Compositions and devices for systemic delivery of uridine

Abstract

A solution of uridine in saline, which may also contain β-hydroxybutyrate, can be administered subcutaneously. A reservoir capable of holding the uridine in saline solution, fluidly connected to one or more infusion needles, and a pump configured to move the solution from the reservoir to the subject through the one or more infusion needles are described.

Description

[Background technology]

[0001] Brain injury caused by trauma or oxygen deprivation is a major cause of irreversible morbidity and mortality. Traumatic brain injury (TBI) and hypoxic-ischemic encephalopathy (HIE) exhibit similar courses of damage and recovery. Once the initial injury (whether traumatic or ischemic) stabilizes, a metabolic crisis phase often follows, resulting in secondary injuries that can begin within minutes to days after the initial injury, including mitochondrial dysfunction, edema (resulting in impaired cerebral blood flow due to increased intracranial pressure), seizures, excitotoxic injury, cell death, and inflammation. This secondary phase often contributes to long-term or irreversible consequences, including mortality after the onset of HIE and TBI, but also provides a time frame in which neuroprotective agents or procedures (e.g., therapeutic hypothermia) can mitigate accumulated damage and promote recovery.

[0002] Intraperitoneal injection of uridine has been shown to reduce cell death and brain swelling in rodent models of TBI and HIE, exhibiting some degree of neuroprotective effect. However, the majority or all of such studies have been conducted in rats, which rapidly and extensively catabolize uridine (using cytidine instead of inter-organ pyrimidine transport) and therefore do not predict the potential activity (or lack thereof) of uridine in humans or other species. Uridine also induces hypothermia in rats as a result of its degradation, but interpretation of protective outcomes is potentially difficult because hypothermia induced by cooling of the head or body may exhibit some neuroprotective activity as it can reduce tissue energy demand.

[0003] In humans, systemic administration of uridine was abandoned after clinical studies were thwarted following problems encountered in attempts to administer uridine intravenously. Intravenous uridine in humans resulted in hyperthermia as a dose-limiting toxicity (in contrast to its hypothermic effect in rodents), and hyperthermia is counterproductive in subjects with brain injury, potentially exacerbating damage in acute traumatic brain injury or ischemia. Furthermore, uridine infusion into accessible peripheral veins in humans caused local phlebitis, requiring administration via a central venous catheter to overcome this side effect. For these reasons, parenteral administration of uridine was abandoned in the early 1990s, and uridine triacetate, an orally bioavailable uridine prodrug, was chosen as a method for systemic delivery of uridine.

[0004] Alternative fuels have been proposed to support brain energy metabolism after TBI or HIE, but the results in animal models have been ambiguous. Beta-hydroxybutyrate (BHB), a ketone body that the brain can directly utilize in situations of reduced or impaired glucose utilization, has shown both positive effects and adverse effects, including damage to the blood-brain barrier, when administered intravenously to rats at neuroprotective doses, e.g., 30 mg / kg BHB over 6 hours.

[0005] Various other magnesium-containing agents have been tested to reduce secondary brain damage after TBI or HIE, but magnesium has shown ambiguous, sometimes adverse, results in preclinical animal models and little to no benefit in human clinical studies. Its usefulness may be limited by magnesium transport across the blood-brain barrier, and it is unclear whether magnesium deficiency observed in the brain and other pathological conditions after TBI is the cause or effect of cellular damage or energy deficiency. A review of the clinical experience with magnesium treatment in TBI concluded that "there is no evidence to support the use of magnesium salts in patients with acute traumatic brain injury" (Arango MF and Bainbridge D, "Magnesium for acute traumatic brain injury (Review). Cochrane Database of Systematic reviews 2008, Issue 4, Article NO. CD005400"). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Arango MF and Bainbridge D, "Magnesium for acute traumatic brain injury (Review). Cochrane Database of Systematic reviews 2008, Issue 4, Article NO. CD005400 [Overview of the Initiative]

[0007] The present invention provides a solution containing a neuroprotective amount of uridine dissolved in a physiological saline solution.

[0008] The present invention provides a method for treating an acute brain injury condition in a subject, comprising the step of administering to the subject a composition containing a neuroprotective amount of uridine dissolved in physiological saline solution.

[0009] The present invention provides a system for the delivery of uridine, comprising a reservoir capable of holding a solution containing uridine dissolved in physiological saline, which is fluid-connected to one or more injection needles, and a pump configured to move the solution from the reservoir to a target through one or more injection needles.

[0010] The present invention provides a method for administering uridine to a mammalian subject requiring uridine therapy, comprising the step of subcutaneously administering to the subject a pharmaceutical composition containing uridine dissolved in physiological saline solution in an amount sufficient to maintain a plasma uridine concentration of 50 to 150 μM (micromolar) in the subject. [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows the effect of uridine and racemic magnesium β-hydroxybutyrate (MgBHB) on mortality in mice treated with 60 mg / kg of 3-nitropropionic acid (3-NP,3-nitropropionic acid). It also shows the survival percentages at various time points in a 3-NP mouse model of mitochondrial dysfunction for mice treated with physiological saline (negative control), subcutaneous uridine solution, MgBHB·3H2O, or both subcutaneous uridine solution and MgBHB·3H2O. [Figure 2] This figure shows the survival of mice that received 60 mg / kg / day of 3-nitropropionic acid and were treated with subcutaneous uridine 200 mg / kg tid and Na(D-BHB) vs. Na(DL-BHB) 328 mg / kg tid, both individually and in combination. [Figure 3]Figure showing the change in body weight (% of baseline weight) in mice treated with 3-nitropropionic acid at 60 mg / kg / day, alone and in combination with subcutaneous uridine at 200 mg / kg t.i.d. and Na(D-BHB) vs. Na(DL-BHB) at 328 mg / kg t.i.d. [Figure 4] Figure showing the survival of mice treated with 3-nitropropionic acid at 65 mg / kg / day, alone and in combination with subcutaneous uridine at 200 mg / kg t.i.d. and Na(D-BHB) at 985 mg / kg t.i.d. [Figure 5] Figure showing the survival of mice treated with 3-nitropropionic acid at 70 mg / kg / day, alone and in combination with subcutaneous uridine at 200 mg / kg t.i.d. and Na(D-BHB) at 985 mg / kg t.i.d. [Figure 6] Figure showing plasma uridine after subcutaneous administration of uridine or uridine + Na(D-BHB). [Figure 7] Figure showing plasma uracil after subcutaneous administration of uridine or uridine + Na(D-BHB). [Figure 8] Figure showing plasma [uridine + uracil] after subcutaneous administration of uridine or uridine + Na(D-BHB). [Figure 9] Figure showing plasma D-β-hydroxybutyrate after subcutaneous administration of uridine or uridine + Na(D-BHB).

MODE FOR CARRYING OUT THE INVENTION

[0012] In traumatic brain injury (TBI) and hypoxic-ischemic encephalopathy (HIE) resulting from perinatal asphyxia, or in emergencies such as resuscitation after cardiac arrest or drowning, it is essential to initiate neuroprotective therapy as quickly as possible. In such situations, especially when other injuries associated with TBI often require immediate treatment, the placement of a central venous catheter is problematic and dangerous.

[0013] When a victim of TBI or HIE is unconscious or otherwise in a state of functional loss, oral uridine prodrugs such as uridine triacetate can be difficult to administer appropriately, especially in emergencies where treatment time is critical.

[0014] There is a need for rapid, controlled systemic administration of uridine at a concentration and duration sufficient to protect the brain and other tissues, without harmful side effects such as hyperthermia or phlebitis and without the need to reach the central vein for administration.

[0015] Uridine has been reported to provide neuroprotection in some models of mitochondrial dysfunction and acute hypoxia and ischemia when delivered as oral uridine triacetate or as intraperitoneal injection of uridine itself. For several emergency clinical indications such as neonatal asphyxia, stroke or traumatic brain injury, the subcutaneous route of delivery is potentially advantageous, but it has not been established so far that systemic and cerebral delivery of uridine quickly enough and in sufficient amounts for neuroprotection is feasible by this route of administration. The efficacy of subcutaneous uridine in models of severe energy failure has not been reported so far. Furthermore, so far the therapeutic activity of other putative neuroprotective agents has not been tested in parallel with uridine and combinations of such agents co-administered in subcutaneous formulations have not been tested to determine whether there is an added or supra-additive benefit of such combinations.

[0016] Subcutaneous uridine infusion device Despite the problems associated with intravenous administration of uridine to humans in the case of emergency systemic administration of uridine, subcutaneous injection of uridine dissolved in saline is effective and practical. Unlike many drugs and biological agents, uridine rapidly distributes throughout the total body water and does not bind to subcutaneous tissue.

[0017] In one embodiment, an infusion device dispenses a liquid at a controlled rate through one or more short, small-diameter injection needles, and in some embodiments, the needles are aligned with an adhesive patch that secures the needles in place after insertion into the skin. The patch is applied, for example, to the skin of the abdomen, and an appropriate concentration of uridine is injected at a rate that results in a sufficient concentration in the plasma to produce a neuroprotective effect in the central nervous system. The circulating uridine crosses the blood-brain barrier via nucleoside transporters at a rate sufficient to produce a neuroprotective effect, provided that a sufficient plasma concentration is maintained for a sufficient amount of time.

[0018] For emergency use, infusion devices that do not require an external power source are advantageous in certain situations, particularly outside of hospitals. Infusion devices that do not require electricity are known in the art. In one embodiment, uridine is infused via a mechanical ("spring-driven") or elastic infusion device that provides a controlled dosing rate over a sufficient period of time. Electric pumps, including battery-powered pumps, are included within the scope of the present invention.

[0019] Pumps with adjustable flow rates, or alternatively, pumps with reservoirs of different sizes, allow for optimization of infusion rates to treat patients of varying body sizes, ranging from neonates (and even premature infants) to adults, enabling the maintenance of plasma uridine in the appropriate therapeutic zone to protect the brain and other tissues.

[0020] In some embodiments, a single injection rather than uridine infusion, for example, an injection given by a first responder under emergency conditions before hospitalization, is an important option. Under such conditions, a subcutaneous injection that can be easily administered from a small, portable device such as a pre-filled syringe may provide neuroprotection, reducing cellular dysfunction and death during the time required for transport to the hospital or for a more complete assessment to determine the course of treatment. A stroke that deprives the brain of blood or oxygen can be ischemic or hemorrhagic. Thrombus-dissolving agents are suitable for ischemic strokes but are potentially lethal in people with hemorrhagic strokes. Distinguishing between ischemic and hemorrhagic strokes generally requires imaging equipment in a hospital. The compositions of the present invention can be safely administered, provide neuroprotection regardless of whether the stroke is ischemic or hemorrhagic, and allow tissue to be preserved until the type of stroke is determined and treatment can be performed.

[0021] Subcutaneous injection composition In addition to uridine, other neuroprotective agents may be included in the subcutaneous uridine injection solution. The constraint is that such components must be soluble and readily diffuse from the injection site into the body's total fluids, including circulation.

[0022] Organic anions may also be included in the uridine infusion solution. Such anions include, but are not limited to, β-hydroxybutyrate (also called 3-hydroxybutyrate), threonine, aspartate or other amino acids, gluconate, pyroglutamate, citrate, ketoleucine, acetate, and lactate. β-hydroxybutyrate (BHB), a ketone body that the brain can utilize as an alternative fuel, is a favorable anion because it has been reported to have some neuroprotective effects. One potential consequence of its use is that BHB may inhibit the de novo synthesis of uridine nucleotides, thus affecting its net protective effect. Co-administration of uridine with BHB overcomes this potential problem, and the protective effect of the combination also has the above-mentioned additional effects on survival and morbidity, and has been found to be significantly more effective than either component alone in animal models of acute mitochondrial energy deficiency. Racemic BHB, or preferably the D-enantiomer of BHB, can be used. The D-enantiomer of BHB is also known as the R-enantiomer in the (R)-(S) nomenclature system. The D-enantiomer of BHB may also be called D-BHB, (R)-BHB, (R)-(-)-BHB, (R)-(-)-3-hydroxybutyrate, (R)-(-)-β-hydroxybutyrate, and D-(-)-β-hydroxybutyrate, among other names known to those skilled in the art.

[0023] Organic anions require a cationic counterion because the composition has a pH within a physiologically tolerable range, preferably pH 5 to 7.5. These include sodium ions (Na+) and magnesium ions (Mg 2+) is a suitable cation. Magnesium ions contribute somewhat to neuroprotection and tissue protection, adding to the overall benefits of the composition of the present invention. However, there is an upper limit to the amount of magnesium that can be administered as an acute dose, as plasma concentrations exceeding 4 mEq per liter can cause side effects such as impaired deep tendon reflexes, and higher concentrations can cause hypotension and heart block. Therefore, for single injections for emergency treatment, the sodium salt of BHB is preferred, or a mixture of magnesium and sodium ions is administered, and the magnesium dose is capped at 40 mEq (1 gram of Mg ions) per injection in human adults and 10 mEq (0.25 grams of Mg ions) per injection in human children, with the remainder of BHB buffering with sodium as a counterion, if present. Generally, for prolonged infusions of the compositions of the present invention, which result in lower peak concentrations of the injected drug, sodium BHB or a mixture of sodium BHB and magnesium BHB is administered, in which case the Mg dose is capped by the remainder of the BHB counterions containing sodium ions at 80 mEq per day (40 mEq per 12 hours) in adults or 40 mEq per day (20 mEq per 12 hours) in children.

[0024] The injectable solution does not need to be isotonic; it is known that solutions with an osmotic pressure of approximately 800 milliosmoles per liter are acceptable for subcutaneous administration in humans. Hypertonic solutions up to the acceptable limit minimize the total volume of liquid that must be administered.

[0025] Local anesthetics, including but not limited to lidocaine or lignocaine, may be added to the composition of the present invention at a maximum of 2% w / v, preferably 0.4-1%, to minimize local discomfort or pain after injection or infusion via the subcutaneous route.

[0026] Hypothermia induced by surface cooling via intravascular heat exchange catheters or by infusion of cold fluids has neuroprotective effects in subjects after TBI, HIE, or cardiac arrest. Pharmacological hypothermia induced by the administration of agents that affect thermoregulation and lower body or brain temperature can also protect the brain and other tissues after trauma or ischemic injury. Pharmacological hypothermia is particularly important in situations where precise cooling devices are not available. A hypothermic agent may be incorporated into the compositions of this disclosure. Suitable hypothermic agents include A3 adenosine receptor agonists (selective or nonselective), 5-HT (serotonin) 1A receptor agonists, activators of "warming" transient receptor potential channels, particularly activators of TRPV1 (e.g., dihydrocapsaicin or rinvanil) and TRPV3 (e.g., carvacrol), and may also include inhibitors of "cooling" TRP channels, such as TRPM8. Such drugs are administered at doses and infusion rates that lower body or brain temperature within an acceptable safety and tolerable profile. Concurrent magnesium infusion may reduce compensatory tremor responses, thereby improving both the thermoregulatory effect and patient comfort.

[0027] Concentration and volume The concentrations of uridine and other components of the infusion are correlated with flow rate, diffusion from the subcutaneous space, and the size of the reservoir in the infusion device.

[0028] The pharmacokinetic goal of subcutaneous uridine injection is to maintain plasma uridine concentrations within the 50–150 μM range, at a rate of 0.5–2 grams / m³ per hour. 2Subcutaneous injection of dissolved uridine is suitable for maintaining plasma uridine within this therapeutic zone. In some embodiments, the infusion delivery is pulsatile, with a periodically increasing infusion rate, and correspondingly, over approximately one hour, plasma uridine and BHB increase to a concentration range of 200–500 μM, more specifically 300–500 μM, with 2–4 such peaks during each 24-hour infusion. Similarly, in embodiments in which the composition of the present invention is administered by subcutaneous injection rather than prolonged infusion, the dose and concentration are measured to achieve a peak plasma uridine concentration of 200–500 μM, or more specifically 300–500 μM.

[0029] Uridine is extremely water-soluble (solubility at room temperature greater than 50%, or greater than 0.5 g / ml), and can be administered in relatively small volumes if desired. For example, a typical adult (BSA approximately 1.6 m³) 2 ) In this case, 1 gram / m² is used for body surface area (BSA) per hour. 2 The 12-hour delivery of uridine can be achieved by a total injection volume of less than 40 or 50 ml, which is an amount that can be easily introduced into the subcutaneous space and absorbed thereover during this time.

[0030] The magnesium ion concentration in the infusion is based on an infusion rate determined to provide optimal exposure to plasma uridine. In embodiments where magnesium ions are used as counterions for β-hydroxybutyrate, for adults, preferred doses of magnesium ions are 10–40 milliequivalents per 12 hours, favorably 20–30 mEq per 12 hours, or twice these amounts per 24-hour infusion. For children, preferred doses of magnesium ions are 10–20 milliequivalents per 12 hours, or twice these amounts per 24-hour infusion.

[0031] Following the initial 12-hour infusion, additional 12-hour infusions may be initiated sequentially as needed, depending on the patient's condition, brain injury, or clinical signs assessed by appropriate neurological monitoring techniques using suitable equipment, and by plasma or serum markers.

[0032] For single-dose injections as an alternative to prolonged infusions, the composition of the present invention contains a total concentration not exceeding 800 milliosmoles per liter of uridine or a metal salt of uridine or uridine plus BHB sufficient to deliver 10-40 mg of uridine and 0-100 mg / kg of BHB, preferably 50-100 mg / kg, per kg of body weight in a single dose. In a 70 kg adult, these dose ranges are considered equivalent to 700-2800 mg of uridine and 0-7000 mg of D-BHB, more specifically 4000-7000 mg of D-BHB. In a 3 kg newborn infant, a single dose of the composition of the present invention is considered to contain 30-120 mg of uridine and 0-300 mg of D-BHB, more specifically 100-300 mg of D-BHB. In an embodiment of the injection according to the present invention, in which magnesium ions are used as counterions for β-hydroxybutyrate, the preferred dose of magnesium ions is 10 to 40 milliequivalents for adults and 5 to 10 milliequivalents for children.

[0033] Peripheral tissue damage in hypoxic-ischemic encephalopathy Hypoxic-ischemic encephalopathy (HIE) is defined as brain injury and functional impairment caused by perinatal brain asphyxia or ischemia, which can be caused by umbilical cord compression, placental abruption, respiratory failure immediately after birth, or perinatal infection. HIE is a leading cause of infant mortality and long-term persistent physical disability, including cerebral palsy, developmental delay, and paroxysmal disorders.

[0034] Infants, especially premature babies, may not be able to receive immediate oral medication due to either immature intestines or a risk of developing intestinal complications such as necrotizing enterocolitis.

[0035] Subcutaneous infusion of uridine solution presents a method for rapidly introducing uridine into the body, which can reach the brain via the circulation of neonatal infants subjected to asphyxia at birth. Furthermore, HIE is very commonly associated with peripheral organ dysfunction or failure due to hypoxia, ischemia, or inflammation. Uridine protects peripheral tissues from hypoxia and inflammation, thereby providing further benefits beyond its neuroprotective effects in HIE when administered to neonates recovering from asphyxia. Similarly, TBI often involves damage to peripheral tissues due to trauma, hemodynamic problems, or inflammation. The uridine-containing infusions of this disclosure protect peripheral tissues during recovery from TBI.

[0036] Embodiments of the pharmaceutical composition of the present invention further comprise neuroprotective amounts of β-hydroxybutyrate and a cationic counterion. In preferred embodiments, the β-hydroxybutyrate and counterion are in the same solution as uridine. In embodiments of the present invention, the β-hydroxybutyrate is a D-enantiomer of β-hydroxybutyrate or a mixture of D and L-enantiomers, for example, racemic-β-hydroxybutyrate. According to the present invention, any conventional cationic counterion can be used. In one embodiment, the cationic counterion is Na + In another embodiment, Na + and Mg 2+ Both exist as counterions.

[0037] In embodiments of the present invention, the solution further comprises an organic anion selected from the group consisting of amino acids, glucons, pyroglutamates, citrates, acetates, and lactates. Examples of suitable amino acids include threonine or aspartate. In embodiments of the present invention, the solution further comprises a thermoregulator. In embodiments of the present invention, the saline solution is isotonic or hypertonic saline. In embodiments of the present invention, the pharmaceutical composition is formulated for subcutaneous administration to a subject.

[0038] In embodiments of the present invention, the pharmaceutical composition is formulated for subcutaneous injection. In more specific embodiments, the amount of uridine is sufficient to maintain plasma uridine at a concentration of 50 - 150 μM in a human subject to whom the pharmaceutical composition is administered. In another embodiment, the amount of uridine is sufficient to maintain plasma uridine at a concentration of 200 - 500 μM for about 1 hour in a human subject to whom the pharmaceutical composition is administered. Embodiments of the pharmaceutical composition for subcutaneous injection further comprise 100 - 300 mg of the D-enantiomer of β-hydroxybutyrate and a cationic counterion. In more specific embodiments, the subject is a human adult, and the cationic counterion is Mg in an amount that delivers 10 - 40 milliequivalents of Mg 2+ over a 12-hour delivery period 2+ Alternatively, the subject is a human infant, e.g., a neonate, and the cationic counterion is Mg in an amount that delivers 10 - 20 milliequivalents of Mg 2+ over a 12-hour delivery period 2+ and contains it.

[0039] In embodiments of the present invention, the pharmaceutical composition is formulated for subcutaneous injection. In more specific embodiments, the amount of uridine is sufficient to maintain plasma uridine at a concentration of 200 - 500 μM for about 1 hour in a human subject to whom the pharmaceutical composition is administered.

[0040] In embodiments of the pharmaceutical composition formulated for subcutaneous injection, the subject is a human adult, and the amount of uridine is 10 - 40 mg / kg. More specific embodiments comprise 50 - 100 mg / kg of the D-enantiomer of β-hydroxybutyrate and a cationic counterion. In more specific embodiments, the cationic counterion is Mg in an amount of 10 - 40 milliequivalents 2+ and contains it.

[0041] In another embodiment of a pharmaceutical composition formulated for subcutaneous injection, the subject is a human child, and the amount of uridine is 30 to 120 mg. A more specific embodiment further comprises 100 to 300 mg of a D-enantiomer of β-hydroxybutyrate and a cationic counterion. In a more specific embodiment, the cationic counterion contains 5 to 10 milliequivalents of Mg2+.

[0042] In embodiments of the present invention, the pharmaceutical composition is for the treatment of patients with acute brain injury. In more specific embodiments, the acute brain injury is selected from the group consisting of traumatic brain injury, birth asphyxia, hypoxic-ischemic encephalopathy, stroke, brain injury due to cardiac arrest, and brain injury due to drowning.

[0043] Embodiments of the method for treating acute brain injury of the present invention further include the step of administering a neuroprotective amount of β-hydroxybutyrate and a cationic counterion. In preferred embodiments, the β-hydroxybutyrate and counterion are in the same solution as uridine. In embodiments of the present invention, the β-hydroxybutyrate is a D-enantiomer of β-hydroxybutyrate or a mixture of D and L-enantiomers, for example, racemic-β-hydroxybutyrate. According to the present invention, any conventional cationic counterion can be used. In one embodiment, the cationic counterion is Na + In another embodiment, Na + and Mg 2+ Both exist as counterions.

[0044] In embodiments of the method of the present invention, the solution further comprises an organic anion selected from the group consisting of amino acids, glucons, pyroglutamates, citrates, acetates, and lactates. Examples of suitable amino acids include threonine or aspartate. In embodiments of the present invention, the solution further comprises a thermoregulator. In embodiments of the present invention, the physiological saline solution is isotonic or hypertonic physiological saline. In embodiments of the present invention, the pharmaceutical composition is administered subcutaneously.

[0045] In embodiments of the present invention, administration is by subcutaneous injection. In a more specific embodiment, the amount of uridine is sufficient to maintain plasma uridine at a concentration of 50-150 μM. In another embodiment, the amount of uridine is sufficient to maintain plasma uridine at a concentration of 200-500 μM for about 1 hour. Embodiments of this method further include the step of administering 100-300 mg of a D-enantiomer of β-hydroxybutyrate and a cationic counterion. In a more specific embodiment, the subject is a human adult, and the cationic counterion is 10-40 milliequivalents of Mg over a 12-hour delivery period. 2+ The amount of Mg that delivers 2+ This includes, or the subject is human children, for example, neonatal infants, and the cationic counterion is 10-20 milliequivalents of Mg over a 12-hour delivery period. 2+ The amount of Mg that delivers 2+ Includes.

[0046] In embodiments of the present invention, administration is by subcutaneous injection. In more specific embodiments, the amount of uridine is sufficient to maintain plasma uridine at a concentration of 200-500 μM in human subjects for about 1 hour.

[0047] In embodiments where administration is by subcutaneous injection, the subject is a human adult, and the amount of uridine is 10-40 mg / kg. More specific embodiments further include the step of administering 50-100 mg / kg of the D-enantiomer and cationic counterion of β-hydroxybutyrate. In more specific embodiments, the cationic counterion is Mg in an amount of 10-40 milliequivalents. 2+ Includes.

[0048] In another embodiment where administration is by subcutaneous injection, the subject is a human child, and the amount of uridine is 30-120 mg. A more specific embodiment further includes the step of administering 100-300 mg of the D-enantiomer and cationic counterion of β-hydroxybutyrate. In a more specific embodiment, the cationic counterion contains 5-10 milliequivalents of Mg2+.

[0049] According to the present invention, any acute brain injury condition to be treated can be any acute brain injury. In a more specific embodiment, the acute brain injury condition is selected from the group consisting of traumatic brain injury, birth asphyxia, hypoxic-ischemic encephalopathy, stroke, brain injury due to cardiac arrest, and brain injury due to drowning.

[0050] The present invention will be better understood by referring to the following examples, which illustrate but do not limit the present invention as described herein. [Examples]

[0051] Protection against mortality caused by 3-nitropropionic acid, an inhibitor of mitochondrial respiration. 3-nitropropionic acid (3-NP) is an inhibitor of mitochondrial respiratory chain complex II. Administration of 3-NP to experimental animals has been used as a model for the pathological consequences of mitochondrial diseases, Huntington's disease, and hypoxia. Daily administration of 3-NP induces energy deficiency in the brain, heart, and other tissues, leading to death within a few days. A 3-NP administration regimen that resulted in 100% mortality in mice approximately one week later was selected for further evaluation of mortality, demonstrating the neuroprotective and cardioprotective effects of the present invention's composition in a model of impairment caused by mitochondrial dysfunction. The protective effect of the test drug was quantified using survival time (median survival time; when 50% of the animals in the group died) and survival percentage at the end of the 13-day study.

[0052] Female CD-1 mice, approximately 16 weeks old, were divided into groups of 10 mice each, matched for body weight. All mice received daily intraperitoneal injections of 3-NP (60 mg / kg) at a volume of 0.01 ml per gram of body weight. 3-NP was administered at 2 PM each day for 13 consecutive days of the experiment. Body weight was recorded each day between repeated 3-NP treatments, and the drug dose was adjusted according to the mice's weight loss.

[0053] The efficacy of the uridine and MgBHB combination was evaluated, as well as the efficacy of uridine alone (VWR International) and the magnesium salt of racemic-β-hydroxybutyrate (magnesium β-hydroxybutyrate trihydrate; MgBHB·3H2O) (Nutra Planet). All treatments were administered by subcutaneous injection twice daily at 7 AM and 1 PM at a volume of 0.02 ml per gram of body weight.

[0054] group: 1. Control medium - 0.9% physiological saline bid 2. Uridine 300mg / kg bid 3. MgBHB 300mg / kg bid 4. Uridine 300mg / kg + MgBHB 300mg / kg bid

[0055] result: In the medial control group, mice began dying on day 4 after the start of 3-NP administration, and all mice in all groups died by day 7. The final survival percentage and median survival time at the end of the 13-day study are shown in the table and Figure 1 below.

[0056] [Table 1]

[0057] These results indicate that subcutaneous administration of uridine alone provided substantial protection against the lethal toxicity of repeated doses of 3-NP. MgBHB provided some protection, but less than that of uridine. The combination of uridine and MgBHB was superior to either uridine or MgBHB alone. [Examples]

[0058] Protection against mortality caused by 3-nitropropionic acid by uridine and sodium β-hydroxybutyrate alone and in combination. Both uridine and β-hydroxybutyrate (BHB) independently exhibit protective effects against the toxicity and mortality caused by repeated daily dosing of 3-nitropropionic acid, an inhibitor of mitochondrial respiratory chain complex II. BHB is an endogenous ketone body that the brain can use as metabolic fuel. The D-isomer of BHB is the form produced as a product of lipid and amino acid metabolism. When BHB is produced industrially, it is often manufactured as a DL-enantiomer mixture, which is simpler to synthesize than the pure D-isomer. Since L-BHB can be enzymatically converted to D-BHB in vivo, both D- and DL-BHB can provide isocaloric usable energy. However, the relative activity of D- versus DL-BHB for protection against energy deficiency in impairments associated with impaired mitochondrial function, particularly when either form of BHB is combined with a uridine source, has not been established.

[0059] Mitochondrial dysfunction was induced by daily administration of 3-nitropropionic acid (60 mg / kg / day) via intraperitoneal injection. The mouse population was also administered saline (control), uridine, DL-BHB, D-BHB, and combinations of uridine, D-BHB, and DL-BHB, as shown below. Both D- and DL-BHB were administered as water-soluble sodium salts: Na(D-BHB) (Toronto Research Chemicals, catalog #H833025) and Na(DL-BHB) (VWR International, catalog #200012-200).

[0060] group: 1. Control medium - 0.9% physiological saline bid 2. Uridine 200 mg / kg tid 3. Na(DL-BHB) 328 mg / kg tid 4. Na(D-BHB) 328 mg / kg tid 5. Uridine 200 mg / kg + Na (DL-BHB) 328 mg / kg tid 6. Uridine 200 mg / kg + Na(D-BHB) 328 mg / kg tid

[0061] The protective effects of the test drugs were quantified and compared using survival time (median survival time; when 50% of the animals in the group died) and the survival percentage at the end of the 12-day study.

[0062] Female CD-1 mice, approximately 16 weeks old, were divided into groups of 10 mice each, matched for body weight. All mice received an intraperitoneal injection of 3-NP (60 mg / kg) at a volume of 0.01 ml per gram of body weight daily at 5 PM.

[0063] The treatment was administered by subcutaneous injection three times a day at 7 AM, 12 PM, and 4 PM at a volume of 0.01 ml per gram of body weight. 3-NP was administered at 5 PM on each consecutive day of the experiment. Body weight was recorded each day between repeated treatments with 3-NP, and the drug dose was adjusted according to the weight loss of the mice. Body weight was graphed for each group of mice until the group size decreased to less than 3.

[0064] result: The final survival percentage and median survival time at the end of the study are shown in the table and Figure 2 below.

[0065] [Table 2]

[0066] These results indicate that subcutaneous administration of uridine alone provided substantial protection against the lethal toxicity of repeated doses of 3-NP. Both Na(DL-BHB) and Na(D-BHB) individually provided some protection against mortality, not significantly different from each other, with median survival times of 7.5 days (20% final survival) and 8 days (10% final survival), respectively. Uridine alone enabled 50% survival with a median survival time of 12 days. Na(DL-BHB) did not enhance the protective effect of uridine at the end of the experiment, and the final survival rate with the uridine and Na(DL-BHB) combination was 50%, with a median survival time of 10 days. In contrast, Na(D-BHB) administered with uridine resulted in 90% survival at the end of the study. Furthermore, the combination of uridine and Na(D-BHB) provided better protection against weight loss than either the single agent or the uridine and Na(DL-BHB) combination (Figure 3). [Examples]

[0067] Protection of uridine and sodium β-hydroxybutyrate alone and in combination against mortality caused by 3-nitropropionic acid Both uridine and β-hydroxybutyrate exhibit protective effects against toxicity and mortality caused by 3-nitropropionic acid (3-NP), an inhibitor of mitochondrial respiratory chain complex II used in brain and other tissue damage models involving mitochondrial energy production deficiency.

[0068] Subcutaneous administration of therapeutic agents is an appropriate route of administration for emergency drugs intended for use in individuals with traumatic brain injury, stroke, or asphyxia, when patients may be unable to take oral medication or when it may be difficult to place an intravenous catheter. While subcutaneous drug administration is practical, the composition must adhere to known volume and osmolality values ​​that are compatible with this route of administration, as excessively high osmolality can cause local edema, and the volume must be low enough for injection without problematic back pressure. Compositions containing uridine, sodium β-hydroxybutyrate, and combinations thereof, with an osmolality limited to a maximum of 800 milliosmoles / liter (a known value acceptable for subcutaneous liquid administration), were tested in a model of systemic energy failure induced by daily intraperitoneal injection of 3-nitropropionic acid at two dose levels: 65 mg / kg / day and 70 mg / kg / day.

[0069] The protective effects of the test drugs were quantified and compared using survival time (median survival time; when 50% of the animals in the group died) and the survival percentage at the end of the 13-day study.

[0070] Female CD-1 mice, approximately 16 weeks old, were divided into groups of 10 mice each, matched for body weight. All mice received an intraperitoneal injection of 3-NP (65 mg / kg or 70 mg / kg) at a volume of 0.01 ml per gram of body weight daily at 5 PM.

[0071] The treatment was administered by subcutaneous injection three times a day at 7 AM, 12 PM, and 4 PM at a volume of 0.02 ml per gram of body weight. 3-NP was administered at 5 PM on each consecutive day of the experiment. Body weight was recorded each day between repeated treatments with 3-NP, and the drug dose was adjusted according to the weight loss of the mice. D-BHB was administered as the water-soluble sodium salt Na(D-BHB) (Toronto Research Chemicals, catalog #H833025).

[0072] group: 1. Control medium - 0.9% physiological saline tid 2. Uridine 200 mg / kg tid 3. Na(D-BHB) 985 mg / kg tid 4. Uridine 200 mg / kg + Na(D-BHB) 985 mg / kg tid

[0073] result: Mice in the medial control group began dying on day 4 after the start of 3-NP administration. The final survival percentage and median survival time at the end of the study are shown in the table below and in Figures 4 and 5.

[0074] [Table 3]

[0075] [Table 4]

[0076] These results indicate that subcutaneous administration of uridine alone provided substantial protection against the lethal toxicity of repeated doses of 3-NP. NaBHB at the doses used in this study provided similar protection to that of uridine. The combination of uridine and NaBHB was superior to either uridine or NaBHB alone, and the benefit was significantly greater (greater than that of the additive) with higher doses of 3-nitropropionic acid (70 mg / kg / day). [Examples]

[0077] Pharmacokinetics of plasma uridine and Na(D-BHB) after subcutaneous administration Subcutaneous injection of a combination of uridine and Na(D-BHB) (sodium salt of the D-isomer β-hydroxybutyrate) provided protection against morbidity and mortality resulting from impaired mitochondrial energy production in a mouse model. Transitioning this protective effect to larger animals, including humans, depends on the ability to achieve plasma protective concentrations of uridine and BHB through practical doses and volumes for subcutaneous injection.

[0078] If the solute concentration is too high, there is a maximum osmotic pressure limit for the solution for subcutaneous administration due to the possibility of local osmotic edema. Approximately 800 milliosmoles / liter is considered the upper limit for the concentration of therapeutic subcutaneous fluids. In a study conducted in mice receiving a daily dose of 3-nitropropionic acid, a mitochondrial complex II inhibitor, a subcutaneous composition containing uridine and D-BHB sodium salts at a total solute concentration of less than 800 milliosmoles was found to provide significant protection against mortality caused by mitochondrial dysfunction. Three daily doses were found to provide excellent protection. Pharmacokinetic studies were conducted to determine the plasma concentrations of uridine and D-BHB achieved after administration of therapeutic doses of the composition, and to determine the exposure concentrations and durations that need to be achieved for replacement of protective effects in other species, including humans.

[0079] A group of eight mice (female CD-1 mice, approximately 16 weeks old) were treated subcutaneously with uridine or uridine plus sodium (D-BHB) as shown in the table below. D-BHB was administered as water-soluble sodium salt Na(D-BHB) (Toronto Research Chemicals, catalog #H833025).

[0080] [Table 5]

[0081] 1. Group 1 (200 mg / kg uridine + Na(D-BHB)) received 10 mg / ml uridine and 49.26 mg / ml Na(D-BHB) (0.02 ml per gram of body weight). 2. Group 2 (300 mg / kg uridine + Na(D-BHB)) received 15 mg / ml uridine and 49.26 mg / ml Na(D-BHB) (0.02 ml / g per gram of body weight). 3. Group 3 (400 mg / kg uridine + Na(D-BHB)) received 20 mg / ml uridine and 49.26 mg / ml Na(D-BHB) (0.02 ml / g per gram of body weight). 4. Group 4 (400 mg / kg uridine) received 20 mg / ml uridine (0.02 ml per gram of body weight).

[0082] Blood samples were obtained from the posterior orbital reticular tissue at 15, 30, 60, and 120 minutes after drug injection. Two blood samples (less than 200 microliters) were collected from each mouse in heparinized polyethylene tubes. Four mice in the group were induced to bleed at 15 and 60 minutes, and the other four mice were induced to bleed at 30 and 120 minutes. Plasma samples were obtained by centrifugation, deproteinized, and analyzed for uridine and uracil by LC / MS. Plasma D-BHB was measured in the same samples using a commercially available enzyme color change assay.

[0083] Rodents degrade uridine to uracil more rapidly and broadly than humans, who have lower activity of the enzyme uridine phosphorylase. Therefore, it is important to consider plasma uracil concentrations in rodents when determining the optimal substitution of rodent pharmacokinetic results for human contexts. The sum of uridine and uracil in rodents better reflects plasma uridine equivalents for substitution in other animal models or human dosing than uridine alone.

[0084] Uridine (and uridine + uracil) rapidly appeared in plasma after subcutaneous administration with a Tmax of 15–30 minutes post-administration (Figures 6, 7, and 8). Plasma D-BHB also rapidly increased, with a Tmax of 15 minutes (Figure 9). Plasma uridine (and uridine + uracil) showed dose-dependent Cmax values ​​(Figures 6, 7, and 8).

[0085] The uridine + Na(D-BHB) dosing regimen in this pharmacokinetic study has been shown to produce a potent protective effect against mortality induced by daily injections of 3-nitropropionic acid. Pharmacokinetic data indicate that periodic transient increases in plasma uridine and BHB are sufficient to induce robust protection against lethal systemic mitochondrial energy deficiency. The maximum plasma uridine + uracil concentration at the highest dose of 400 mg / kg uridine was less than 500 μM, which is below the threshold for hyperthermia and shivering induced in humans, where clinical problems have been reported with intravenous administration of uridine. The maximum D-BHB concentration observed in this study, approximately 3 mmol, is known to be well-tolerated in humans, equivalent to the concentration observed during post-exercise ketosis, and well below the threshold (above 15 mM) for ketoacidosis.

Claims

1. A pharmaceutical composition for use in providing neuroprotection in human subjects, comprising a neuroprotective amount of uridine dissolved in physiological saline solution, a neuroprotective amount of β-hydroxybutyrate, and a cationic counterion, The β-hydroxybutyrate is a D-enantiomer of β-hydroxybutyrate, and The pharmaceutical composition is formulated for administration to a human subject by subcutaneous injection, wherein the amount of uridine is 10 to 40 mg / kg per kg of body weight of the human subject, and the amount of the D-enantiomer of β-hydroxybutyrate is 50 to 100 mg / kg per kg of body weight of the human subject. The aforementioned pharmaceutical composition.

2. Cationic counterions are Na ＋ The pharmaceutical composition according to claim 1.

3. The cationic counterion is Na ＋ and Mg ２＋ A pharmaceutical composition according to claim 1, selected from the group consisting of one or more of the following.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the physiological saline solution further comprises an organic anion selected from the group consisting of amino acids, gluconates, pyroglutamates, citrates, acetates, and lactates.

5. The pharmaceutical composition according to claim 4, wherein the amino acid is threonine or aspartate.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the physiological saline solution further comprises a thermoregulator.

7. The pharmaceutical composition according to any one of claims 1 to 3, wherein the physiological saline solution is isotonic or hypertonic physiological saline.

8. A pharmaceutical composition according to claim 1, The amount of uridine is sufficient to maintain plasma uridine at a concentration of 200-500 μM for approximately one hour in the aforementioned human subjects. The aforementioned pharmaceutical composition.

9. The pharmaceutical composition according to claim 8, wherein the human subject is a human adult.

10. The cationic counterion is Mg in an amount of 10 to 40 milliequivalents. ２＋ The pharmaceutical composition according to claim 9, comprising:

11. The pharmaceutical composition according to claim 8, wherein the human subject is a human child.

12. The cationic counterion is Mg in an amount of 5 to 10 milliequivalents. ２＋ A pharmaceutical composition according to claim 11, comprising:

13. A pharmaceutical composition according to any one of claims 1 to 12 for use in the treatment of a patient having acute brain injury.

14. The pharmaceutical composition for use according to claim 13, wherein the acute brain injury condition is selected from the group consisting of traumatic brain injury, birth asphyxia, hypoxic-ischemic encephalopathy, stroke, brain injury due to cardiac arrest, and brain injury due to drowning.

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