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WO2017055421A1 - Procédés et dispositif pour la prolifération de cellules bêta pancréatiques - Google Patents

Procédés et dispositif pour la prolifération de cellules bêta pancréatiques Download PDF

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Publication number
WO2017055421A1
WO2017055421A1 PCT/EP2016/073221 EP2016073221W WO2017055421A1 WO 2017055421 A1 WO2017055421 A1 WO 2017055421A1 EP 2016073221 W EP2016073221 W EP 2016073221W WO 2017055421 A1 WO2017055421 A1 WO 2017055421A1
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Prior art keywords
cells
beta
human
light
lepts
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Inventor
Harald JANOVJAK
Eva REICHHART
Cathrin Constanze HEIDSIEK
Alexandra-Madelaine TICHY
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Ist Austria
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Ist Austria
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0601—Apparatus for use inside the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A61N5/062—Photodynamic therapy, i.e. excitation of an agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0601—Apparatus for use inside the body
    • A61N2005/0612—Apparatus for use inside the body using probes penetrating tissue; interstitial probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N2005/0658—Radiation therapy using light characterised by the wavelength of light used
    • A61N2005/0662—Visible light

Definitions

  • This invention relates to medical devices suitable for irradiating pancreatic beta- cells and methods for such irradiation.
  • Beta-cells are an important group of cells located in the human pancreas.
  • the key functions of beta-cells are insulin production, storage and release and thereby the control of blood glucose concentration.
  • T1 D also known as insulin dependent diabetes, is believed to be caused by an autoimmune response where the immune system destroys beta-cells [1 ].
  • beta-cell death is implicated in Type 2 diabetes mellitus [2]. Promoting the proliferation of residual or newly-generated beta- cells has the potential to improve patient health in some forms of diabetes, and thus this is pursued and supported by many parties. These parties include industry (e.g. beta-cell proliferation is a beneficial effect of treatment with glucagon-like peptide 1 (GLP1 ) [3]), funding agencies and many academic researchers.
  • GLP1 glucagon-like peptide 1
  • beta-cell proliferation very few methods are currently available to control beta-cell proliferation in patients.
  • Chemical factors promoting the growth of beta-cells such as peptide growth factors including hepatocyte growth factor (HGF) or GLP1 , are efficacious in animal models of diabetes in vitro and in vivo [4, 5].
  • HGF hepatocyte growth factor
  • GLP1 hepatocyte growth factor
  • many of these factors and their cognate cell receptors have widespread tissue distribution, and systemic factor administration may result in non-specific effects, such as undesired proliferation of non-beta-cells and an increased risk of cancer [6-9].
  • many factors exhibit limited half-life in the circulation and thus are difficult to apply to patients.
  • factor delivery using gene therapy is sustained and cannot be withdrawn at later stages or after treatment.
  • Phototherapy the illumination of tissues affected by or causative for disease, has been described for controlling cellular functions.
  • Phototherapy methods either employ photosensitizers (chemical molecules that respond to light and result in a modification of cellular functions [10]) or optogenetic methods and constructs (e.g., engineering microbial opsins into mammalian cells or tissue [1 1 ]).
  • photosensitizers chemical molecules that respond to light and result in a modification of cellular functions [10]
  • optogenetic methods and constructs e.g., engineering microbial opsins into mammalian cells or tissue [1 1 ].
  • Reinbothe et al. and Kushibiki et al. [12, 13] describe optogenetic control of insulin secretion in beta -cells using transgenic cells expressing the light-sensitive cation channel Channelrhodopsin-2 (ChR2), an algal protein.
  • Blue light stimulation of the beta-cells triggered increases in intracellular Ca ++ and enhanced insulin
  • US2012/0197358A1 discloses techniques for managing diabetes and prediabetes.
  • a light generating device is positioned close to a body area rich in adipose tissue, such as the abdominal area, thigh, buttocks, and upper arm of a patient, and a low energy light beam (wavelength ( ⁇ ) ranging from 400 to 1300 nm, power (P) ranging from 1 to 12 mW) is used.
  • ⁇ wavelength
  • P power
  • Such light therapy is described to increase adiponectin synthesis by the adipose tissue and modulate the immune system.
  • a biocompatible device for invasive phototherapy comprising a light emitting phototherapy source (LEPTS) incorporating an outlet or connected with an outlet, suitable for emitting light to a target, wherein the outlet and optionally the LEPTS or the device is suitable to be located implanted in a patient suffering from diabetes into an area within or in close proximity to the pancreas, to deliver adequate light intensity onto pancreatic beta-cells and/or the pancreas, and the LEPTS is suitable to irradiate the target pancreatic beta-cells with an effective energy dose, using at least one wavelength of visible light to activate pancreatic opsin proteins.
  • LPTS light emitting phototherapy source
  • such device is used with the exception of such uses that comprise or encompass an invasive step representing a substantial physical intervention on the body of a human or an animal, which requires professional medical expertise to be carried out and which entail a substantial health risk even when carried out with the required professional care and expertise.
  • the device is applied to a patient by a physician and/or provided for the use by a patient without specific professional medical expertise.
  • the LEPTS is connected with an outlet suitable for emitting light to a target which forms a distant part of the LEPTS.
  • an outlet which is e.g., suitable to illuminate the target cells, and connected to an LEPTS that is situated in a convenient location, such as within the fascia of the pectoral or axiallary region, the fascia of the abdominal and pleural cavities, subcutaneous tissue, etc.
  • the target cells are not of adipose tissue.
  • the LEPTS is extracorporeal and the outlet comprises optical fibers to conduct light to the remote, internal treatment site.
  • the invention further provides for a method of treating a patient suffering from diabetes by invasive or otherwise in vivo phototherapy, comprising activating target beta-cells in the pancreas (herein also referred to as pancreatic beta -cells) of said patient by irradiation using a device comprising a LEPTS incorporating an outlet or connected with an outlet, which is suitable for emitting light to the target beta-cells, comprising the steps:
  • the target beta-cells are repeatedly irradiated according to step b).
  • the outlet is positioned within the patient's body and is connected to the LEPTS that is positioned outside the patient's body.
  • the outlet is part of the LEPTS which is implanted into the patient's body.
  • the method is combined with any anti-diabetic treatment, e.g., by administering an active substance or pharmaceutical preparation for treating diabetes.
  • the invention provides for a method of treating a patient suffering from diabetes by administering an effective amount of a preparation of human pancreatic beta-cells that have been activated by ex vivo irradiation as further described herein, in particular wherein said irradiation has been performed in a manner to promote the pancreatic beta-cells's cell growth, proliferation and/or insulin
  • pancreatic beta -cells are autologous to said patient.
  • the target pancreatic beta-cells are not modified transgenically, but of mammalian origin, such as isolated from a subject e.g., human, dog, pig.
  • pancreatic cells particularly do not comprise microbial gene products or transgenes.
  • the device allows an energy dose which effectively activates opsin proteins in the pancreas and in beta -cells, and specifically in close proximity to the pancreas.
  • the LEPTS is suitable for irradiating the pancreatic beta-cells and/or the pancreas with visible light.
  • one or more wavelengths ranging from 400 to 650 nm, specifically a wavelength of at least 425 nm, or at least 440, or at least 450 nm, and further specifically a wavelength of up to 600 nm, or up to 550 nm may be used.
  • One or more specific wavelengths optionally be selected at 470 nm (+/- 20 nm, or +/- 10 nm); or at 535 nm (+/- 20 nm, or +/- 10 nm); or at both wavelengths, 470 nm (+/- 20 nm, or +/- 10 nm) and 535 nm (+/- 20 nm, or +/- 10 nm), in particular by both wavelengths consecutively.
  • laser diodes include e.g., a laser, superluminous diode, laser diode, or a light-emitting diode (LED), such as laser diodes (including also semiconductor laser diodes) and superluminous diodes which provide for highly directional light that is limited in its frequency range.
  • laser diodes produce a beam of light or radiation that is essentially monochromatic, is sharply collimated and is coherent.
  • Laser diodes are available with continuous wave emission capability and as devices that are pulsed.
  • the LEPTS is suitable for irradiating the pancreatic beta-cells and/or the pancreas with an effective energy dose.
  • the energy dose is ranging from 1 LiW/cm 2 to 10 mW/cm 2 , e.g., at least 1 pW/cm 2 , or at least 10 pW/cm 2 , or at least 100 pW/cm 2 , or at least 1 mW/cm 2 , or at least 2, 3, 4, 5, 6, 7, 8, or 9 mW/cm 2 .
  • the target beta-cells are irradiated with a suitable energy dose, such that the effective energy dose is directly reaching the target cells.
  • the energy dose is effective for phototherapy so to effectively treat the patient's disorder, in particular by stimulation of cell proliferation with light having the selected optical parameters.
  • the patient is suffering from a disorder of diabetes (diabetes type 1 or diabetes type 2), and specifically hyperglycemia, hypoinsulinemia or insulin resistance.
  • diabetes diabetes type 1 or diabetes type 2
  • hyperglycemia hyperglycemia
  • hypoinsulinemia hyperinsulinemia
  • insulin resistance specifically hyperglycemia, hypoinsulinemia or insulin resistance.
  • the device is a medical device which can optionally include one or more of a sensing circuit, a controller circuit, a memory circuit, a communication circuit, a power source such as a battery, a battery status circuit, an activity sensor, configured to sense a physical activity signal of a patient, and a physiologic sensor configured to sense a physiologic signal of the patient.
  • the LEPTS provides for manipulating and controlling the outlet and/or the administration of the effective energy dose to the target, and comprises at least one or more controllers for controlling phototherapy.
  • control of phototherapy is provided via manual, mechanical, electrical, optical, laser, magnetic, hydraulic, pneumatic, motorized, ultrasonic, acoustic, wired and/or wireless technology, or any combination thereof.
  • the device and phototherapy system as described herein may be associated with an auxiliary device such as a catheter, endoscope, trocar, or the like.
  • the device comprises signal communication means for repeatedly transmitting light to the target pancreatic beta -cells with the same or a different energy dose according to a predefined dose regimen.
  • the typical treatment period may be from 0.5 minutes to 20 minutes or 30 minutes, specifically at least 1 minute and up to 15 minutes.
  • treatment is shorter, e.g. 1 millisecond or longer, such as at least 1 millisecond or at least 10 milliseconds, up to 0.5 minutes.
  • Treatment may also be continuous for a longer period, e.g. comprising illumination for 0.5 hours or longer, such as at least 0.5 hours, or at least 1 hour, up to 2, 3, 4, or 5 hours.
  • the dose regimen may include regular intervals of treatment or follow a treatment schedule on demand within a predefined range, depending on the patient's physical condition or diet. Specifically, the activation of pancreatic beta-cells is reversible, thus, repeated treatment is indicated at least once in regular intervals.
  • pancreatic beta-cells Because of activating endogeneous pancreatic beta-cells (or those which originate from allogenic or heterologous sources and have previously been implanted and considered as endogenous), the likelihood of overdosing is low. Thus, specific dose/treatment schedules comprise a lower limit in regular intervals, and further repeated treatment on demand, to improve the patient's conditions.
  • the patient is treated at least 1 x a day, or at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 1 1 x, or 12x a day, e.g. in regular or irregular intervals.
  • the LEPTS is suitable to be positioned outside the patient's body.
  • the LEPTS is situated outside the patient's body carried by the patient, and the outlet is directly attached to the body, or implanted.
  • the outlet is positioned into an area within the pancreas.
  • the outlet is positioned into an area in close proximity to the pancreas, which is non-adipotic tissue. At least a portion of the device or LEPTS outlet is physically shaped suitable to be situated at a predetermined position. Positioning and operations can be facilitated via a channeling device such as a guiding catheter and/or via a remote visualization device as is known and accepted in the art for example emplyong a scope or an endoscope.
  • a channeling device such as a guiding catheter and/or via a remote visualization device as is known and accepted in the art for example emplyong a scope or an endoscope.
  • the outlet is situated in the inner pancreas organ, or in close proximity (e.g., within a distance of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cm) to the outer organ, such as in the abdominal cavity behind the stomach.
  • the outlet is shaped to physically contact the pancreas.
  • the LEPTS is suitable to be implanted into the patient's body.
  • the device is an implantable device.
  • Specific embodiments have a manual control carried by the patient outside the body e.g., as a remote control or directly connected to implanted LEPTS and/or implanted outlet of the LEPTS.
  • implanted parts can comprise the implantable light source and/or the light outlet, suitable for intracorporeal phototherapy.
  • the method of insertion may optionally include endoscopy with or without ultrasound, stereotactic methods laparoscopy, such as implantation with a laparoscope into abdominal organs, the pancreatic wall and body cavities.
  • the device may optionally be biocompatible as a whole, or at least the implantable parts, such as the LEPTS and/or its outlet, may be biocompatible.
  • the biocompatible device specifically comprises those parts that are in direct contact with the patient's body or tissue which at least partially (or completely) consist of or are at least partially (or completely) coated with a biocompatible inert material, in particular a non-degradable or non-absorbable material that is designed and engineered to provide for the necessary stability and flexibility of specific device parts.
  • a biocompatible inert material can be based on a metal, alloy, polymer, biologic scaffolding, or a combination comprising at least one of the foregoing .
  • the biocompatible material is a biocompatible polymer.
  • the use of a biocompatible material helps to prevent inflammation, toxic or allergic reactions and ensures that the implant system causes no complications after implantation.
  • Exemplary biocompatible materials are conventional linear or crossl inked block copolymers or other thermoplastic polymers e.g., (crosslinked) polyurethane and block copolymer of polyethylene terephthalate and polyethyleneoxide.
  • a pharmaceutical composition comprising an active substance for treating diabetes in a patient, for use in combination with the device as described herein.
  • the invention particularly further refers to a combination treatment, wherein a subject is treated both, by standard treatment with a pharmaceutical composition, in combination with the irradiation treatment as described herein.
  • Specific active substances as used for combination purposes can be any of an antidiabetic agent, such as preferably any of insulin, sulfonylureas, incretins, other secretagogues, glitazones, metformin, GLP-1 agonists, DPP4 inhibitors, glucosidease inhibitors, amylin analogs, or SGLT2 inhibitors.
  • an active substance retinal may be used.
  • the active substance can as well be an immunomodulatory drug, including vaccine-based approaches using beta-cell autoantigens, anti-CD3 antibodies, anti- CD20 antibodies, anti-CTLA4 antibodies, nicotinamide, rapamycin, cyclosporine A, azatiopirine, anti-thymocyte globulin (ATG), or prednisolone.
  • the pharmaceutical composition of an active substance may be formulated for oral, parenteral, systemic, mucosal, topic, rectal, sublingual, buccal or implant use.
  • Such preparation typically comprises a pharmaceutically acceptable carrier appropriate for a desired route of administration, preferably wherein the pharmaceutical preparation is a tablet, dermal or transdermal formulation, ointment, gel, cream, lotion, patch, solution, injectable, ophtalmic solution, disperse system, emulsion, microencapsulated drug system, osmotic pump, subdermal implant, granule, microsphere, modified release system, targeted release system, granules, or pill.
  • Pharmaceutically acceptable carriers generally include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, sustained release agents, and the like that are physiologically compatible with an active agent or related composition. Further examples of pharmaceutically acceptable carriers include sterile water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations of any thereof.
  • Examplary formulations as used for parenteral administration include subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension.
  • Formulations for topical application include a number of forms such as creams or ointments, patches, pastes and gels.
  • Specific pharmaceutically acceptable carriers and pharmaceutical compositions are known in the art and described in, e.g. REMINGTON'S PHARMACEUTICAL SCIENCES.
  • a method of ex vivo treatment of a population of human pancreatic beta -cells to promote cell growth, proliferation and/or insulin production comprising
  • the population of human pancreatic beta-cells may be obtained from a human donor, in particular a healthy (or dead) subject or else from a patient suffering from diabetes.
  • Specific populations are obtained from cell culture, wherein the beta -eel Is are cultivated e.g., to obtain a well-characterized standard population.
  • Specific culture techniques employ handling and/or cultivating cells in a suited growth medium supplemented with additives to promote survival and/or growth of beta -cells and at a suited temperature under sterile conditions.
  • a specific aspect relates to the cultivation of human pancreatic beta -cells in a cell culture, wherein the cells are exposed to light treatment as described herein, thereby obtaining a cell culture of pancreatic beta-cells with improved proliferation and insulin production.
  • the light treatment specifically is employed such that the opsin proteins located close to or within the pancreas and/or pancreatic beta -cells (i.e. the pancreatic opsin proteins) are activated.
  • the energy dose is effective for phototherapy so to effectively stimulate pancreatic beta-cells for an improved proliferation and insulin production by stimulation with light having the selected optical parameters.
  • the beta-cells are treated in a medium which is suited for maintenance of the cells.
  • the treatment period mainly depends on the type of light and power of energy, to obtain the effective energy dose required for the opsin activation.
  • the selection of the wavelength and the effective energy dose directly reaching the target beta-cells is as described above.
  • the light emitting source is placed in close proximity to the beta-cells but without physical contact, and the opsin activation and/or beta -eel I proliferation controlled.
  • beta-cells are stimulated, such as by excitatory stimulation resulting in a high proliferation rate over at least two generations as measured by assessing number, metabolic activity or composition of cells.
  • the treated pancreatic beta-cells have preserved and/or protected beta-cell function and are characterized e.g., by the following expression of insulin and response to glucose with insulin secretion.
  • the expression of insulin is at least 10% of the control beta-cells, where control cells are defined as untreated cells, and preferably at least 25%, 50% or 75%, up to 100% of the control cells, or more than 100%.
  • the glucose concentration that triggers insulin secretion in treated cells is less than 5-fold the concentration that triggers insulin sectration in control cells, where control cells are defined as untreated cells, preferably less than 4, 3 or 2-fold or equal than of the effective concentration at the provided cells.
  • the opsin proteins are one or more of
  • the treatment is such that at least one, two, three, four, five, six, or all of the opsins are activated. In some instances, it may be sufficient to activate less than 7, 6, 5, 4, 3, 2, or only one of the opsins, e.g. at least 1 , 2, 3, or all of the opsins identified by SEQ ID 1 to 5.
  • the invention further provides for a preparation of human pancreatic beta-cells with preserved and/or protected beta-cell function, obtainable by a method as described herein.
  • Such preparation can be provided as isolated cell culture and optionally be provided in the context of an implantable material (such as tissue) or device.
  • the cultivated cells may be provided as research tool, e.g., for analytical, diagnostic or medical purposes.
  • pancreatic beta-cells are further used e.g., for medical purposes freshly prepared, such as within 24 hours upon irradiating.
  • the preparation treated pancreatic beta-cells may be provided as a medical device, a medicinal product, or a pharmaceutic product.
  • the preparation is provided for use in a method of treating a patient suffering from diabetes.
  • Specific preparations may be heterologous (e.g., allogenic), or autologous to a patient, preferably wherein the pancreatic beta-cells are autologous to said patient.
  • the patient is treated e.g., by implanting the beta-cells, and by further combination treatment, such as an additional immunosuppressive regimen.
  • the preparation comprises the treated beta -cells as such, or those which are obtained from a further cell culture to increase the amount of implantable material.
  • the invention further provides for the use of a LEPTS for ex vivo treatment of isolated pancreatic beta-cells to control cell growth , proliferation and/or insulin production, by exposing the cells to light with an effective energy dose, using at least one wavelength of visible light to activate pancreatic opsin proteins.
  • Figure 1 Light stimulation (arrow from left to right) supports the proliferation of beta-cells (increased transition from left to right) and protects beta-cells against attack (reduced transition from right to left).
  • the modified thymidine analogue EdU is incorporated into newly synthesized DNA and fluorescently labeled. Increased proliferation upon light stimulation is observed.
  • FIG. 6 LEPTS (1 ) suitable for emitting light targeted to the pancreas.
  • Extracorporeal LEPTS is connected to an outlet that is placed close to the target pancreas and emitting light to the target pancreas (left);
  • Intracorporeal LEPTS is placed into the body close to the target pancreas and included in a housing or device incorporating both, the LEPTS and the outlet (right).
  • Figure 7 Schematic construction of a LEPTS (1 ) incorporating an outlet (3) capable of emitting light to a target, comprises the following parts: connector (2) connecting the light source (4) and the outlet (3); light source (4); controller (5); power source (6); circuit (7) (e.g. memory circuit or communication circuit); activity/physiological/physical sensor (8); auxiliary device (9) (e.g. catheter, endoscope, or trocar).
  • the modified thymidine analogue EdU is incorporated into newly synthesized DNA and fluorescently labeled. Increased proliferation upon light stimulation is observed in two independent experiments.
  • beta-cells express several light-sensitive proteins that are members of the opsin protein superfamily. Members of the opsins are well-known for their role in vision (four of the seven human opsins are used as the primary photoreceptors for dim light and three color vision) but also play a role outside of visual light detection [14-16]. Also, cellular signaling cascades exist that should allow opsins to activate proliferative signals via, e.g.
  • G-protein coupled receptor kinases [17], arrestins [18], tyrosine-protein kinase Src, receptor tyrosine kinases and phosphoinositide 3-kinase (PI3K) [19, 20].
  • beta-cells such as the experimental INS-1 E cell line model [21 ]
  • the proliferation of beta-cells can be controlled e.g., by blue light, which is a color of light that should activate many opsins (in particular, both blue and green-light absorbing opsins).
  • Light effectively augmented the proliferation of beta-cells Fig. 2, 3 and 8) and augmentation was comparable to that achieved e.g. with the potent chemical factor HGF in this cell model in a previous study [22].
  • cellular signaling pathways particularly the Mitogen-activated protein kinase/Extracellular signal-regulated kinase (MAPK/ERK) and Phosphoinositide 3-kinase/Protein kinase B (PI3K/AKT) pathways, be activated (Fig. 4 and 9), and beta-cell function be protected against attack (e.g., toxins or stress factors, such as by the toxin streptozotocin, cytokines or H2O2.
  • MAPK/ERK Mitogen-activated protein kinase/Extracellular signal-regulated kinase
  • PI3K/AKT Phosphoinositide 3-kinase/Protein kinase B
  • the present phototherapy or treatment of beta-cells for controlling cell proliferation has the following advantages: - the treatment can be local by applying/focusing the light to/on the tissue of interest. This is not possible with chemicals as these diffuse in tissues and act in the circulation;
  • - light control can be started and stopped quickly and with varying repetition rates, as light can be switched on and off quickly and precisely from the outside;
  • beta-cells in contrast to replacement of beta-cells using outside sources of cells [23], which involves a surgical transplant procedure, internal regeneration of beta-cells is more direct and does not require donor tissue the supply of which is limited.
  • Example 1 Light activation of opsins endoqenously expressed in a pancreatic beta-cell line and their effects on proliferation and signaling pathways.
  • Type I diabetes Type I diabetes
  • replacement of insulin-producing tissue by transplantation of allogenic islets together with immune suppression has corrected hyperglycemia in a number of TID patients.
  • a major limitation of this approach is the limited number of donor tissue and the non-proliferative status of beta-cells ex vivo.
  • Current evidence indicates that the activation of pro-proliferative signaling pathways like MAPK/ERK and PI3K/AKT pathways play a crucial role in beta-cell survival and growth [4, 5, 24].
  • INS-1 E cells were used as a model cell line [21 ]. This cell line is able to secrete insulin in response to elevated glucose concentration and their concentration dependence curve is similar to that of rat pancreatic islets.
  • INS-1 E cells are an isolated clone from INS-1 cells which are derived from a rat insulinoma induced by x-ray irradiation [27].
  • MTT 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • INS-1 E cells were seeded at a density of 3 x 10 4 cells in each well of a 96-well plate in complete medium (RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 pg/ml streptomycin, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 ⁇ beta-mercaptoethanol).
  • cells were starved in reduced serum medium (RPMI 1640 supplemented with 0.1 % fetal bovine serum, 100 units/ml penicillin, 100 pg/ml streptomycin, 10 mM HEPES, 2 mM L- glutamine, 1 mM sodium pyruvate, and 50 ⁇ beta-mercaptoethanol) for 16 hours before 6 hour pre-treatment with 10 ⁇ 9-cis retinal (Sigma) or 10 ⁇ retinoic acid (Sigma) or only vehicle followed by blue light stimulation for 16 hours.
  • MTT assay was performed by incubating the cells with 0.5 mg/ml MTT for 2 hours at 37°C in 5% CO 2 .
  • Formazan produced in the cells was dissolved with acidic isopropanol containing 0.1 N HCL (Sigma), and absorbance was read at wavelengths of 570 nm and 690 nm with a microplate reader (Synergy H1 , BioTek, Bad Friedrichshall, Germany). Increased proliferation was observed upon light stimulation and retinal addition (Fig. 2). After depletion of cellular and medium retinal, only cells treated with retinal but not untreated or retinoic acid treated cells respond to blue light with an increase in proliferation.
  • an immunoblot was performed to investigate downstream signaling activation in INS-1 E cells upon light stimulation.
  • the secondary antibody, HRP-coupled goat anti-rabbit (#170-6515; BioRad Vienna, Austria) was applied at a dilution of 1 :10,000 in TBST for 1 hour at room temperature. Chemiluminescence was developed with Clarity Western ECL Substrate (Biorad) and signals recorded with Molecular Imager® VersaDocTMMP Substrate (Biorad).
  • Example 2 Light activation of opsins endogenously expressed in human pancreatic islets and their effects on proliferation and signaling pathways.
  • IEQ islet equivalents
  • 250 lEQs per condition were cultured in a 35 mm dish in complete medium (CMRL 1066 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 pg/ml streptomycin and 2 mM L-glutamine) for 24 hours at 37°C in 5% CO 2 .
  • complete medium CMRL 1066 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 pg/ml streptomycin and 2 mM L-glutamine
  • medium was changed to fresh complete medium supplemented with 5 ⁇ EdU followed by blue and green light illumination for 96 hours (the control dish was kept in the dark).
  • 1 ml fresh complete medium was added to each dish.
  • cytospin slides were prepared with dispersed cells. Cells were fixed with 4% paraformaldehyde for 8 minutes followed by washing three times with blocking buffer (5% BSA in PBST). Subsequently, newly synthesized DNA was stained with Click-iT ® Plus EdU Alexa Fluor ® 488 Imaging Kit following the manufacturer's protocol. For visualizing cell nuclei, the slides were also stained with DAPI. After washing with PBST, the slides were mounted with Mowiol ® 4- 88 (Sigma).
  • lEQs were collect in a 15 ml falcon tube, centrifuged for 5 minutes at 200 rpm and resuspended in A+AB buffer (135 mM NaCI, 5.6 mM KCI, 1 .2 mM MgCI 2 , 1 .28 mM CaCI 2 , 10 mM HEPES, 3 mM glucose, 1 % (v/v) Penicillin/Streptomycin, 0.1 mg/ml BSA; pH 7.4). 330 lEQs per condition were transferred into separate microcentrifuge tubes and incubated for 2 hours in the dark.
  • the blot was incubated with the primary antibodies, phospho-p44/42 MAPK (Erk1 /2) (Thr202/Tyr204) (#9101 ; Cell Signaling Technology) and Akt (#9272; Cell Signaling Technology), or phosph-Akt (Ser473)(D9E) (#4060; Cell Signaling Technology) and ERK2 (K-23) (sc-153; Santa Cruz Biotechnology, Heidelberg, Germany), diluted 1 :1 ,000 in blocking solution (5% BSA in TBST) overnight at 4°C.
  • the secondary antibody, HRP-coupled goat anti-rabbit (#170-6515; BioRad) was applied at a dilution of 1 :10,000 in TBST for 1 hour at room temperature. Chemiluminescence was developed with Clarity Western ECL Substrate (Biorad) and signals recorded with Molecular Imager® VersaDocTMMP Substrate (Biorad).
  • HGF Hepatocyte growth factor
  • pancreatic beta cell in Islet Cell Growth Factors, R.N. Kulkarni, Editor. 201 1 , Austin, TX. p. 85-102.

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Abstract

L'invention concerne un dispositif biocompatible pour la photothérapie invasive comprenant une source (LEPTS) d'émission de lumière photothérapeutique connectée à une sortie conçue pour émettre de la lumière vers une cible, la sortie convenant pour être implantée dans un patient souffrant de diabète, dans une zone se situant à l'intérieur ou à proximité immédiate du pancréas, afin d'administrer une intensité lumineuse adéquate sur les cellules bêta pancréatiques et/ou sur le pancréas, la LEPTS convenant pour exposer les cellules bêta pancréatiques cibles à un rayonnement comportant une dose d'énergie efficace, à l'aide d'au moins une longueur d'onde de la lumière visible qui active les protéines opsines du pancréas ; et une méthode de traitement ex vivo d'une population de cellules bêta pancréatiques humaines pour activer la croissance cellulaire, la prolifération cellulaire et/ou la production d'insuline, ladite méthode consistant à : a) fournir une préparation de cellules bêta pancréatiques humaines ; b) traiter les cellules en les exposant à de la lumière comportant une dose d'énergie efficace, en utilisant au moins une longueur d'onde de la lumière visible afin d'activer les protéines opsines du pancréas dans les cellules traitées.
PCT/EP2016/073221 2015-09-29 2016-09-29 Procédés et dispositif pour la prolifération de cellules bêta pancréatiques Ceased WO2017055421A1 (fr)

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US20210079323A1 (en) * 2019-09-15 2021-03-18 Board Of Regents, The University Of Texas System Device and uses thereof for treating diabetes
RU2787071C2 (ru) * 2017-11-13 2022-12-28 Те Риджентс Оф Те Юниверсити Оф Калифорния Композиции и способы улучшения зрительной функции

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019094904A1 (fr) * 2017-11-13 2019-05-16 The Regents Of The University Of California Compositions et méthodes pour améliorer la fonction visuelle
CN111417339A (zh) * 2017-11-13 2020-07-14 加利福尼亚大学董事会 用于增强视功能的组合物和方法
US10745453B2 (en) 2017-11-13 2020-08-18 The Regents Of The University Of California Compositions and methods for enhancing visual function
RU2787071C2 (ru) * 2017-11-13 2022-12-28 Те Риджентс Оф Те Юниверсити Оф Калифорния Композиции и способы улучшения зрительной функции
US20210079323A1 (en) * 2019-09-15 2021-03-18 Board Of Regents, The University Of Texas System Device and uses thereof for treating diabetes
US11834640B2 (en) * 2019-09-15 2023-12-05 Board Of Regents, The University Of Texas System Device and uses thereof for treating diabetes

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