonic irradiation devices that can be used to deliver the compositions of the invention are described below. In addition, the following ultrasonic irradiation apparatus can be used not only for delivering a bioactive agent prepared as the above composition, but also for delivering other forms of bioactive agents.

  The eyeball is a spherical organ of about 2.5 cm located in a space surrounded by an orbital bone with a posterior cone on the front upper part, a nasal bone on the inner side, and the back. The cornea in front of the eyeball is covered with upper and lower eyelids (eyelids) (FIG. 7a). In other words, it is an organ located in a small bag with a narrow entrance. Therefore, in order to irradiate the eyeball with ultrasonic waves, it is preferable to change the shape of the ultrasonic probe according to the target site.

  For example, the intraocular ultrasound probe 1 is preferred when targeting the internal tissue of the eyeball such as the lens, uvea, retina, optic nerve, anterior chamber or vitreous cavity (FIG. 7b), and the cornea, part of the sclera, conjunctiva, When the eyeball surface tissue such as the eyelid and the skin around the eyeball (FIG. 7a) is the main target site, the ultrasonic probe 2 for external use is preferable.

  The above two types of ultrasonic probes have different irradiation target sites, but are common in that they irradiate a narrow area called an eyeball. As a structure common to both, the above-described two types of ultrasonic probes are both members that irradiate the target site with ultrasonic waves (referred to herein as “ultrasonic irradiation unit” or simply “irradiation unit”) 11, 21. And members (referred to herein as “supporting portions”) 12 and 22 for the operator to hold the ultrasonic probe. In addition, since the shape of the ultrasonic irradiation part is usually an elongated shape, in order to efficiently transmit ultrasonic energy to the tissue, the ultrasonic generation parts 101 and 201 must be located at the tip of the irradiation part. Is preferred.

The ultrasonic energy to be irradiated is preferably 0.5 to 10 MHz and 2 w / cm ² or less. If it is this range, it is rare that a target site | part becomes high heat by ultrasonic irradiation. However, it is necessary to consider the possibility of causing tissue damage due to heat generated at the same site when the irradiated part comes into contact with the tissue strongly. It is preferable that both the intraocular ultrasound probe and the extraocular ultrasound probe include a cooling unit for cooling the irradiation unit. In particular, in an intraocular ultrasonic probe, it is highly necessary to provide a cooling means.

  A particularly important point in the intraocular ultrasound probe is the size of the irradiation part. In order to insert the irradiation part into the vitreous cavity and the anterior chamber, the ultrasonic irradiation part 11 is made after puncturing the ciliary flat part sclera and the limbal cornea (FIGS. 7a and 7b) which are part of the outer wall of the eyeball. Need to be inserted. Since punctures other than the same site cause complications such as retinal detachment, the puncture can only be performed at this site. If the width of the puncture is too large, the eyeball collapses and the tissue is damaged. Therefore, the width of the puncture usually has to be kept within 1.5 mm. Therefore, it is preferable that the thickness 16 of the irradiation part is a size that can pass through the puncture part, that is, within 1.5 mm. Since the length 17 of the irradiation part is about 20 mm to the optic nerve (FIG. 7b) located farthest from the puncture part, it is preferably at least 30 to 40 mm.

  In order to insert the irradiation part 11 of the intraocular ultrasonic probe 1 into the vitreous cavity or the anterior chamber (FIG. 7b) and confirm the state of the target site in detail, a means for observing the target site is further provided. It is preferable that it is provided. As such observation means, for example, it is preferable to mount an observation system using a light source or an endoscope on an intraocular ultrasonic probe.

  Since the target site of the ultrasonic probe 2 for external use is a surface tissue, unlike the intraocular 1, it can reach the target site directly without puncturing. Therefore, the thickness of the ultrasonic irradiation unit can be appropriately selected so as to match the size of the irradiation destination. Considering that the irradiation target site of the ultrasonic probe 2 for external use is mainly the cornea and the conjunctiva (FIG. 7a), and that the ultrasonic wave is also applied to the site other than the target if it is too large, the thickness of the irradiation unit 26 is preferably about 3 to 9 mm. In addition, it is preferable to prepare a plurality of types of thickness ultrasonic irradiation units that can be selected according to the size of the target site.

  In the ultrasonic probe 2 for external use, when irradiating a part of the target cornea, conjunctiva, sclera (FIG. 7b), etc., the upper and lower eyelids (FIG. 7a) press the tip of the irradiated part by blinking. There is a possibility that accurate irradiation cannot be performed. Therefore, it is preferable that the extracorporeal ultrasonic probe 2 further includes a heel contact prevention member for preventing the heel from coming into contact with the distal end portion thereof. It is preferable that the heel contact preventing member is a member 4 formed so as to be widened toward the ultrasonic wave irradiation direction.

  In order to achieve efficient administration of the bioactive agent to the target site, the ultrasonic probes 1 and 2 further apply the bioactive agent to the target site (usually near the tip of the ultrasonic irradiation unit). It is preferable to provide a supply means for supplying. In the intraocular ultrasonic probe, as described above, since there is a demand to reduce the size of the ultrasonic irradiation unit, it is preferable that the supply unit is disposed inside the ultrasonic irradiation unit. Since the size of the irradiating unit is not greatly limited in the extra-ocular ultrasonic probe, the supply unit may be provided outside the irradiating unit.

  Usually, the ultrasonic frequency, output, output time, ultrasonic on / off ratio per fixed time, and the like are controlled using the control unit 6 (FIG. 6). Further, since the ultrasonic irradiation unit is a clean area, it is preferable to manage the start of ultrasonic irradiation, perfusion, and the like with the foot switch 69. A foot switch 69 is also connected to the control unit 6.

  The present invention will be described below with reference to an embodiment. The present invention is not limited to the embodiments described below, and it goes without saying that various modifications can be made within the scope of the technical idea described in the claims.

FIG. 1 is a perspective view of an ultrasonic probe (FIG. 1a: for intraocular use, FIG. 1b: for extraocular use).
The intraocular ultrasonic probe 1 includes an ultrasonic irradiation unit 11 inserted into the eye and a support unit 12 for an operator to operate the ultrasonic irradiation unit, and has a cord 13 connected to the control unit 6. The support part 12 has a connector 14 connected to a tube through which the perfusate is circulated in order to perform perfusion cooling.

  The intraocular ultrasonic probe 1 is used by being inserted into an eyeball having a diameter of about 25 mm (vitreous cavity, anterior chamber) (FIG. 7b). The irradiation unit 11 preferably has a substantially rod shape (for example, a columnar shape) having a length 17 of 30 mm or more, for example, about 30 to 40 mm, and a thickness 16 of 1.5 mm or less, for example, about 1 to 1.5 mm. is there. The support portion 12 can be appropriately selected according to the operator's preference. However, in consideration of the operability of the irradiation portion, a cylindrical shape having a length 19 of 40 to 50 mm and a thickness 18 of 10 mm or less is appropriate. .

  FIG. 2 a shows a cross-sectional view along the central axis of the intraocular ultrasonic probe 1. FIG. 2B is a view of the distal end portion of the intraocular ultrasonic probe 1 as seen from the ultrasonic irradiation direction. FIG. 2 c shows an enlarged cross-sectional view from the direction of the arrow along the broken line in FIG. 2 b near the tip of the intraocular ultrasound probe 1. In addition to the ultrasonic wave generator 101, the intraocular ultrasonic probe 1 includes a light guide 106, an endoscope lens 102, an image guide 105 for image transfer, and a supply path 103 for administering various physiologically active agents. I have. The light guide 106, the endoscope lens 102, the image transfer image guide 105, and the endoscope system connected thereto correspond to the observation means in the present invention. The supply path 103 for administering various physiologically active agents, the drug delivery tube 110, and the drug delivery system connected to them correspond to the supply means in the present invention. Examples of the drug delivery system include a syringe and a pump. Further, the drug delivery system such as a syringe and the supply path 103 may be directly connected without passing through the tube 110. Since the supply path 103 for administering the bioactive agent does not appear on the cross section passing through the plane indicated by the broken line in FIG. 2b, the supply path 103 is shown in phantom in FIGS. 2a and c. The ultrasonic generator 101 preferably has a diameter of 0.7 to 1 mm. A tube 104 is connected to the back of the ultrasonic wave generation unit 101, and the tube 104 is connected to a high frequency transmission source in the control unit via a cord 13. A cylindrical objective lens 102 having the same diameter as that of the image guide is disposed on the tip side of the image guide 105 for image transfer. The outer diameter of the image guide 105 is preferably about 0.3 mm. The material is not particularly limited, and quartz-based optical fiber, plastic-based optical fiber, multi-component glass-based optical fiber or the like can be used. In addition to the rod lens, the objective lens 102 may be an optical lens unit in which a plurality of lenses are combined. The supply path 103 is a small-diameter tube having a predetermined thickness and can distribute a bioactive agent. The irradiation unit 11 is covered with a mantle 109. The outer jacket 109 is a tube made of a corrosion-resistant material such as stainless steel. A plurality of illumination light guides 106 are disposed along the long axis direction together with the image guide 105, the objective lens 102, and the supply path 103 so as to fill a gap in the outer jacket. The number of light guides is not particularly limited, and the material can be arbitrarily selected from known materials such as quartz and plastic.

  The ultrasonic probe 1 has a perfusate channel 107 for perfusion cooling. The opening 108 of the flow path 107 is provided at a transition portion between the irradiation unit 11 and the support unit 12. The flow path 107 is branched from the connector 14 behind the support section to the opening 108 (the branching mode of the flow path 107 is the same as the flow path 207 in the probe 2. Details of the flow path 207 See the description in the text and FIGS. 3a and b). The connector 14 is connected to 64 parts of the control unit and connected to the perfusion device. The perfusate is supplied from the perfusion bottle connected to the perfusion apparatus, and is discharged from the opening 108 via the flow path 107. The discharged perfusate flows on the outer surface of the irradiation unit 11 and cools the same part. Further, for efficient cooling, it is preferable to use a sleeve 51 (see below for the sleeve). The flow path 107, the opening 108, the perfusion device, the perfusion bottle, the sleeve 51, and the like correspond to the cooling means in the present invention.

  FIG. 3 a is a cross-sectional view taken along the central axis of the extraocular ultrasonic probe 2. FIG. 3B is a cross-sectional view seen from the direction of the arrow in the broken line in FIG. Similar to the intraocular ultrasound probe 1, the extraocular ultrasound probe 2 includes an irradiation unit 21 and a support unit 22. The ultrasonic generator 201, the tube 209, the cord 23, the flow channel 207 of the perfusate, the opening 208 of the flow channel 207, and the connector 24 are the ultrasonic generator 101, the tube 104, and the cord in the intraocular ultrasonic probe 1, respectively. 13 corresponds to the perfusate flow path 107, the opening 108 of the flow path 107, and the connector 14. For the function and configuration of each member, refer to the description of the intraocular ultrasonic probe 1. The flow path 207 is branched into two near the connector 24 at the rear of the support part, one is formed to reach the opening 108 along the longitudinal direction of the support part 22, and the other is bridged from the branch point. After being detoured in the opposite direction with respect to the central axis of the support part via the flow path 207a (FIG. 3b), it is formed so as to reach the opening 108 along the longitudinal direction of the support part 22 (flow path 107 in the probe 1). The same applies to. In FIG. 3a, the bridging channel 207a does not exist on a cross section passing through the central axis, and is drawn with an imaginary line. The bioactive agent supply path 25 provided along the longitudinal direction of the extraocular ultrasound probe 2 corresponds to the supply path 103 of the intraocular ultrasound probe 1 (in FIG. 3a, the supply path 25 is a cross section passing through the central axis). Since it doesn't exist above, draw with imaginary lines). However, the supply path 25 is different from the above 103 in that it is provided so as to pass through the inside of the support portion 22 and on the outer surface of the side wall portion of the irradiation portion 21. The reason why the supply path 25 is provided so as to pass on the outer surface of the side wall portion of the irradiation unit 21 is to ensure a large surface for transmitting ultrasonic waves. The supply path 25 has an inner diameter of about 19 to 26 gauge. The supply path 25, the drug delivery tube 205, and the drug delivery system connected to the drug delivery tube 205 correspond to the supply means in the present invention. Examples of the drug delivery system include a syringe and a pump. The drug delivery system such as a syringe and the supply path 25 may be directly connected without passing through the tube 205. Since the external probe 2 can observe the target site with the naked eye, observation means such as an endoscope system is not necessarily required.

  Irradiation target sites by the extraocular ultrasonic probe 2 are mainly the cornea, conjunctiva, and eyelid (FIG. 7a). Since these tissues are on the body surface, they need not be as small as the intraocular ultrasonic probe 1. For the purpose of enabling the surgeon to stably hold the ultrasonic irradiation part, the support part 22 is preferably a cylindrical shape having a length 29 of about 10 cm and a thickness 28 of about 1.5 to 2 cm. The length 27 of the irradiation part is about 2 to 3 cm, and the thickness 26 is different depending on the size of the planned irradiation site, but is preferably in the range of about 3 mm to 9 mm considering the corneal diameter. More preferably, a plurality of ultrasonic probes for external use having different irradiation part thicknesses 26 (for example, three types of irradiation part thickness 26 in 3 mm increments: 3 mm, 6 mm, and 9 mm) are prepared and can be appropriately selected. Keep it.

  The problem when using the ultrasonic probe 2 for external use is that the patient's eyelids come into contact with the irradiating part due to blinking, and the positional deviation of the irradiation occurs. Accordingly, it is preferable that the ultrasonic probe 2 for external use further includes a wrinkle contact blocking member 4 for blocking the wrinkle from coming into contact with the tip portion thereof. It is preferable that the heel contact preventing member 4 is a member 4 formed so as to be widened toward the ultrasonic irradiation direction where the target site exists. FIG. 4A is a perspective view of the heel contact preventing member 4, and FIG. 4B is a longitudinal sectional view of a surface passing through the central axis of the member. The eyelid contact blocking member 4 is mounted substantially coaxially on the extraocular ultrasound probe 2 by inserting the tip of the extraocular ultrasound probe 2 from above in FIG. The heel contact blocking member 4 is configured such that the entire or part of the cone-shaped wall 41 is made of a substantially transparent material (for example, a transparent acrylic resin), or a part of the wall 41 It is preferable that the operator can visually recognize the eyeball surface by providing the opening 44. The upper and lower eyelids (FIG. 7a) sandwich the edge of the wall 41 on the opened side, and the eyelids are prevented from coming into direct contact with the irradiation part. The circle formed by the edge of the wall 41 on the open side is preferably about 7 mm in radius. This is because it is easy to put the edge portion of the wall portion 41 between the ridges and it is difficult to come off.

  The heel contact blocking member 4 has a wall portion 43 formed at the end on the side where the irradiation portion of the ultrasonic probe is inserted so as to protrude in the direction of the central axis of the member and having an opening in the center. Yes. The wall 43 serves as a stopper that prevents the tip of the ultrasonic irradiation unit from being pressed too strongly against the cornea or the conjunctiva during irradiation. Since the stepped portion between the irradiation unit 21 and the support unit 22 abuts against the wall 43, when the irradiation unit 21 of the extraocular ultrasonic probe 2 is inserted from above in FIG. It is forbidden to be inserted in the downward direction in FIG. For example, when the thickness of the support portion 22 is 1.5 to 2 cm and the maximum thickness of the irradiation portion 21 is 9 mm, the inner diameter of the opening surrounded by the wall portion 43 is set to 1 cm. Can have a function.

  When using both the ultrasonic probes 1 and 2, it is preferable to attach a sleeve 51 for cooling. As shown in FIG. 5, the sleeve 51 has a hollow structure having a portion 52 having a large inner diameter, a portion 54 having a small inner diameter, and a transition portion 53 that connects both the portions and the inner diameter continuously changes along the central axis direction. Have Since the ultrasonic irradiation units 11 and 21 are inserted from the open end of the portion 52 having a large inner diameter (see FIG. 1), the ultrasonic irradiation units 11 and 21 are illustrated in FIG. 5 for the purpose of facilitating the insertion. As described above, the portion 52 having a large inner diameter preferably has a shape in which the inner diameter continuously expands from the connecting portion with the transition portion 53 toward the open end. When the sleeve 51 is attached to the ultrasonic probes 1 and 2, as shown in FIG. 5, the portion 52 having a large inner diameter is in close contact with the support portions 12 and 22, and the transition portion 53 is in the perfusion discharge direction of the openings 108 and 208. A slight gap is formed between the inner wall surface of the portion 54 having a small inner diameter and the outer wall surfaces of the irradiation units 11 and 21 that constitute the gap. When the perfusate is discharged from the openings 108 and 208 through the flow paths 107 and 207, the perfusate passes through a slight gap between the inner wall surface of the portion 54 having a small inner diameter and the outer wall surfaces of the irradiation units 11 and 21. Thus, the perfusate flows out in the direction of ultrasonic irradiation. Since the outer surface of the irradiation unit is cooled by the flow of the perfusate and the cooled liquid is supplied to the irradiation surface, it is possible to cope with unexpected heat generation of the ultrasonic irradiation unit. The portion 52 having a larger inner diameter of the sleeve 51 preferably includes a portion having the same inner diameter as the outer diameter of the support portions 12 and 22, and the inner diameter of the portion 54 having a smaller inner diameter is larger than the outer diameter of the irradiation portions 11 and 21. It is preferably about 0.1 to 1 mm larger. The length along the central axis of the portion 54 having a small inner diameter is preferably about 1 to 2 cm. The length of the transition portion 53 along the central axis is preferably within 5 mm. The sleeve 51 is preferably made of flexible silicon. By making the sleeve 51 made of silicon, it is possible to adjust the length by appropriately excising the irradiation portion sleeve according to the target site.

  Both the intraocular and extraocular ultrasonic probes 1 and 2 preferably have the shape of cords 13 and 23 having a common connector shape, and the cords 13 and 23 are connected to the connection port 65 of the control unit 6. The control unit 62 of the control unit 6 can control the frequency, energy, irradiation time, and ultrasonic on / off ratio per fixed time. The control unit 6 also has a monitor 61 that displays those parameter settings. In addition, a flow path from the perfusion bottle in which the perfusate for perfusion cooling is stored is connected to the portion 64 of the control unit 6, and the perfusion pressure is controlled by the perfusion device inside the control unit 6 to flow through the connectors 14 and 24. Perfusion is supplied to the channels 107,207. Ultrasonic on / off, perfusion flow, and the like can be adjusted by a foot switch 69 connected to the control unit.

  Moreover, it is preferable that the control unit 6 includes an endoscope light source and a CCD camera inside thereof, and can capture an image from the intraocular ultrasonic probe 1 and supply light to the endoscope. Moreover, the thing which can project an image | video on the existing television monitor from an external output terminal may be used.

  Next, a procedure when using the intraocular ultrasonic probe 1 will be described. For ease of understanding, the case where the retina is used as a target site and ultrasonic irradiation is performed in the vitreous cavity will be described as an example. However, the following procedure is basically used when the apparatus of the present invention is used in another mode. Is also true.

  First, before starting the treatment, the irradiation conditions suitable for the target site are set using the control unit 6. The sleeve 51 is attached to the ultrasonic probe 1 and it is confirmed that the perfusion cooling system is functioning. The supply path 103 passes purified water once and confirms that it is normal, and then replaces the supply path 103 with air. A syringe containing a bioactive agent to be introduced is attached to the tube 110, and the syringe is slowly advanced to push out the air in the supply path 103. Thereafter, the ciliary body flattened 3.5 mm away from the cornea ring portion The sclera is punctured approximately 1 mm (see FIGS. 7 and 8), and the irradiation part 11 of the intraocular ultrasonic probe 1 is inserted. While observing the irradiated part with an endoscopic monitor, proceed to the target site. Push the syringe slowly to dispense the required amount into the eye. Start ultrasound irradiation while confirming that the dose spreads on the monitor. After the irradiation, it is confirmed with an endoscope that there is no abnormality in the irradiated part and the surrounding tissue, and the ultrasonic irradiation part is pulled out. Thereafter, the scleral puncture portion is sutured with a thread having a thickness of about 8-0 to complete the treatment.

  In addition, since this method is based on the conventional vitrectomy, it can be applied to a combination therapy of vitrectomy. For example, as an additional treatment after surgery for diabetic retinopathy and age-related macular degeneration, it is possible to introduce an angiogenesis inhibitor or the like into the retina by ultrasound.

  As described above, by using the ultrasonic device according to the present invention, it is possible to safely administer genes, drugs, and proteins specifically to eye tissues. A new therapeutic possibility for hereditary eye diseases has been developed, and it can be applied to drug delivery in the eye, which has been attracting attention in recent years. The choices can be increased, and improvement in treatment results can be expected.

  Hereinafter, although the Example demonstrates the composition for bioactive agent introduction | transduction to the eyeball tissue based on this invention, this invention is not limited to the range described in the Example.

In vitro gene introduction pEGFP plasmid (Clontech) and Optison (Molecular Biosystems Inc.) were mixed at a ratio of 5: 1 (weight ratio). Rat corneal epithelial cells (RC-1) were added to a 24-well plate so that the number of cells was 8 × 10 ⁴ cells / 24 wells, and 0.6 μl of the above mixture was added to each well. Samples in each well were adjusted to 1000 μl with MEM medium.

As an ultrasonic irradiation apparatus, Sonitron 2000 (trade name: Rich mar Inc.) was used. The frequency of the ultrasonic wave was 1 MHz, the intensity was 1 W / cm ² , the duty ratio was 50%, and the irradiation time was 60 seconds. 48 hours after irradiation, GFP positive cells were counted using a fluorescent stereomicroscope to calculate the introduction efficiency.

For comparison, a system without ultrasonic irradiation (US (-)), a system with ultrasonic irradiation (frequency 1 MHz, 1 W / cm ² , duty ratio 50%, irradiation time 60 seconds), but no addition of Optison (US (+ The same experiment was conducted for)).

  In this specification and the drawings, US means ultra sonic, that is, ultrasound, and MB means micro bubble, that is, microbubble-containing microspheres.

  The results are shown in FIG. It has been clarified that the gene transfer efficiency is increased when ultrasonic irradiation is performed in the presence of Optison (US (+), MB).

Subsequently, the relationship between the intensity of ultrasonic irradiation and the occurrence of cell damage was examined. Cytotoxicity was assessed using the LDH assay. In a 96-well plate, 4 × 10 ⁴ rat corneal epithelial cells (RC-1) were prepared in 200 μl of MEM medium, and ultrasonic waves were irradiated by the same method as that for measuring the GFP introduction efficiency. Experiments were performed by changing the ultrasonic irradiation intensity to 0.5, 1, 1.5, and ² W / cm ² . Moreover, as a control group, the TRI group (high control group) which is a group mixed with 1% TRITON X in which the cell membrane is highly disturbed, and the low control group which is a group to which only cells and a medium are added without performing ultrasonic irradiation Was provided. Three hours after the ultrasonic irradiation, reagents necessary for measurement were added and reacted for 30 minutes, and then LDH activity was measured using an absorptiometer.

The results are shown in FIG. When the ultrasonic energy is relatively low (0.5 or 1 W / cm ² ), the degree of cytotoxicity is significantly higher than when the ultrasonic energy is relatively high (1.5 or 2 W / cm ² ). It was low.

Gene transfer to the cornea (in vivo)
Using the same mixture prepared in Example 1, the introduction efficiency of the GFP gene into the corneal stroma of white rabbits was measured. Ultrasonic irradiation was performed using Sonitron 2000 (trade name: Rich mar Inc.). Irradiation conditions were a frequency of 1 MHz, a duty ratio of 50%, an irradiation time of 120 seconds, and the irradiation intensity was changed to 1, 1.5 and ² W / cm ² .

  A 30 G needle was used for the substantially shallow layer at the center of the cornea and 12 μl of a mixed solution of 10 μl of pEGFP plasmid and 2 μl of physiological saline was injected. Optison (Molecular Biosystems Inc.) was used as the microbubble (MB). In the MB combination group, the amount of MB added was 20% by weight of the plasmid, 10 μl of pEGFP plasmid and 2 μl of MB were mixed, and the cornea was injected. After corneal injection, ultrasonic irradiation was performed under the above-mentioned conditions by directly applying a Sonitron 2000 probe to the corneal surface.

Three days after the ultrasonic irradiation, the cornea was removed, and GFP fluorescence was observed using a fluorescent stereomicroscope. In the group treated with ultrasonic irradiation alone without adding Optison, only GFP fluorescence was observed slightly, but in the group using both ultrasonic irradiation and Optison addition, GFP fluorescence became positive as the ultrasonic intensity increased. Many cells were observed. FIG. 11 shows a fluorescent stereomicroscope image of the cornea under the condition of Optison addition when the irradiation conditions are a frequency of 1 MHz, 2 W / cm ² , a duty ratio of 50%, and an irradiation time of 120 seconds. The region where GFP fluorescence was observed was consistent with the region irradiated with ultrasound.

  In addition, the presence or absence of cytotoxicity of the isolated cornea was observed using an electron microscope. The left figure of FIG. 12 is the endothelium side of the cornea, and the right figure is the epithelial side. Cell damage due to ultrasonic irradiation was not observed from images of chairs.

Gene transfer to the retina (in vivo)
A reporter gene assay using luciferase was performed to examine whether or not gene transfer into the retina was possible by using both Optison addition and ultrasonic irradiation.

First, a solution was prepared by mixing luciferase plasmid (pCMV-GL3), Optison (Molecular Biosystems Inc.), and PBS (phosphate buffered saline) at a ratio (weight ratio) of 2: 1: 2. 20 μL of this solution was injected into the vitreous body of a rat with a 30-gauge sharp needle under a microscope, and the probe was directly applied to the cornea using a Sonitron 2000 (trade name: Rich mar Inc.) to transretinally reach the retina. Sonication was performed. Irradiation conditions were a frequency of 1 MHz, 2 W / cm ² , a duty ratio of 50%, and an irradiation time of 240 seconds. Then, the introduction efficiency of the luciferase gene into the retina and lens was measured.

  Two days after the ultrasonic irradiation, the eyeball is removed, the eyeball is separated into five parts: retina, cornea, lens, pleura, iris, and each tissue is ground using a homogenizer, and then the retina and lens (Lens), the amount of luciferase was quantified (unit: RLU) with a luminometer. In order to correct the difference depending on the tissue, the protein contained in the homogenized solution was simultaneously quantified (after correction: RLU / mg).

  As a comparative experiment, a solution in which luciferase plasmid and PBS were mixed at a ratio of 1: 1 without ultrasonic irradiation was injected into the vitreous, and then luciferase was quantified by the same method as above (P + non-US).

  The results are shown in FIG. In this experiment, it was possible to effectively introduce the luciferase gene into the retina by using ultrasonic irradiation and adding Optoison together. Further, the luciferase gene was specifically introduced into the retina and hardly introduced into the lens.

  The observation results of cell injury were also performed using an electron microscope and an optical microscope. In any of the observation methods, no tissue damage was observed under the above conditions (not shown).

FIG. 1 a is a perspective view of the intraocular ultrasonic probe 1 and the sleeve 51. FIG. 1 b is a perspective view of the ultrasonic probe 1 and the sleeve 51 for external use. FIG. 2 a is a cross-sectional view along the central axis of the intraocular ultrasonic probe 1. FIG. 2B is a view of the distal end portion of the intraocular ultrasonic probe 1 as seen from the ultrasonic irradiation direction. FIG. 2 c is an enlarged cross-sectional view from the direction of the arrow along the broken line in FIG. 2 b near the tip of the intraocular ultrasonic probe 1. FIG. 3 a is a cross-sectional view taken along the central axis of the extraocular ultrasonic probe 2. 3b is a cross-sectional view from the direction of the arrow along the broken line in FIG. 3a. FIG. 4 a is a perspective view of the heel contact blocking member 4. FIG. 4B is a longitudinal sectional view taken along a plane passing through the central axis of the member. FIG. 5 a is a perspective view of the sleeve 51. FIG. 5 b is a cross-sectional view when the sleeve 51 is attached to the ultrasonic probes 1 and 2. FIG. 6 is a schematic view schematically showing the configuration of the control unit 6. FIG. 7a is a diagram showing the ocular surface tissue. FIG. 7b is a cross-sectional view of the ocular tissue. FIG. 8 is a view showing a state where the intraocular ultrasonic probe 1 is inserted from the puncture portion of the ciliary flat portion sclera. It is a figure which shows the relationship between the presence or absence of ultrasonic irradiation (US), the presence or absence of addition of microbubble containing microspheres (MB), and the gene introduction efficiency. It is a figure which shows the relationship between the intensity | strength of ultrasonic irradiation, and the grade of cell damage. It is a photograph which shows the image by the fluorescence stereomicroscope of a cornea after inject | pouring a GPF gene into a corneal stroma and irradiating with an ultrasonic wave. It is a photograph which shows the image by the electron microscope of a cornea after inject | pouring a GPF gene into a corneal stroma and irradiating with an ultrasonic wave. It is a figure which shows the amount of luciferase in a retina and a lens after inject | pouring a luciferase gene into a vitreous body and irradiating with an ultrasonic wave.

Claims (3)

An ultrasonic irradiation device for introducing a bioactive agent into the internal tissue of the eyeball,
An ultrasonic irradiation unit including an ultrasonic generation unit, which is inserted into the internal tissue of the eyeball and irradiates the target site with ultrasonic waves;
A cooling means for cooling the ultrasonic irradiation section;
A supply means for supplying the bioactive agent to the target site;
An observation means for observing the target site ;
Bei to give a,
The member included in the supply unit and directed to the target site, and the member included in the observation unit and directed to the target site are arranged such that the tip of each member is positioned at the tip of the ultrasonic irradiation unit said device being arranged inside the ultrasonic wave irradiation unit. The apparatus according to claim 1, wherein the cooling means is means for perfusing the perfusate so as to flow on the outer surface of the ultrasonic irradiation unit. The apparatus according to claim 1, wherein the ultrasonic irradiation unit has a substantially rod shape having a thickness of 1.5 mm or less and a length of 30 mm or more.

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