Neuroprotective mixture for mitigating the harmful effects of ischemic stroke

Abstract

A neuroprotective mixture includes Phosphocreatine (PCr), Magnesium Gluconate (MgG), and ß-hydroxybutyrate (BHB). The PCR, MgG, and BHB are combined in a 2:1:2 molar ratio of PCr:MgH:BHB. The neuroprotective mixture is a useful preventative and prophylactic supplement, which also can function to extend the post-stroke treatment window.

Claims

1. A composition for prophylactically mitigating the harmful effects of stroke, the composition comprising a mixture of creatine, magnesium salt, and one or more ketone bodies selected from the groups consisting of (1) creatine monohydrate, creatine ethyl ester, creatine ethyl ester malate, creatine gluconate, creatine hydrochlorate, tri creatine malate, tri creatine orotate, and phosphocreatine, (2) magnesium gluconate, magnesium chloride, magnesium sulfate, and magnesium citrate, and (3) β-Hydroxybutyrate (BHB) and acetoacetate; and further wherein the creatine, magnesium salt, and one or more ketone bodies are present in a 2:1:2 ratio.

2. The composition of claim 1, wherein the creatine is phosphocreatine.

3. The composition of claim 1, wherein the magnesium salt is magnesium gluconate.

4. The composition of claim 1, wherein the one or more ketone bodies is BHB.

5. The composition of claim 1, wherein the one or more ketone bodies and magnesium salt are combined.

6. The composition of claim 5, wherein the one or more ketone bodies and magnesium salt are combined as magnesium beta-hydroxbybutyrate.

7. The composition of claim 5, wherein the ratio of creatine:magnesium salt:ketone body remains 2:1:2.

8. A method of mitigating the harmful effects of stroke in a person, comprising administering to that person a composition comprising a mixture of a creatine, a magnesium salt, and one or more ketone bodies, selected from the groups consisting of (1) creatine monohydrate, creatine ethyl ester, creatine ethyl ester malate, creatine gluconate, creatine hydrochlorate, tri creatine malate, tri creatine orotate, and phosphocreatine, (2) magnesium gluconate, magnesium chloride, magnesium sulfate, and magnesium citrate, and (3) β-Hydroxybutyrate (BHB) and acetoacetate; and further wherein the composition is administered orally on a daily basis prior to the occurrence of a stroke.

9. The method of claim 8, wherein the creatine, magnesium salt, and one or more ketone bodies are present in a 2:1:2 ratio.

10. The method of claim 8, wherein the creatine is phosphocreatine.

11. The method of claim 8, wherein the magnesium salt is magnesium gluconate.

12. The method of claim 8, wherein the one or more ketone bodies is BHB or acetoacetate.

13. The method of claim 8, wherein the one or more ketone bodies is BHB.

14. The composition of claim 5, wherein the one or more ketone bodies and magnesium salt are combined as magnesium beta-hydroxbybutyrate, and the ratio of creatine:magnesium salt:ketone body remains 2:1:2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawing will be provided by the USPTO upon request and payment of the necessary fee.

(2) The foregoing and other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which:

(3) FIG. 1 shows reductions in cortical infarct area using TCP in a rat model of ischemic stroke;

(4) FIG. 2 shows improvement in functional outcomes with TCP in a rat model of ischemic stroke;

(5) FIG. 3 shows improved blood brain barrier integrity with TCP administration in a rat model of ischemic stroke;

(6) FIG. 4 shows extravasation of Evans Blue in 5 separate treatment groups using an ischemic stroke model in rats;

(7) FIG. 5 shows reduced expression of HSP25 using TCP in a hemorrhagic stroke model in rats;

(8) FIG. 6 shows reduced expression of HSP25 using TCP in a hemorrhagic stroke model;

(9) FIG. 7 shows reductions in HSP25 expression using TCP pre-treatment in an ischemic stroke model in gerbils;

(10) FIG. 8 shows reductions in HSP25 expression with TCP pre-treatment in an ischemic stroke model in gerbils; and

(11) FIG. 9 shows reductions in HSP25 expression when pre-treatment with TCP is provided in an ischemic stroke model in gerbils.

DETAILED DESCRIPTION

(12) The present invention is a neuroprotective mixture that protects against the devastating effects of strokes and aberrant cerebral perfusion states. In one of many possible embodiments, the neuroprotective mixture includes: 1) creatine, 2) a magnesium salt, and (3) one or more ketone bodies. In one of many possible embodiments of the invention, the creatine can be phosphocreatine (PCr), the magnesium salt can be magnesium gluconate (MgG), and the one or more ketone bodies can be ß-hydroxybutyrate (BHB). Optionally, but preferably, the creatine, magnesium salt, and one or more ketone bodies are present in the mixture in a 2:1:2 molar ratio of creatine:magnesium salt:ketone body. In one of many alternative embodiments, the magnesium salt and one or more ketone bodies can be combined. A non-limiting example is magnesium beta-hydroxbybutyrate, which, when combined with creatine, maintains the desired 2:1:2 molar ratio of creatine:magnesium salt:ketone body.

(13) This neuroprotective mixture represents a novel approach to neuroprotection in several respects. First, the neuroprotective mixture is a combination of three separate classes of chemical 5 compound that favorably affect major adverse events involved in neuronal death. Together these three classes of compounds create a tri-functional neuroprotective system that protects the brain, the blood vessels, and the blood brain barrier following stroke. These classes of chemical compounds have several mechanisms of action, which, when combined in the proper ratio, yield surprisingly good results. This represents a significant departure from earlier neuroprotective trials that were one-dimensional in nature.

(14) The neuroprotective mixture further provides a novel method of administration. The neuroprotective mixture can be administered as a daily oral supplement. By providing the neuroprotective mixture on a continuous daily basis, individuals receive neuronal protection in a preventative/prophylactic manner. Currently, there are no efficacious prophylactic neuroprotective agents for acute ischemic/hemorrhagic stroke victims or patients that sustain aberrant cerebral perfusion states. The different classes of compounds comprising the mixture are discussed below.

(15) Creatine. Creatine is an organic compound with the nominal formula CNCH.sub.2CO.sub.2H. This species exists in various modifications in solution. Creatine is found in vertebrates where it facilitates recycling of adenosine triphosphate (ATP), the energy currency of the cell, primarily in muscle and brain tissue. Recycling is achieved by converting adenosine diphosphate (ADP) back to ATP via donation of phosphate groups. Creatine acts as an energy buffer.

(16) Phosphocreatine is the phosphorylated version of creatine and as a high energy phosphate phosphocreatine can be used to buffer the ATP concentration. Phosphocreatine is ionic and can be purchased and administered as a sodium salt, potassium salt, acid and similar salt formulations. Creatine is readily converted into phosphocreatine in numerous cells included brain cells. Creatine comes in numerous formulations that have been established to be biologically active.

(17) Phosphocreatine (PCr) contains one phosphate and one creatine molecule and typically is marketed as a sodium salt. It stabilizes membranes and protects against apoptosis by influencing the mitochondrial creatine kinase. It also plays a critical role in energy management and protection against free oxygen radicals. Clearance from the blood stream has a fast and slow half-life, and it can be broken down to creatine and eventually to phosphate. Creatine and phosphate both enter cells and can be reconverted to phosphocreatine in the cell and therefore can be beneficial to the cells following stroke. The neuroprotective mixture optionally but preferably contains about 4 mM of phosphocreatine. The slow clearance of phosphocreatine comes from the spontaneous formation of creatinine. Creatinine is distinct from creatine and is excreted in the urine at a clearance rate of about 3% per day.

(18) In one of many alternative embodiments of the invention, forms of creatine other than phosphocreatine can be used. Non-limiting examples of acceptable forms of creatine can include creatine monohydrate, creatine ethyl ester, creatine ethyl ester malate, creatine gluconate, creatine hydrochlorate, tri creatine malate, and tri creatine orotate.

(19) Magnesium Salt. Magnesium is a divalent metal cation. It is critical to numerous biological activities in the ionized and non-ionized (bound) forms. It is available in oral and intravenous (IV) formulations. It is utilized in medicine as the chloride, sulfate and citrate salts. Other forms include gluconate and other anions. There are multiple formulations of magnesium that have biological activity and bioavailability.

(20) Recent evidence suggests that low levels of free magnesium (Mg++) in the blood may be correlated with poor neurological outcome in patients with strokes, head injury, and brain tumors. Mg++ has also been used as an anti-convulsant for pregnant women with pre-eclampsia and/or eclampsia, and may play a role in the treatment or prevention of cerebral vasospasm after subarachnoid hemorrhage.

(21) Magnesium gluconate (MgG) is a potent calcium ion (Ca.sup.++) channel blocker and Ca.sup.++ antagonist. It can decrease Ca.sup.++ overload in the cell and thus help decrease mitochondrial mediated apoptosis. MgG is safe and well-tolerated in humans and is a first line cerebral vascular protectant for pregnant women at risk for eclampsia. MgG can be separated into the magnesium ion and the gluconate ion.

(22) In one of many alternative embodiments of the invention, the magnesium salt in the mixture can be magnesium chloride. Intraperitoneal injections of MgCl.sub.2, at a dose of 1 mmol/kg given immediately after middle cerebral artery (MCA) occlusion in a rat model of ischemic infarct decreased the size of the measured infarct. MgCl.sub.2 given via an intraperitoneal injection has been shown to reduce the average size of infarcts in a rat model of ischemia from 165 mm.sup.3 to a 122 mm.sup.3.

(23) In yet another alternative embodiment, the magnesium salt can be magnesium sulfate. Magnesium sulfate has been successfully used in a number of animal models for neuron protection. It has been demonstrated in a fluid percussion model of head trauma that magnesium chloride limits the extent of neuronal injury in this model of diffuse neuronal injury. It also has been demonstrated that magnesium chloride has a similar effect in models of spinal cord ischemia. In a study utilizing magnesium therapy, imaging and spectroscopy, both magnesium sulfate and magnesium chloride were able to enter the cerebrospinal fluid (CSF) as well as alter the amount of neuronal injury and a diffuse anoxia injury model in rats.

(24) Ketone Bodies. The human brain is dependent upon the presence of glucose for its metabolic needs, as the brain has essentially no glycogen reserves. The only exception to the brain's dependence upon glucose is that it can derive a major portion of its metabolic needs from the catabolism of ketone bodies, such as β-Hydroxybutyrate (BHB) and acetoacetate, during periods when glucose is not available.

(25) BHB is a reduced form of acetoacetate and the combination of BHB and acetoacetate are often referred to as ketone bodies. The ketone bodies are often and quickly interconverted in the body. BHB is an anion and is often found in salt forms. The salt forms of BHB include, but are not limited to, sodium salt, potassium salt, free acid, etc. These salts and the interconversion of the ketone bodies are extremely well established with documented biological activity and pharmacokinetics.

(26) BHB was first utilized as a cerebral protectant in an indirect manner. Since the early 1920's the use of a ketogenic diet has been known to influence the severity and frequency of seizures. The preliminary data supporting the beneficial effects of BHB as a cerebral protectant or neuroplegic solution following surgery: 1) the clinical efficacy of the ketogenic diet as an antiepileptic therapy, 2) the neuroprotective effects seen in vitro, and 3) the neuroprotective effects seen in the ketogenic state. These benefits are not likely to be mutually exclusive.

(27) There are several hypotheses for the antiepileptic effects of ketone bodies. One is that the ketone bodies may have an energetic benefit (as discussed previously); the second is that the ketone bodies, specifically BHB, may decrease neural excitability; and the third is that the extracellular fluid composition is changed with increased ketone bodies and that this impacts on epileptic discharges. As has been suggested previously, ketone bodies may benefit the brain, neural and vascular tissue by providing an energetic benefit.

(28) BHB is a naturally occurring ketone body that normally circulates in the blood stream at levels of 0.1 to 10 mM. BHB is readily utilized by the brain as an energy source when glucose is limited. Inside the cell, BHB normalizes mitochondrial function and decreases mitochondrial derived reactive oxygen species. Its putative antiepileptogenic activity is also important, as seizures following surgery are a relatively common complication. BHB may prevent seizures by antagonizing some of the excitotoxic effects of glutamate.

(29) An alternative embodiment of the invention utilizes acetoacetate in combination with, or instead of, BHB. Acetoacetate is synthesized in the liver from acetyl-CoA. Acetoacetate is a true ketone and is reduced to BHB in a typical redox reaction using NADH as the reducing equivalent. This investment in reducing power has two important beneficial effects for the BHB in its role as a neuroprotective. The first is that BHB is more stable than acetoacetate and is less likely to cause acidosis. Ketoacidosis occurs in diabetic patients from the production of acetone via a spontaneous, irreversible and non-enzymatic decarboxylation of acetoacetate. The production of acetone and the acidosis associated with the liberated CO.sub.2 makes acetoacetate a poor choice for a ketone based neural protectant. BHB, however, is more thermodynamically stable and is able to produce NADH in the brain when it is oxidized to acetoacetate. Therefore, BHB is able to quickly provide energy for the brain, without the risk of ketoacidosis.

EXAMPLES

(30) Ischemic Stroke Model Studies

(31) As shown in FIG. 1, the mixture of the present invention (TCP) has been shown to be protective against ischemic damage in the cortical region. Control animals demonstrated cerebral cortical infarctions comprising a mean infarct area of 62.5±8.5%, while animals receiving the TCP neuroprotectant at ninety (90) minutes post-occlusion showed a reduction in infarct area to 30±3.5%.

(32) As shown in FIG. 2, functional outcomes based on a modified neurologic severity scale were statistically improved in animals treated with TCP.

(33) Evidence also demonstrates that TCP can reduce disruption to the blood brain barrier following an ischemic insult. Blood brain barrier extravasation studies utilizing Evan's Blue dye demonstrated a significant decrease in extravasation within groups receiving the invention alone as well as a synergistic effect in animals receiving both the invention and tPA (FIG. 3).

(34) Additional studies using a validated rat model for ischemic stroke have also been conducted. These studies made comparisons in stroke volumes between five different treatment groups:

(35) a. Ischemic stroke model in aged rats; no reperfusion; no neuroprotection

(36) b. Ischemic stroke model in aged rats; no reperfusion; neuroprotection administered

(37) c. Ischemic stroke model in aged rats; reperfusion (tPA) given; no neuroprotection

(38) d. Ischemic model in young rats; no reperfusion; no neuroprotection

(39) e. Ischemic stroke model in young rats; reperfusion (tPA) given; no neuroprotection

(40) Table 1 provides data on the infarct volumes in these five separate treatment groups. As reflected in the table, neuroprotection with TCP alone not only demonstrated reductions in infarct volume, but it also shows a favorable synergistic effect when combined with the thrombolytic agent, tPA.

(41) TABLE-US-00001 TABLE 1 Infarct volumes by treatment groups in a rat model of ischemic stroke. Aged Aged + TCP Aged + IPA Infarct Contra Infarct Contra Infarct Contra 10.86 1.18 11.27 1.15 7.85 0.81 16.99 1.62 8.2 1.23 8.95 1.39 11.3 1.52 8.43 1.43 10.7 1.07 11.27 0.66 9.31 0.59 9.58 1.29 14.17 2.08 10.52 1.36 7.26 0.87 Average 12.918 1.412 9.546 1.152 8.868 1.086 Std Dev 2.63 0.53 1.33 0.33 1.37 0.25 Std Err 1.175173504 0.23627841 0.591990209 0.14848029 0.610966492 0.113185271 Ipsilateral 12.9 9.5 8.9 Contralateral  1.4 1.2 1.1 Aged + TCP + IPA Young Young + IPA Infarct Contra Infarct Contra Infarct Contra 4.38 0.94 7.06 0.84 3.75 1.07 5.39 0.88 8.69 1.03 4.35 1.28 5.77 1.18 8.29 1.02 3.64 1.66 3.47 1.5 7.85 0.89 4.1 0.52 6.01 0.74 8.17 1.36 5.2 1.36 Average 5.004 1.048 8.012 1.028 4.208 1.178 Std Dev 1.06 0.30 0.61 0.20 0.62 0.42 Std Err 0.472999446 0.133272201 0.272877068 0.090582106 0.277745312 0.189466726 Ipsilateral 5  8   4.2 Contralateral  1.1 1   1.2

(42) These same animal cohorts were evaluated for blood brain barrier integrity by staining with Evans Blue dye. FIG. 4 shows the favorable impact of the invention alone on blood brain barrier integrity as well as its combined administration with tPA. This data provides evidence of a favorable synergistic effect when the invention is combined with thrombolytic therapy.

(43) Hemorrhagic Stroke Model Studies

(44) Further studies in a hemorrhagic stroke model were performed using the neuroprotective mixture containing phosphocreatine, beta-hydroxybutyrate, and magnesium gluconate. A buffered solution of 50:25:50 mM PCr:MgG:BHB was prepared. This solution, which is buffered with NaOH and iso-osmotic with NaCl, is sufficient for increasing MgG to a target dose, which preferably is not to exceed 2 mM. The mixture was dissolved and buffered to a neutral pH, freeze dried for storage, and resuspended for IV use. The solution was administered intraperitoneally (IP) in rats.

(45) As shown in FIG. 5, the IP administration of the neuroprotective mixture was neuroprotective following a model of hemorrhagic stroke. The neuroprotection was assessed by a decrease in stress gene expression. Specifically, heat shock protein 25 (HSP25) was measured since it is a well-documented marker of neuronal stress. BOXes stands for bilirubin oxidation products. There are two BOXes; BOX-A and BOX-B. These two BOXes are isomers derived from the two ends of the bilirubin molecule. BOXes are thought to be produced post brain hemorrhage, such as subarachnoid hemorrhage. When produced, they have neurologic toxic and vasoactive activity in the tissue surrounding the hemorrhage. As an established toxic metabolite produced post brain hemorrhage, BOXes are often used as a mechanism of pathology post hemorrhaging in the brain.

(46) Using a hemorrhagic stroke model, IP injections of TPC during the stroke lead to significant neuroprotection. The protection observed in these figures is largely in the glial cells via decreased heat shock protein expression (FIG. 6). This has been studied in rats with the TCP neuroprotective mixture and rats without the mixture. The neuroprotective benefits of the TCP mixture are shown consistently in the animals studied to date. Safety studies also were performed on rats, and the TCP neuroprotective mixture caused no detectible detrimental effects.

(47) The data demonstrate that the TCP neuroprotective mixture benefits vascular and neural tissues intracellularly and extracellularly against various models of ischemic and hemorrhagic stroke. These data also demonstrate that the TCP neuroprotective mixture can protect the brains in vivo from stress as assessed by stress gene expression (HSP25). Moreover, the target doses for the TCP neuroprotective mixture have no detectible detrimental effects in the rats.

(48) Prophylactic Neuroprotection in the Ischemic Model

(49) Additional studies have been conducted where the TCP neuroprotective mixture was administered prior to the neurologic insult in the animal model. A buffered solution of 50:25:50 mM PCr:MgG:BHB was prepared. This solution, which is buffered with NaOH and iso-osmotic with NaCl, is sufficient for increasing MgG to our target dose which is not to exceed 2 mM. We have demonstrated previously that this solution is buffered and does not significantly change human blood pH using a target dose in vitro of 6 mM.

(50) Ten (10) minutes of hemispheric ischemia was preceded by IP administration of the TCP neuroprotective mixture in gerbils. The TCP neuroprotective mixture was administered at the same dose as for the hemorrhagic rat experiments in a 2:1:2 ratio of phosphocreatine:magnesium gluconate:beta-hydroxybutyrate. Three animals received the TCP neuroprotective mixture and three control animals received saline injections. One of the control animals died within 10 minutes of the procedure, whereas none of the animals that received the TCP neuroprotective mixture died.

(51) During recovery, the TCP neuroprotective mixture animals appeared to begin walking, grooming and investigating their surroundings faster than the saline control animals. Twenty-four (24) hours after the ischemic insult, all of the TCP neuroprotective mixture animals were alive and behaving normally. The two surviving control animals appeared normal as well. At 24 hours post ischemia, the gerbils were anesthetized and the brains perfusion fixed. Upon histological examination we assessed stress gene expression (HSP-25, HO-1, and HSP-70) in the brains. There was greater stress gene expression in the ischemic hemisphere of the saline control animals as compared to the non-ischemic hemisphere. FIGS. 7 and 8 show the expression of HSP-25 in the ischemic hemisphere of a representative saline control and neuroprotective mixture brain. There is greater expression of HSP25 in the saline control animal as indicated with the dark staining (arrow) compared to the neuroprotective mixture animal. This was a consistent result with all of the surviving animals. Thus, the TCP neuroprotective mixture can protect the brain from ischemic events.

(52) Furthermore, as shown in FIG. 9, we have quantified the reductions in stress gene expression when pre-treatment with the TCP neuroprotective mixture is administered in the ischemic stroke model in gerbils.

CONCLUSION

(53) While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention. Thus, the breadth and scope of the invention should not be limited by any of the above-described exemplary embodiments.