WO2016048942A1 - Systèmes et procédés pour améliorer la biogenèse des plaquettes et augmenter la durée de vie de plaquettes, en circulation et en stockage - Google Patents

Systèmes et procédés pour améliorer la biogenèse des plaquettes et augmenter la durée de vie de plaquettes, en circulation et en stockage Download PDF

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Publication number
WO2016048942A1
WO2016048942A1 PCT/US2015/051323 US2015051323W WO2016048942A1 WO 2016048942 A1 WO2016048942 A1 WO 2016048942A1 US 2015051323 W US2015051323 W US 2015051323W WO 2016048942 A1 WO2016048942 A1 WO 2016048942A1
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Prior art keywords
lll
platelet
platelets
generator
parameter
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PCT/US2015/051323
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English (en)
Inventor
Mei X. WU
Qi Zhang
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The General Hospital Corporation Dba Massachusetts General Hospital
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Application filed by The General Hospital Corporation Dba Massachusetts General Hospital filed Critical The General Hospital Corporation Dba Massachusetts General Hospital
Publication of WO2016048942A1 publication Critical patent/WO2016048942A1/fr
Priority to US15/465,924 priority Critical patent/US20170246470A1/en
Priority to US16/875,111 priority patent/US11623103B2/en

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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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0278—Physical preservation processes
    • A01N1/0294—Electromagnetic, i.e. using electromagnetic radiation or electromagnetic fields
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/02—Blood transfusion apparatus
    • A61M1/0272—Apparatus for treatment of blood or blood constituents prior to or for conservation, e.g. freezing, drying or centrifuging
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2202/00—Special media to be introduced, removed or treated
    • A61M2202/04—Liquids
    • A61M2202/0413—Blood
    • A61M2202/0427—Platelets; Thrombocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2205/00—General characteristics of the apparatus
    • A61M2205/05—General characteristics of the apparatus combined with other kinds of therapy
    • A61M2205/051—General characteristics of the apparatus combined with other kinds of therapy with radiation therapy

Definitions

  • the present disclosure relates generally to platelet biogenesis and storage and, more specifically, to systems and methods that employ low level light (1 ) to enhance either in vivo or in vitro platelet biogenesis and/or (2) to extend platelet lifespan in either in vivo circulation or in vitro storage.
  • Platelets are irregular, disc-shaped elements in the blood that assist in blood clotting.
  • An abnormally low level of platelets in the blood is referred to as thrombocytopenia.
  • thrombocytopenia can occur after ⁇ -irradiation exposure, after trauma, after platelet donation, as a side effect of cancer
  • thrombocytopenia can exhibit an increased risk of hemorrhage and death due to uncontrollable bleeding.
  • the only effective means to manage thrombocytopenia is platelet transfusion because, although there are several biological agents and drugs that are either FDA-approved (e.g., IL-1 1 ) or in clinical trials, the effects of these drugs are modest and cause side effects, particularly with high dosages.
  • Platelet transfusions are expensive and frequently associated with complications, including transfusion-transmitted diseases, allergic reactions, graft- versus-host reactions, infections, and platelet refractoriness. Accordingly, platelet transfusion is relatively rare, often limited to patients in critical condition, because the benefits of these transfusions must always outweigh the potential risks to the patient.
  • a safe, convenient, and cost-effective modality to augment platelet regeneration in vivo would greatly reduce the need of platelet transfusions and confer primary or secondary prophylaxis of thrombocytopenia.
  • the present disclosure relates generally to platelet biogenesis and storage. More specifically, the present disclosure relates to employing low level light (LLL) to facilitate platelet biogenesis, prolong platelet life in circulation and in storage.
  • LLL low level light
  • One aspect of the present disclosure is directed to employing LLL (e.g., alone or in combination with other drugs and/or biological agents) to facilitate both in vitro and in vivo platelet biogenesis.
  • a further aspect of the present disclosure is directed to employing LLL (e.g., alone or in combination with other agents), to prolong the shelf-life of stored platelets and the lifespan of circulating platelets within a patient's blood.
  • the present disclosure can include a device configured to apply LLL to platelets and/or platelet precursors (e.g., megakaryocytes).
  • the LLL can be used to facilitate platelet biogenesis, prolong platelet life in in vivo circulation, and/or in in vitro platelet storage.
  • the device can include a controller, comprising a non-transitory memory and a processor, that can be configured to generate a control signal comprising a parameter for LLL application.
  • the device can also include a LLL generator that can be configured to receive the control signal, generate the LLL based on the parameter, and apply the generated LLL to the platelets and/or platelet precursors.
  • the parameter can be at least one of an on time, an off time, a light density, a power, a power density, and an output characteristic.
  • the present disclosure can include a system that can include a controller device and a LLL generator device.
  • the controller device can include a non-transitory memory and a processor and be configured to generate a control signal comprising a parameter for the LLL application.
  • the LLL generator device can be configured to receive the control signal, generate the LLL based on the parameter, and apply the generated LLL to platelets and/or platelet precursors.
  • the LLL generator device can include a monochromatic laser and/or a light emitting diode (LED) configured to generate the LLL comprising a wavelength from 600 nm to 1 100 nm.
  • LED light emitting diode
  • the parameter can include an on time, an off time, a light density, a power, a power density, and/or an output characteristic.
  • the LLL can be delivered continuously and/or in a pulsed mode of operation.
  • the LLL generator device can include one or more Gallium Arsenide (GaAs) super pulsed diodes that emit radiation for an extremely short duration (e.g., 100 to 300 nanoseconds) at a wavelength from 600 to 1 1 00 nm.
  • GaAs Gallium Arsenide
  • the present disclosure can include a method that can be performed by a LLL generator device.
  • the method can include receiving a control signal from a controller device that can include a parameter for the LLL application.
  • the method can also include obtaining the parameter from the control signal and applying LLL to at least one of platelets and platelet precursors according to the parameter.
  • the parameter can include an on time, an off time, a light density, a power, a power density, and/or an output characteristic.
  • the energy of LLL can be delivered in vivo, while in other instances, the energy of LLL can be delivered in vitro.
  • the method can include administering LLL non-invasively over an area of a patient's body that includes one or more platelet-making bones.
  • LLL can be applied directly to the platelet-making bone marrow by an invasive technique, such as a laser probe catheter.
  • a method of enhancing platelet biogenesis in a patient can include administering thrombopoietic agents in conjunction with LLL to increase the platelet quantity, such as interleukin 1 1 (IL-1 1 ), thrombopoietin (TPO), TPO peptide or non-peptide mimics.
  • IL-1 1 interleukin 1 1
  • TPO thrombopoietin
  • TPO TPO peptide or non-peptide mimics.
  • a method of extending platelet storage can include adding platelet additive solution (PSA) configured to enhance platelet function during long-term storage.
  • PSA platelet additive solution
  • a method of extending platelet storage can include using ultraviolet-A, ultraviolet-C, or blue light (405-495 nm) radiation in conjunction with LLL to reduce pathogen contamination during long-term storage.
  • FIG. 1 is a schematic diagram illustrating a mechanism by which low level light (LLL) can act on cells
  • FIG. 2 is a schematic diagram showing a path of platelet generation
  • FIG. 3 is a schematic block diagram of a system that can apply LLL to facilitate platelet biogenesis, according to an aspect of the present disclosure
  • FIG. 4 is a process flow diagram illustrating a method for delivering LLL to facilitate platelet biogenesis that can be employed by the low level light (LLL) generator of FIG. 3;
  • FIG. 5 is an experimental result showing that LLL increases the ATP synthesis in murine thrombopoietic cells
  • FIG. 6 is an experimental result showing that LLL enhances HSC differentiation ex vivo, in particular, after stress;
  • FIG. 7 is an experimental result showing that LLL preferably promotes the differentiation of MKs to platelets
  • FIG. 8 is an experimental result showing that LLL enhances protoplatelet formation (PPF) ex vivo;
  • FIG. 9 is an experimental result showing that LLL enhances platelet formation from LLL-treated MKs in vivo
  • FIG. 10 is an experimental result showing that LLL improves platelet regeneration in mice after ⁇ -irradiation
  • FIG. 1 1 is an experimental result showing that LLL enhances platelet biogenesis in vivo in the presence of anti-CD41 antibody
  • FIG. 12 is a schematic block diagram of a system that can apply LLL to enhance platelet storage, according to another aspect of the present disclosure
  • FIG. 13 is a process flow diagram illustrating a method for delivering LLL to enhance platelet storage that can be employed by the LLL generating incubator of FIG. 14;
  • FIG. 14 is an experimental result showing that LLL increases ATP synthesis in fresh platelets
  • FIG. 15 is an experimental result showing that LLL does not activate platelets
  • FIG. 16 is an experimental result showing that LLL significantly sustains ATP synthesis during platelet storage
  • FIG. 17 is an experimental result showing LLL significantly blunts the decrease in the pH value of stored platelets over time
  • FIG. 18 is an experimental result showing that LLL significantly improves viability of stored platelets
  • FIG. 19 is an experimental result showing that LLL significantly sustains mitochondrial membrane potential of stored platelets
  • FIG. 20 is an experimental result showing that LLL reduces the production of reactive oxygen species (ROS) during platelet storage.
  • ROS reactive oxygen species
  • the term “low level light (LLL)” can refer to a procedure that involves exposing cells (e.g., stem cells, other types of platelet precursor cells, platelets, etc.), tissue and/or at least a portion of a patient's body (e.g., platelet- making bone) to low levels of red and near infrared (NIR) light at energy densities that are low compared to other forms of laser therapy (e.g., ablation, cutting, thermal coagulation, etc.).
  • NIR near infrared
  • LLLT low level light therapy
  • platelet can refer to a blood cell that contains fragments of cytoplasm and no nucleus that contributes to homeostasis by contributing to the process of stopping bleeding (or clotting).
  • biogenesis can refer to the synthesis of a biological substance.
  • platelet biogenesis can refer to the synthesis of platelets, platelet precursors, etc.
  • the terms "subject” and "patient” can refer to any warmblooded living organism including, but not limited to, a human being, a pig, a rat, a mouse, a dog, a cat, a goat, a sheep, a horse, a monkey, an ape, a rabbit, a cow, etc.
  • low level light can be applied (in one dose or in multiple doses) to cells (e.g., stem cells, megakaryocytes, other platelet precursor cells, platelets, etc.), tissue, and/or at least a portion of a patient's body at energy densities that are low compared to other forms of laser therapy (e.g., ablation, cutting, thermal coagulation, etc.).
  • LLL low level light
  • the present disclosure relates to the application of LLL to enhance both in vivo and in vitro platelet biogenesis and to extend platelet lifespan in both in vivo circulation and in vitro storage.
  • LLL is a simple, non-invasive, safe, convenient, and cost-effective modality that has been clinically employed for decades for pain relief and other applications.
  • LLL can be employed to enhance both in vivo and in vitro platelet biogenesis and to extend platelet lifespan in both in vivo circulation and in vitro storage at least because LLL can enhance ATP synthesis within cells and/or platelets. It is believed that mitochondria are likely site for the initial effects of LLL. As shown schematically in FIG. 1 , the LLL can excite several protein complexes (e.g., I, III, and/or IV,) in the mitochondrial respiratory chain (MRC). Normally, the MRC can produce more than 90% of the ATP in the cell, but the level of ATP synthesis would be reduced in a cell under stress, so with LLL, the amount of ATP within the cell can increase. In some instances, the LLL can lead (additionally or alternatively to the increased of ATP synthesis) to enhanced oxidative phosphorylation, enhanced mitochondrial membrane potential, reduced oxidative stress, and anti-apoptosis.
  • MRC mitochondrial respiratory chain
  • LLL can promote both in vivo and in vitro platelet biogenesis.
  • platelets 12 can be formed from mature
  • MKs megakaryocytes
  • HSCs hematopoietic stem cells
  • platelets can be formed from MKs 14 in a patient's bone marrow and then enter the patient's blood for circulation through the patient's body.
  • LLL can act on the HSCs 16, the MKs 14, the platelets 12, and/or other progenitors.
  • the LLL can be applied as a single dose. In other instances, the LLL can be applied in multiple doses. Moreover, in some instances, the LLL application can be non-invasive. In other instances, the LLL application can be invasive. For instance, LLL can be administered transcutaneously and/or subcutaneously, so that it is transmitted into bone marrow, via one or more optical fibers (biodegradable/non-biodegradable, flexible/rigid, etc.) and/or via a mesh formed over an area of marrow-containing bone. Regardless of the source or the output, the LLL energy at the bone marrow can be between 1 and 20 J/cm 2 .
  • the LLL can be applied in conjunction with the administration of one or more pharmaceutical treatments. Examples of
  • agents that can promote megakaryopoiesis e.g., thrombopoietin and its derivatives, any activators or stimulators wa thrombopoietin receptor, IL-6, IL- 1 1 , etc.
  • agents or chemical compounds or diet supplements that can enhances mitochondrial activity or improve mitochondrial functions (e.g., CoQ, metabolic substrates like pyruvate, lactate, glucose, vitamin D, etc.), growth factors and their analogs or peptides that can enhance the generation of megakaryocytes or myeloid progenitors, like colony stimulating factor (CSF), GM-CSF, platelet-derived growth factor (PDGF), megakaryocyte growth and development factors (MGDF).
  • CSF colony stimulating factor
  • GM-CSF GM-CSF
  • PDGF platelet-derived growth factor
  • MGDF megakaryocyte growth and development factors
  • clinically-available agents that can be used in connection with LLL include, but are not limited to: Nplate/Romiplostim (Amgen)-fusion protein analog of thrombopoeitin; Promacta/Eltrombopag (Ligand/GSK) for ITP, a small molecule agonist to the c-mpl (human TPO receptor), the target of thrombopoeitin; Neumega/Oprelvekin
  • application of the LLL can be controlled by a system 40 that can include a LLL generator 42, a controller 44, and a power source 46.
  • the power source 46 can be configured to supply an operating power to the controller 44 and/or the LLL generator 42.
  • the controller 44 and/or the LLL generator 42 can be electrically coupled to the power source 46.
  • the power source 46 can be a device that is configured to generate a power signal (e.g., including enough power to power up the controller 44 and/or the LLL generator), such as a battery power source, a line power source (e.g., a plug), or the like.
  • the power source can include at least two power sources (e.g., one to power the LLL generator 42 and one to power the controller 44).
  • the controller 44 can be configured to generate and transmit a control signal (e.g., including dosage parameters for LLL) to the LLL generator 42.
  • the controller 44 can be a computing device (e.g., a general purpose computer, special purpose computer, and/or other programmable data processing apparatus) that can include or be otherwise associated with a non-transitory memory storing instructions (e.g., computer program instructions) that, upon execution by a processor, can create a mechanism for implementing the functions of the controller 44 (e.g., generating and transmitting the control signal to the LLL generator 42).
  • one or more of the dosage parameters can be pre-set within the controller 44.
  • one or more of the dosage parameters can be input by a user via a user interface associated with the controller 44.
  • the controller 44 and the LLL generator 42 can be communicatively coupled (e.g., via a wired connection and/or a wireless connection) to facilitate the transmission of the control signal.
  • An example of a method 70 that the LLL generator 42 can utilize to apply the LLL according to the control signal is shown in FIG. 4.
  • the LLL generator 42 can receive the control signal from the controller 44.
  • the desired power density can be selected from 0.1 mW/cm 2 to 50 mW/cm 2 to be directly delivered to platelet-making bones.
  • a relatively higher surface power density of LLL may be required, depending on patient's skin pigmentation, the location of the target bone or vessel, and the depth of the bone or vessel relative to the skin surface.
  • super pulsed GaAs laser may be employed to generate super pulses of LLL with extremely short duration (100 to 300 nanoseconds), that can penetrate into tissue depths of 3 to 13 cm and deeper.
  • the LLL generator 42 can obtain the dosage parameters for the LLL from the control signal.
  • the LLL generator 42 can deliver the LLL according to the dosage.
  • the dosage can include a desired power density (e.g., .selected from 0.1 mW/cm 2 to 50 mW/cm 2 ) to be delivered to target cells (e.g., MKs inside the bone marrow cavities, circulating platelets in the blood vessels, and stored platelets in storage bags).
  • target cells e.g., MKs inside the bone marrow cavities, circulating platelets in the blood vessels, and stored platelets in storage bags.
  • a relatively higher surface power density of LLL may be required, depending on patient's skin pigmentation, the location of the target bone or vessel, and the depth of the bone or vessel relative to the skin surface.
  • super pulsed GaAs lasers can be employed to generate super pulses of LLL with extremely short duration (100 to 300 nanoseconds), that can penetrate into tissue depths of 3 to 13 cm and deeper to illuminate plate
  • the LLL generator 42 can be configured to apply LLL to a patient's body (e.g., the whole body or a portion of the body) and/or to an in vitro preparation based on the control signal.
  • the LLL generator 42 can be configured to apply the LLL to the patient's body according to the control signal.
  • the LLL can be applied to a portion of the patient's body (e.g., vertebrae, pelvis, ribs, sternum, femur, tibia, etc.).
  • a portion of the patient's body e.g., vertebrae, pelvis, ribs, sternum, femur, tibia, etc.
  • One example of the LLL generator 42 that can deliver the LLL to the portion of the patient's body is a LLL blanket.
  • the LLL blanket can be wrapped around a portion of the patient's body to deliver the LLL according to the control signal.
  • Another example of the LLL generator 42 is a LLL vest.
  • the LLL vest can cover the patient's vertebrae, sternum, and ribs to deliver LLL according to the control signal.
  • Yet another example of the LLL generator 42 is an LLL chair.
  • LLL chair which can deliver the LLL to the patient's vertebrae, pelvis, ribs and/or sternum according to the control signal.
  • an LLL bed can be arranged similarly to a tanning bed to deliver LLL to at least a portion of the patient's body.
  • the LLL can provide a safe and effective mechanism that can promote platelet regeneration and extend the life of circulating platelets in vivo.
  • LLL can provide a primary or secondary prophylaxis that can increase the number of platelets in the blood of a patient with thrombocytopenia, potentially reducing the need for platelet transfusions.
  • the LLL generator 42 can be configured to apply the LLL to an in vitro preparation according to the control signal to facilitate the generation of platelets. Due to the increasing demand of platelet transfusions each year, platelet shortages are common due to both limited donors and the short (5-day) shelf life for the stored platelets. To alleviate these shortages, much effort has been put into the development of donor-independent platelet generation in vitro using newly developed stem cell technologies. However, these stem cell technologies often lead to growing-limited processes for in vitro platelet generation. For example, in vitro platelet generation from CD34 + stem cells is a relatively low rate of platelet production.
  • LLL can significantly increase the rate of platelet production in vitro, either alone or in combination with other agents, enhancing the yield of platelet biogenesis from MKs or various stem cell technologies. In some instances, additional factors that facilitate platelet growth or mitochondrial activity, also can be added to the in vitro preparation to further promote the biogenesis of platelets in vitro.
  • Platelets can be generated in vivo from various stem cells, including both natural occurring and synthesized stem cells. LLL can be applied alone and/or in combination with one or more agents to induce platelet biogenesis in vitro. In some instances, LLL can promote platelet production from umbilical cord stem cells (ESC), induced pluripotent stem cells (iPSC), or other types of induced stem cells that can differentiate into myeloid progenitors and/or platelets.
  • ESC umbilical cord stem cells
  • iPSC induced pluripotent stem cells
  • LLL increases the ATP synthesis in murine thrombopoietic cells.
  • the graph 92 illustrates how LLL enhances ATP synthesis in these thrombopoietic cells.
  • Megakaryocytes (MKs), hematopoietic stem cells (HSCs), and bone marrow (BM) cells were prepared from 8-week-old C57BL/6 mice, and exposed to 81 0-nm diode laser at a power density of 10 mW/cm 2 for 5 min at an energy density (also called fluence) of 3 J/cm 2 .
  • the LLL treatment increased ATP formation immediately in these cells, which peaked at 60 min before returning to a base level in 90 min after LLL.
  • LLL enhances HSC differentiation ex vivo.
  • Murine HSCs were prepared from 8-week-old C57BL/6 mice and treated with or without 3- Gy ⁇ -irradiation (IR), followed by a single dose of 3 J/cm 2 LLL at 6 hr after ⁇ - irradiation.
  • the treated HSCs were cultured in methylcellulose-based media supplemented with optimal cytokines at 1 x10 3 cells per 35 mm dish for 14 days.
  • These HSCs produced heterogeneous populations of actively dividing hematopoietic progenitors, forming discrete colonies that can be enumerated and characterized as colony-forming units (CFUs).
  • CFUs colony-forming units
  • IR Compared with non-IR-treatment controls, IR severely reduced the number of Granulocyte-Erythrocyte-Monocyte-Megakaryocyte (CFU- GEMM, chart 96) and Granulocyte-Macrophage (CFU-GM, chart 96), as well as Burst-Forming Units of Erythroid (BFU-E, chart 100), in agreement with myeloid suppression by ⁇ -irradiation. Strikingly, the colony-forming ability of radiated HSCs was significantly restored by LLL (charts 96, 98, 100), with little effect on the differentiation of non-IR-HSC controls.
  • LLL preferably promotes the differentiation of MKs to platelets.
  • a single dose of 3 J/cm 2 LLL was given to ⁇ - ⁇ -irradiated (as shown in chart 106) and 3-Gy v- irradiated (as shown in chart 108) HSCs at 6 hr (DO), 6 days (D6), or 12 days (D12), respectively, after initial culture.
  • DO 6 hr
  • D6 6 days
  • D12 12 days
  • LLL enhances proplatelet formation (PPF).
  • Proplatelets are decorated with multiple swellings of a similar size to a platelet, on which the individual platelet develops (pictures 1 10, 1 1 2, 1 14, and 1 1 6). The complex of these branches and the length and number of swellings on each proplatelet determine the quantity of platelets produced from a single MK.
  • murine MKs were sorted from control mice or ⁇ -irradiated mice in 6 hr after 3-Gy ⁇ -irradiation.
  • the MKs that were exposed to a single dose of 3 J/cm 2 LLL were differentiated into platelets in medium supplied with 100 ng/ml thrombopoietin (TPO) for 24 hr.
  • TPO thrombopoietin
  • Control MKs formed branched proplatelets with abundant swellings on them, and the terminally mature MKs were ⁇ 100- ⁇ in diameter on average (picture 1 10).
  • 3-Gy ⁇ -irradiation gave rise to much shorter branches with a fewer swellings on the proplatelets, and the terminally mature MKs were only a half of the normal size (picture 1 14).
  • LLL enhances in vivo platelet production by treatment of mature MKs.
  • CD41 + MKs were sorted from donor mice, treated with a single dose of 3 J/cm 2 LLL, and then labeled with a vital fluorescent dye, carboxyfluorescein succinimidyl ester (CFSE).
  • CFSE carboxyfluorescein succinimidyl ester
  • the labeled MKs were intravenously infused into recipient mice at 1 x10 5 MKs per mouse.
  • the infused MKs produced a higher level of platelets in recipients with LLL than without it.
  • LLL improves platelet regeneration in mice after ⁇ - irradiation.
  • C57BL/6 mice at 8-week-old were subjected to 3-Gy whole body ⁇ - irradiation, followed by a whole body exposure to LLL at 6 hr post-Y-irradiation.
  • Two LLL regimens were one exposure with a 808-nm light-emitting diode (LED) at a fluence of 10 J/cm 2 (I R+1 xLLL); and a total of 4 exposures performed once per day up to 4 days with 1 0 J/ cm 2 at each point (IR+4xLLL).
  • LED light-emitting diode
  • mice that received 4 x LLL reduced a tail bleeding time to a normal level, significantly shorter than that in the absence of LLL (P ⁇ 0.001 ) when examined 3 weeks post-Y-irradiation (graph 124).
  • control mice treated with 4XLLL showed little effect on their platelet counts, underscoring a safety of the approach since there would be few concerns about thrombosis even after repeated LLL uses.
  • LLL enhances platelet biogenesis in vivo in the presence of anti-CD41 antibody.
  • Immune thrombocytopenia occurs frequently after repeated platelet transfusions, cancer patients receiving chemotherapy, and patients with autoimmunity against platelets.
  • mice were given anti-CD41 antibody every other day for a week at a dose of 68 ⁇ g/kg body weight.
  • Anti-CD41 antibody can bind to platelets, megakaryocytes, and their precursors and triggers platelet depletion in 24 hr after injection.
  • mice receiving anti-CD41 antibody were illuminated with 1 0 J/cm 2 LLL daily for 4 days with an initial illumination at 4 hr after first anti-CD41 injection.
  • the LLL-illuminated mice displayed a much less severe decline in platelet counts in the first 2 days as compared to non-LLL-treated controls. This was followed by accelerating platelet recovery starting at as early as day 3 after anti-CD41 antibody injection.
  • graph 122 again, there was no effect of LLL on platelet counts in control mice.
  • n 6, * P ⁇ 0.05, ** P ⁇ 0.01 , *** P ⁇ 0.001 compared to mice receiving anti-CD41 antibody only.
  • LLL can be used to increase the shelf life of the stored platelets (e.g., applied before storage in an incubator and/or while stored in the incubator). Platelets require special storage conditions, and their storage time is limited due to storage-related mitochondrial injury (e.g., the U.S. Food and Drug Administration has set an expiration date of 5 days for stored platelets). While not wishing to be bound by theory, it is believed that because LLL can sustain mitochondrial functionality under a variety of stresses, LLL can preserve the quality of stored platelets over an extended period of time. For example, LLL can extend a shelf life of stored platelets beyond the FDA-mandated expiration date of 5 days (e.g., to 8 days or more). Increasing the shelf life of platelets can increase the availability of platelets and help to meet an increasing demand of platelet transfusion. Additionally, in some instances, LLL can increase the efficacy of platelet transfusions even with a reduced number of platelets in each transfusion.
  • LLL can be controlled by a system 180 that can be included in incubator 182 with a LLL generator 1 84, a controller 188, and a power source 190.
  • the LLL generator 184 is illustrated as being housed within the incubator 182, in some instances, the LLL generator 184 can be independent from the incubator 182. In both instances, the LLL generator 184 can be configured to deliver the LLL to the stored platelets.
  • the power source 190 can be configured to supply an operating power to the controller 188, the incubator 182, and/or the LLL generator 184.
  • the controller 188, the incubator 1 82, and/or the LLL generator 1 84 can be electrically coupled to the power source 190.
  • the power source 190 can be a device that is configured to generate a power signal (e.g., including enough power to power up the controller 188, the incubator 1 82, and/or the LLL generator 184), such as a battery power source, a line power source (e.g., a plug), or the like.
  • the controller 188 can be configured to generate and transmit a control signal (e.g., including parameters for LLL and/or incubation parameters) to the LLL generator 184 and/or the incubator 182.
  • a control signal e.g., including parameters for LLL and/or incubation parameters
  • the instructions can create a mechanism for implementing the functions of the controller 188 (e.g., generating and transmitting the control signal to the LLL generator 184 and/or the incubator 182).
  • one or more of the parameters can be pre-set within the controller 188.
  • one or more of the dosage parameters can be input by a user via a user interface associated with the controller 188.
  • the controller 188 can be a computing device (e.g., a general purpose computer, special purpose computer, and/or other programmable data processing apparatus) that can include a non- transitory memory that stores instructions (e.g., computer program instructions) and a processor that can be configured to execute the instructions.
  • a computing device e.g., a general purpose computer, special purpose computer, and/or other programmable data processing apparatus
  • the controller 188, the incubator 182, and/or the LLL generator 1 84 can be communicatively coupled (e.g., via a wired connection and/or a wireless connection) to facilitate the transmission of the control signal.
  • An example of a method 1 50 that the LLL generator 184 can utilize to apply the LLL according to the control signal is shown in FIG. 13.
  • the platelets can be stored in the incubator 182.
  • the LLL generator 184 can receive the control signal from the controller 186.
  • the LLL generator 1 84 can deliver the LLL to the platelets according to the parameters.
  • the LLL generator 184 can be configured to apply LLL to stored platelets based on the control signal.
  • LED light emitting diode
  • the LLL generator 184 can be integrated into the current platelet storages mechanisms.
  • the LLL generator 184 e.g., a LED array
  • the flatbed can be integrated within an incubator 182 to apply the LLL to the stored platelets throughout their storage period.
  • Platelet-rich plasma were prepared from 8-week-old C57BL/6 mice, and illuminated with an 808-nm LED at 1 0 mW/cm 2 .
  • ATP synthesis in platelets was enhanced significantly by LLL at energy densities from 0.5 J/cm 2 to 1 0 J/cm 2 .
  • n 6, *p ⁇ 0.05, **p ⁇ 0.01 , ***p ⁇ 0.001 , ns, not significant.
  • LLL does not activate platelets. Platelet storage is often associated with platelet activation that reduces efficacy of transfusion and should be avoided.
  • PRP illuminated by various doses of LLL (diagrams 1 62, 1 64, 1 66) and non-treated control (diagram 1 60) were stained with platelet marker CD41 and activation biomarker CD62p.
  • LLL at energy densities from 0.5 J/cm 2 to 4 J/cm 2 did not affect platelet activation (p>0.05).
  • LLL significantly sustains ATP synthesis during platelet storage. Platelet storage-associated mitochondrial injury reduces ATP production over time during storage.
  • murine PRP was prepared from 8-week-old C57BL/6 mice and stored for 8 days in a standard condition (22 °C with constant flatbed agitation at 70 cycles per minute). LLL was administered only once at 1 hr after PRP collection at an energy density of 0.5, 2 or 4 J/cm 2 . ATP levels were determined every other day during storage. As shown in graph 1 68, ATP levels in PRP treated with LLL at 0.5, 2 or 4 J/cm 2 were significantly higher than control at all times tested.
  • the ATP level in 8-day-stored platelets treated with 0.5 J/cm 2 LLL was similar to that measured in 5-day stored platelets.
  • the latter is a standard time of current platelet storage and the result clearly suggests a great potential to extend a platelet storage time to 8 days from 5 days.
  • n 6, ***p ⁇ 0.001 , compared to non-treated control.
  • Murine PRP was prepared and treated with LLL similarly to the data shown in FIG. 20. pH values of each sample were determined every other day during storage.
  • LLL significantly retains pH values over controls during the 8 days of platelet storage. On day 8 of storage, platelets treated with LLL at all energy densities tested had a pH value as same as that in non-treated PRP stored for 5 days under similar conditions. n 6, *** p ⁇ 0.001 , compared to non-treated control.
  • LLL significantly improves the viability of stored platelets.
  • Murine PRP was prepared and treated with LLL similarly to the data shown in FIG. 20.
  • graph 172 illustrates that platelet counts were significantly higher with LLL treatment at 0.5 or 2 J/cm 2 than non-treated control at all time points.
  • platelet counts on day 8 of the storage with 0.5 or 2 J/cm 2 LLL were similar as those platelets stored for 5 days in the absence of LLL.
  • LLL significantly sustains mitochondrial membrane potential of stored platelets.
  • Murine PRP was prepared d from 8-week-old C57BL/6 mice and stored as above. A single dose of 0.5 J/cm 2 LLL was administered at 1 hr after PRP collection. Changes of mitochondrial membrane potential were
  • LLL reduces the production of reactive oxygen species (ROS) during platelet storage. Increased ROS production secondarily to mitochondrial injury can damage platelets and mitochondria, reducing the viability of stored platelets.
  • ROS levels were determined by cell-permeant fluorescent dye, 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (also known as dichlorofluores cin diacetate or DCF) in platelets stored for 5 days.
  • H2DCFDA 2',7'-dichlorodihydrofluorescein diacetate
  • DCF dichlorofluores cin diacetate

Abstract

La présente invention concerne des systèmes et des procédés qui peuvent appliquer une lumière de faible niveau (LLL) à des plaquettes et/ou des cellules précurseurs de plaquettes. Sans vouloir se limiter à la théorie, on pense que la LLL peut améliorer la synthèse d'ATP par les mitochondries dans les plaquettes et/ou les cellules précurseurs de plaquettes pour contribuer à améliorer la biogenèse des plaquettes et augmenter la durée de vie des plaquettes. Dans certains cas, la LLL peut faciliter la biogenèse des plaquettes in vitro et/ou in vivo. Dans d'autres cas, la LLL peut augmenter la durée de vie des plaquettes en circulation. Dans encore d'autres cas, la LLL peut être utilisée pour prolonger la durée de conservation des plaquettes stockées.
PCT/US2015/051323 2014-09-24 2015-09-22 Systèmes et procédés pour améliorer la biogenèse des plaquettes et augmenter la durée de vie de plaquettes, en circulation et en stockage WO2016048942A1 (fr)

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WO2018005417A1 (fr) * 2016-06-27 2018-01-04 The General Hospital Corp Dba Massachusetts General Hospital Stimulation de génération de plaquettes par activation de la biogenèse mitochondriale
CN110087643A (zh) * 2016-06-27 2019-08-02 综合医院公司以麻省总医院名义经营 通过激活线粒体生物发生的血小板生成刺激
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