CN111777671A

CN111777671A - Long-acting PSD-95 inhibitor

Info

Publication number: CN111777671A
Application number: CN202010799838.7A
Authority: CN
Inventors: 黄晓兵; 周述靓; 代景莹
Original assignee: Chengdu Aoda Biotechnology Co ltd
Current assignee: Chengdu Aoda Biotechnology Co ltd
Priority date: 2019-09-25
Filing date: 2020-08-11
Publication date: 2020-10-16
Anticipated expiration: 2040-08-11
Also published as: CN111777671B; CN110627877A; WO2022033047A1

Abstract

The invention relates to the field of medicine synthesis, and discloses a long-acting PSD-95 inhibitor. The long-acting PSD-95 inhibitor is used for preparing a pharmaceutical composition for treating diseases and an application of the pharmaceutical composition in preparing neuroprotective drugs after ischemic brain injury.

Description

Long-acting PSD-95 inhibitor

Technical Field

The invention relates to a long-acting PSD-95 inhibitor and application thereof.

Background

Acute ischemic stroke is one of the diseases with the highest fatality rate and disability rate all over the world, and an effective treatment method is lacking clinically at present. Cerebral ischemia causes a cascade of energy deprivation to cell death, with early events mainly involving excitotoxicity and oxidative stress, and late events dominated by inflammatory responses and apoptosis. At present, a large number of basic and clinical studies are devoted to the intervention of signaling molecules associated with stroke pathology in order to develop effective neuroprotective therapies. Excitotoxicity is used as the initial link of stroke pathology and is the primary target of stroke treatment.

After cerebral ischemia, excitatory glutamate is excessively released and can continuously act on N-methyl-D-aspartate receptors, so that a series of downstream harmful signal pathways, such as intracellular Ca2+ overload, free radical generation and the like, are initiated, and finally, nerve cells are killed. Recent studies have shown that PSD-95 protein in postsynaptic dense region (PSD) can bridge NMDA receptor with multiple toxic signaling molecules in cells, and that inhibition of PSD-95 can dissociate NMDA receptor from PSD-95 protein, which can reduce excitotoxicity without affecting NMDA receptor activity and synaptic function.

Nerinetide (NA-1) is a PSD-95 inhibitor that interferes with postsynaptic density protein 95(PSD-95) by terminating the production of intracellular NO free radicals, reduces infarct size of cerebral ischemia reperfusion in preclinical ischemic stroke models, and improves functional prognosis.

Because the in vivo half-life period of the Nerinetide is short, a patient needs to take a large dose of medicine every day, the patient compliance is poor, and the clinical cost is high. The invention aims to provide long-acting PSD-95 inhibitor for patients, reduce the administration frequency and reduce the cost of the patients.

Disclosure of Invention

The invention provides a long-acting PSD-95 inhibitor and application thereof.

To achieve the above object, the present invention provides, in a first aspect, a compound of structure I, a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex thereof, a prodrug based on the compound, or any mixture thereof.

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-AA1(R)-AA2

Structure I

AA1 in structure I is Lys, or Dap, or Orn, or Dab, or Dah;

AA2 in Structure I is NH₂Or is OH;

r in the structure I is succinic acid cholesterol monoester, or is 2-cholesteryl acetic acid, or is 2-cholesteryl propionic acid, or is 3-cholesteryl propionic acid, or is HO₂C(CH₂)_n1CO-(γGlu)_n2-(PEG_n3(CH2)_n4CO)_n5-。

Wherein: n1 is an integer from 10 to 20;

n2 is an integer from 1 to 5;

n3 is an integer from 1 to 30;

n4 is an integer from 1 to 5;

n5 is an integer from 1 to 5;

the invention also provides pharmaceutical compositions comprising a compound according to the invention and the use of a pharmaceutical composition comprising a compound of the invention for the preparation of a medicament for the treatment of a disease.

Preferably, the pharmaceutical composition is used for preparing neuroprotective drugs after ischemic brain injury.

Further details of the invention are set forth below, or some may be appreciated in embodiments of the invention.

Unless otherwise indicated, the amounts of the various ingredients, reaction conditions, and the like used herein are to be construed in any case to mean "about". Accordingly, unless expressly stated otherwise, all numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the standard deviation found in the respective experimental conditions.

Herein, when a chemical structural formula and a chemical name of a compound are ambiguous or ambiguous, the compound is exactly defined by the chemical structural formula. The compounds described herein may contain one or more chiral centers, and/or double bonds and the like, and stereoisomers, including isomers of double bonds (e.g., geometric isomers), optical enantiomers, or diastereomers, may also be present. Accordingly, any chemical structure within the scope of the description, whether partial or complete, including similar structures as described above, includes all possible enantiomers and diastereomers of the compound, including any stereoisomer alone (e.g., pure geometric isomers, pure enantiomers, or pure diastereomers), as well as any mixture of such stereoisomers. Mixtures of these racemates and stereoisomers may also be further resolved into the enantiomers or stereoisomers of their constituent members by those skilled in the art using non-stop separation techniques or methods of chiral molecular synthesis.

The compounds of formula I include, but are not limited to, optical isomers, racemates and/or other mixtures of these compounds. In the above case, a single enantiomer or diastereomer, such as an optical isomer, can be obtained by asymmetric synthesis or racemate resolution. Resolution of the racemates can be accomplished by various methods, such as conventional recrystallization from resolution-assisting reagents, or by chromatographic methods. In addition, the compounds of formula I also include cis and/or trans isomers with double bonds.

The compounds of the present invention include, but are not limited to, the compounds of formula I and all of their pharmaceutically acceptable different forms. The pharmaceutically acceptable different forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above and any mixtures of these forms.

The prodrug comprises an ester or amide derivative of the compound shown as the structural formula I contained in the compound.

The compound shown in the structure I provided by the invention has stable property, is a long-acting PSD-95 inhibitor, and has a remarkable protection effect on nerves after ischemic brain injury.

Detailed Description

The invention discloses a PSD-95 inhibitor and application thereof, and a person skilled in the art can appropriately improve related parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the process of the present invention has been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the compounds and processes described herein, as well as other changes and combinations of the foregoing, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

The Chinese names corresponding to the English abbreviations related in the invention are shown in the following table:

EXAMPLE 1 preparation of Compound 1

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-Lys (AEEA-AEEA-gamma Glu-18 alkanedioic acid) -NH₂

The preparation method comprises the following steps: preparing peptide resin by adopting a solid-phase polypeptide synthesis method, carrying out acidolysis on the peptide resin to obtain a crude product, and finally purifying the crude product to obtain a pure product; the step of preparing the peptide resin by the solid-phase polypeptide synthesis method is to sequentially insert corresponding protective amino acids or fragments in the following sequences on a carrier resin by the solid-phase coupling synthesis method to prepare the peptide resin:

in the preparation method, the dosage of the Fmoc-protected amino acid or the protected amino acid fragment is 1.2-6 times of the total mole number of the charged resin; preferably 2.5 to 3.5 times.

In the preparation method, the substitution value of the carrier resin is 0.2-1.0 mmol/g resin, and the preferable substitution value is 0.3-0.5 mmol/g resin.

In a preferred embodiment of the present invention, the solid-phase coupling synthesis method comprises: and (3) after the Fmoc protecting group of the protected amino acid-resin obtained in the previous step is removed, carrying out coupling reaction with the next protected amino acid. The deprotection time for removing Fmoc protection is 10-60 minutes, and preferably 15-25 minutes. The coupling reaction time is 60-300 minutes, and preferably 100-140 minutes.

The coupling reaction needs to add a condensation reagent, and the condensation reagent is selected from one of DIC (N, N-diisopropyl carbodiimide), N, N-dicyclohexylcarbodiimide, benzotriazole-1-yl-oxy tripyrrolidinophosphonium hexafluorophosphate, 2- (7-aza-1H-benzotriazole-1-yl) -1,1,3, 3-tetramethylurea hexafluorophosphate, benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate or O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroborate; n, N-diisopropylcarbodiimide is preferred. The molar consumption of the condensation reagent is 1.2-6 times of the total molar number of amino groups in the amino resin, and preferably 2.5-3.5 times.

The coupling reaction needs to add an activating reagent, wherein the activating reagent is selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, and 1-hydroxybenzotriazole is preferred. The amount of the activating agent is 1.2 to 6 times, preferably 2.5 to 3.5 times of the total mole number of the amino groups in the amino resin.

As a preferable scheme of the invention, the reagent for removing Fmoc protection is PIP/DMF (piperidine/N, N-dimethylformamide) mixed solution, and the piperidine content in the mixed solution is 10-30% (V). The dosage of the Fmoc protection removing reagent is 5-15 mL per gram of amino resin, and preferably 8-12 mL per gram of amino resin.

Preferably, the peptide resin is subjected to acidolysis while removing the resin and side chain protecting groups to obtain a crude product:

more preferably, the acidolysis agent used in the acidolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1, 2-Ethanedithiol (EDT) and water, and the volume ratio of the mixed solvent is as follows: 80-95% of TFA, 1-10% of EDT and the balance of water.

More preferably, the volume ratio of the mixed solvent is: 89-91% of TFA, 4-6% of EDT and the balance of water. Optimally, the volume ratio of the mixed solvent is as follows: TFA 90%, EDT 5%, balance water.

The dosage of the acidolysis agent is 4-15 mL per gram of the peptide resin; preferably, 7-10 mL of acidolysis agent is required per gram of peptide resin.

The time for cracking by using the acidolysis agent is 1-6 hours, preferably 3-4 hours at room temperature.

Further, the crude product is purified by high performance liquid chromatography and freeze-dried to obtain a pure product, and the specific method comprises the following steps:

adding water into the crude product, stirring, adjusting pH value to completely dissolve, filtering the solution with 0.45 μm mixed microporous membrane, and purifying;

purifying by high performance liquid chromatography, wherein the chromatographic packing for purification is 10 μm reversed phase C18, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a chromatographic column of 77mm × 250mm is 90mL/min, eluting by a gradient system, circularly sampling for purification, sampling the crude product solution in the chromatographic column, starting the mobile phase for elution, collecting the main peak, and evaporating acetonitrile to obtain a purified intermediate concentrated solution;

filtering the purified intermediate concentrated solution with 0.45 μm filter membrane for use;

performing salt exchange by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the chromatographic packing for purification is reversed phase C18 with 10 μm, and the flow rate of 77mm × 250mm chromatographic column is 90mL/min (corresponding flow rate can be adjusted according to chromatographic columns with different specifications); loading the sample into chromatographic column by gradient elution and cyclic loading method, starting mobile phase elution, collecting main salt exchange peak, detecting purity with analytical liquid phase, combining main salt exchange peak solutions, concentrating under reduced pressure to obtain pure acetic acid aqueous solution, and freeze drying to obtain pure product.

1. Synthesis of peptide resins

Rink Amide BHHA resin is used as carrier resin, and is coupled with protected amino acid shown in the following table in sequence through Fmoc protection removal and coupling reaction to prepare peptide resin. The protected amino acids used in this example correspond to the protected amino acids shown below:

the peptide sequence n ═	Protected amino acids
		1	Fmoc-Lys(Alloc)
2	Fmoc-Val
		3	Fmoc-Asp(OtBu)
4	Fmoc-Ser(tBu)
		5	Fmoc-Glu(OtBu)
6	Fmoc-Ile
		7	Fmoc-Ser(tBu)
8	Fmoc-Ser(tBu)
		9	Fmoc-Leu
10	Fmoc-Lys(Boc)
		11	Fmoc-Arg(Pbf)
12	Fmoc-Arg(Pbf)
		13	Fmoc-Arg(Pbf)
14	Fmoc-Gln(Trt)
		15	Fmoc-Arg(Pbf)
16	Fmoc-Arg(Pbf)
		17	Fmoc-Lys(Boc)
18	Fmoc-Lys(Boc)
		19	Fmoc-Arg(Pbf)
20	Fmoc-Gly
		21	Boc-Tyr(tBu)
Side chain-1	Fmoc-AEEA
		Side chain-2	Fmoc-AEEA
Side chain-3	Fmoc-γGlu-OtBu
		Side chain-4	18 Alkanedioic acid mono-tert-butyl ester

(1) 1 st protected amino acid inserted into main chain

Dissolving 0.03mol of the 1 st protected amino acid and 0.03mol of HOBt in a proper amount of DMF; and adding 0.03mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution for later use.

0.01mol of Rink amide MBHA resin (substitution value about 0.4mmol/g) is taken, deprotected by 20% PIP/DMF solution for 25 minutes, washed and filtered to obtain Fmoc-removed resin.

And adding the activated 1 st protected amino acid solution into the Fmoc-removed resin, performing coupling reaction for 60-300 minutes, and filtering and washing to obtain the resin containing 1 protected amino acid.

(2) 2-35 th protected amino acid connected to main chain

And sequentially inoculating the corresponding 2 nd to 35 th protected amino acids by the same method for inoculating the 1 st protected amino acid of the main chain to obtain the resin containing 35 amino acids of the main chain.

(3) Side chain insertion of the 1 st protected amino acid

Dissolving 0.03mol of the 1 st protected amino acid of the side chain and 0.03mol of HOBt in a proper amount of DMF; and adding 0.03mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution.

Taking 2.5mmol of tetratriphenylphosphine palladium and 25mmol of phenylsilane, dissolving with a proper amount of dichloromethane, deprotecting for 4 hours, filtering and washing to obtain a resin without Alloc for later use.

Adding the activated side chain 1 st protected amino acid solution into the Alloc-removed resin, performing coupling reaction for 60-300 minutes, filtering and washing to obtain the side chain 1 st protected amino acid-containing resin.

(4) 2-3 protective amino acids of grafted side chain

And sequentially inoculating 2 nd to 3 rd protected amino acids and single-protected fatty acid corresponding to side chains by adopting the same method for inoculating the 1 st protected amino acid to the main chain to obtain the peptide resin.

2. Preparation of crude product

Adding a cleavage reagent (10 mL of cleavage reagent/g of resin) with the volume ratio of TFA, water and EDT (95: 5) into the peptide resin, uniformly stirring, stirring at room temperature for reaction for 3 hours, filtering a reaction mixture by using a sand core funnel, collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate with anhydrous ether for 3 times, and drying to obtain white-like powder, namely a crude product.

3. Preparation of the pure product

Dissolving the crude product in water under stirring, filtering the solution with 0.45 μm mixed microporous membrane, and purifying. Purifying by high performance liquid chromatography, wherein the chromatographic packing for purification is 10 μm reversed phase C18, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a 30mm by 250mm chromatographic column is 20mL/min, eluting by a gradient system, circularly sampling for purification, sampling the crude product solution in the chromatographic column, starting the mobile phase for elution, collecting the main peak, and evaporating acetonitrile to obtain a purified intermediate concentrated solution;

filtering the purified intermediate concentrated solution with 0.45 μm filter membrane for use, and performing salt exchange by high performance liquid chromatography with 1% acetic acid/water solution-acetonitrile as mobile phase system, 10 μm reversed phase C18 as purification chromatographic filler, and 20mL/min of 30 mm/250 mm chromatographic column flow rate (corresponding flow rate can be adjusted according to chromatographic columns of different specifications); gradient elution and circular sample loading are adopted, the sample is loaded in a chromatographic column, mobile phase elution is started, a main salt exchange peak is collected, the purity is detected by an analytical liquid phase, main salt exchange peak solutions are combined, reduced pressure concentration is carried out, a pure acetic acid aqueous solution is obtained, and freeze drying is carried out to obtain 8.5g of a pure product, the purity is 98.1%, and the total yield is 25.3%. The molecular weight was 3361.8 (100% M + H).

EXAMPLE 2 preparation of Compound 2

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-Lys (cholesterol succinate monoester) -NH₂

The procedure is as in example 1, using the protected amino acids as in the following table:

7.7g of a pure product is obtained, the purity is 97.1 percent, and the total yield is 24.7 percent. The molecular weight was 3114.6 (100% M + H).

EXAMPLE 3 preparation of Compound 3

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-Lys(PEG₅CH₂CO-gamma Glu-18 alkanedioic acid) -NH₂

9.3g of pure product is obtained, the purity is 98.7 percent, and the total yield is 27.8 percent. The molecular weight was 3349.2 (100% M + H).

EXAMPLE 4 preparation of Compound 4

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-Lys (2-cholesteryl cholesterol acetic acid) -NH₂

7.2g of pure product is obtained, the purity is 97.6 percent, and the total yield is 23.4 percent. The molecular weight was 3072.8 (100% M + H).

Example 4 in vivo Activity assay

In SD rats of 6-10 weeks, central cerebral artery occlusion (MCAO) focal cerebral ischemia of the rats is caused by a wire-embolism method, and the wire-embolism is pulled out 2 hours after ischemia to realize reperfusion. Reperfusion was given immediately, i.v., a single administration, and the solvent control group was given an equivalent amount of physiological saline.

1. Cerebral infarct volume

3 days after MCAO reperfusion, groups of surviving rats were dissected, hearts perfused with pre-cooled PBS, brain tissue was taken, stained with 1% red tetrazolium (TTC), and percent infarct volume was calculated.

The volume percent of cerebral infarction is the right infarct volume/right brain volume x 100%.

Volume percent of cerebral infarction

After 3 days of MCAO reperfusion, all surviving rats were euthanized, brain tissue was TTC stained, and each group of cerebral infarct areas were observed and the percent cerebral infarct volume was calculated: right cerebral infarct volume/right brain volume x 100%. The solvent control group had significantly increased cerebral infarct volume compared to the sham group (. about.p < 0.001). Comparison with solvent control: the cerebral infarction volumes of the compound 1, the compound 2, the compound 3 and the compound 4 are reduced to different degrees by injecting 15mg/kg through tail veins, wherein the remission degree of the cerebral infarction volumes of the compound 1 and the compound 3 is slightly better than that of the compound 1 and the compound 4

2. Exercise capacity test

The rats were given adaptive training (4 rpm for 1 minute) using a wheeled fatigue roller prior to testing. The speed of the rotarod was gradually increased from 4 rpm to 40 rpm within 5 minutes of the test, and the time for the rat to remain on the rotarod until falling was recorded (over 300 seconds, recorded as 300 seconds). Detection time: before molding and 24 hours and 3 days after reperfusion.

Wheel type fatigue rolling bar test

The rat motor ability was tested using a wheeled fatigue rotarod. The speed of the rotor was gradually increased from 4 rpm/s to 40 rpm/s over a test period of 5 minutes, and the time the rat remained on the rotor was recorded (over 300 seconds, recorded as 300 seconds). Before modeling, the motor function of each group of rats is not different (P is more than 0.05). After 24 hours of ischemia reperfusion, the motor function of rats in the solvent control group is obviously lower than that of a sham operation group (P ═ 0.006), the mean values of the rolling rod maintaining time of rats in the compound 1, the compound 2 and the compound 3 administration group are all higher than that of the solvent control group, wherein the compound 1 has the most obvious improvement on the motor ability and is obviously better than that of the compound 4(P ═ 0.021). On day 3 post-ischemia reperfusion, mean time of rotarod maintenance was higher for compound 1, compound 2 and compound 3 than for compound 4.

3. Autonomic activity capability detection

The detection method comprises the following steps: the rat to be tested is placed in a new object recognition box to freely move for 10min, and the total movement path, the number of times of entering the central area and the percentage of movement time of the central area of the rat are recorded. Detection time: before molding and 24 hours and 3 days after reperfusion.

Autonomic activity capability detection

Before MCAO reperfusion modeling, the moving distance of each group of rats in the autonomous activity box within 10min is about 5 meters, and no significant difference exists between groups (P is more than 0.05). After 24 hours of ischemia reperfusion, the mean moving distance of rats in compound 1 group was higher than that in the solvent control group and significantly higher than that in compound 3 group (P ═ 0.041) and compound 14 group (P ═ 0.029). The mean values of the distances traveled by the rats in each group were comparable 72 hours after ischemia reperfusion. The compound 1 group showed significant improvement in rat motor function 24 hours after ischemia reperfusion.

EXAMPLE 5 determination of Primary pharmacokinetic Properties

Each compound was divided into two dosing groups: SD rats, 4 males per group, 8 in total.

Tail vein intravenous injection group: the dose is 1mg/kg, rat orbital veins are respectively bled before (0h) and 30min, 1h, 2h, 4h, 8h, 24h, 48h, 96h and 144h after administration, and plasma samples are centrifugally separated.

Subcutaneous administration group: the dose is 1mg/kg, rat orbital veins are respectively bled before (0h) and 1h, 2h, 3h, 4h, 8h, 24h, 48h, 96h and 144h after administration, and plasma samples are separated by centrifugation.

Plasma concentrations of the corresponding compounds in plasma samples of SD rats were measured by the liquid chromatography-mass spectrometry method, and the half-lives of the compounds after intravenous and subcutaneous administration in SD rats under Subcutaneous (SC) administration are shown in the following table:

compound (I)	t_1/2(h)
		Compound 1	8.3
Compound 2	7.6
		Compound 3	8.8
Compound 4	7.2

Claims

1. A compound represented by structure I:

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-

Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-AA1(R)-AA2

structure I

AA1 in structure I is Lys, or Dap, or Orn, or Dab, or Dah;

AA2 in Structure I is NH₂Or is OH;

r in structure I is DDiacid cholesterol monoester is 2-cholesteryl acetic acid, 2-cholesteryl propionic acid, 3-cholesteryl propionic acid or HO₂C(CH₂)_n1CO-(γGlu)_n2-(PEG_n3(CH2)_n4CO)_n5-。

Wherein: n1 is an integer from 10 to 20;

n2 is an integer from 1 to 5;

n3 is an integer from 1 to 30;

n4 is an integer from 1 to 5;

n5 is an integer from 1 to 5.

2. A compound according to claim 1, comprising a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex of the compound, a prodrug based on the compound, or a mixture of any of the foregoing.

3. A compound according to claim 1 and claim 2 for the preparation of a pharmaceutical composition for the treatment of a disease.

4. The pharmaceutical composition according to claim 3, for use in the preparation of a neuroprotective medicament following ischemic brain injury.

5. A compound of structure I according to claim 1, comprising the compound for use in a method of neuroprotection following ischemic brain injury.