Neuroprotection in hypoxic-ischemic injury of the immature brain using allopregnanolone

Abstract

Allopregnanolone (ALLO), an endogenous neurosteroid essential for brain growth, neuronal and glial cell survival, can ameliorate brain injury through promoting neurogenesis, neuro-regeneration, oligodendrogenesis and reducing neuro-inflammation and glutamate excitotoxicity . Methods of treatment include intrauterine administration of ALLO to the fetus, preferably at 23-38 weeks of gestational age, but may also be administered earlier when spinal damage is first observed, or at the time of birth, to reduce or prevent damage to neurological tissue.

Description

[0001] NEUROPROTECTION IN HYPOXIC-ISCHEMIC INJURY OF THE IMMATURE BRAIN USING ALLOPREGNANOLONE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 721,934, filed November 18, 2024, the entire content of which is hereby incorporated herein by reference for all purposes in its entirety.

[0004] FIELD OF THE INVENTION

[0005] This application is generally in the field of preventing or reducing neurological damage due to oxygen deprivation.

[0006] BACKGROUND OF THE INVENTION

[0007] Hypoxic ischemic (HI) brain injury in premature infants is the most common cause of cerebral palsy (CP). Yearly, 40,000 infants bom prematurely suffer from major cognitive deficits and 7,000 develop CP. Hypoxia- Ischemia (HI) in premature infants is primarily characterized by white matter injury and is associated with high morbidity, mortality and cerebral palsy (CP). Brain development during gestational age 24-34 weeks, mainly axon and dendrite formation, differentiation synaptogenesis, myelination and synaptic pruning and development of circuitry, are attenuated and compromised by any injury in this stage. Such injury can lead to significant developmental delay and retardation. Many factors play a pivotal role in the pathogenesis of HI brain injury. They include neuro-inflammation, microglial activation, susceptibility of preoligodendrocytes to reactive oxidative (ROS), nitrative stress (RNS) and excitotoxicity induced by glutamate accumulation.

[0008] No current therapy can reverse or ameliorate HI injury and its sequelae in premature infants. Therefore, there is an urgent need to develop safe therapeutic treatments to prevent or reduce neurological deficits due to hypoxia.

[0009] It is an object of the present invention to provide a method and formulations to reduce or prevent neurological damage in fetuses or newborn infants arising from hypoxia or its sequela.

[0010] SUMMARY OF THE INVENTION

[0011] Allopregnanolone (ALLO) is an endogenous neurosteroid essential for brain growth, neuronal and glial cell survival. Premature infants born are devoid of a placental supply of ALLO. ALLO administration can ameliorate brain injury through promoting neurogenesis, neuroregeneration, oligodendrogenesis and reducing neuro-inflammation and glutamate excitotoxicity.

[0012] Methods of treatment include intrauterine administration of ALLO to the fetus, preferably at 23-38 weeks of gestational age, but may also be administered earlier when spinal damage is first 1

[0013] 45803005.1 observed, or at the time of birth, to reduce or prevent damage to neurological tissue. Preferably this is administered by injection into the amniotic sac or at or into the neural tissue but can also be administered using a controlled release formulation.

[0014] Efficacy is shown in in vitro studies and a neonate mouse study. ALLO administration ameliorated brain injury in a HI mouse model of the immature brain, through promoting neurogenesis, neuro-regeneration, oligodendrogenesis and reducing neuro-inflammation and improving mitochondrial bioenergetics.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph of ALLO (ALLO, ng / ml) measured from umbilical cord plasma of preterm and full term human infants (from 23 -38 weeks gestational age) reflecting their plasma ALLO concentration levels at birth. Plasma ALLO concentration were quantified using a sensitive liquid chromatography tandem mass spectrometry assay. N = 20 neonates. Bars represent the Mean ±SE.

[0016] FIG. 2 shows serial H&E coronal whole brain sections from anterior (top) to posterior (bottom) of 3 randomly selected animals from each group at Pl 4. Sham RA, HI and HI+ Allo, with rd

[0017] special emphasis on lateral and 3 ventricle size. Scale bar 2 mm. N = 12 animals / group.

[0018] FIG. 3A and 3B arc a graph of activated CNPasc (3A) and cclls / scction of the immunostaining for all Oligodendroglia (CNPase) on P14 for RA, HI, and HI + ALLO (3B). HI causes a significant loss in Oligodendroglia and ALLO partially prevents that loss. Scale Bar = 100 / pm. N = 5 animals / group & 4 sections / animal. Bars represent the Mean +SE.

[0019] Fig 4A and 4B are a graph (4A) of activated caspase 3 / NeuN per section for RA, HI, and HI + ALLO and the immunohistochemistry (4B) of periventricular brain area on P14. Apoptosis (Caspase 3) shown in red (top panel).Neurons (NeuN) shown in green (middle panel). Colocalization is shown in orange indicates neuronal apoptosis (bottom panel). Neuronal apoptosis is significantly decreased in HI + ALLO as compared to HI. Scale bar = 100 pm. N = 5 animals / group & 4 sections / animal. Bars represent the Mean + SE.

[0020] FIG. 5A-5C are PCNA and Doublecortin western blot (5A) showing proliferating cell nuclear antigen (PCNA), a cell proliferation marker and doublecortin (neurogenesis and neuronal migration marker. HI +ALLO causes significant increase in both marker as compared to HI group. N = 6 animals / group (5B, 5C). Bars represent the Mean + SE.

[0021] FIG. 6A-6C are graphs showing long term outcome assessed on P60. 6A shows front and rear grip strength significantly higher in HI+ALLO than HI group. 6B shows significant improved co-ordination through rotarod testing in HI+ALLO group as compared to HI group. 6 C shows higher preference index of novel object recognition as a measure of memory. N=15 animals / group.

[0022] 2

[0023] 45803005.1 Fig 7A-7D is the MRI volumetric analysis using the BioSpec 94 / 30 imaging system is a 9.4T horizontal bore magnet of Coronal (top) and axial (bottom) brain slices (7 A) , and graphs of lateral ventricle volume bilaterally (7B); brain volume including brain volume + lateral ventricle volume (7C); and ratio of bilateral lateral ventricle volume to total brain volume (7D). All volumes were measured by paravision software. Since there was no significant difference in brain volume, increased lateral ventricle volume indicates white matter loss. N = 10 animals / group. Bars represent the Mean ±SE.

[0024] FIG. 8A-8J are a graph of the CD69+ to Ibal + cells per section (8A) and immunostains on P14 for all Microglia (Ibal) in green, CD 68 (marker of activated microglia only) in red (8B-8J). Co-localization in yellow indicating amount of activated microglia. HI causes the activation of microglia. By giving Allo, we have decreased this activation. Scale Bar = 100 pm. N = 5 animals / group & 4 sections / animal. Bars represent the Mean ± SE

[0025] FIG. 9A-9C are graphs of brain pro-inflammatory cytokine levels TNFa (9A) , IL-10 (9B), and IL6 (9C) measured by ELISA on P12 were significantly increased by HI and decreased with ALLO administration. N = 8 animals / group. Bars represent the Mean ± SE.

[0026] FIG. 10 is a graph of the activation of NFKB on P12 showing it is decreased when ALLO is administered after HI as compared to HI alone. N = 8 animals / group. Bars represent the Mean + SE.

[0027] FIG. 11A-11B are a graph (11A) of and immuno-florescent staining (11B) of choline acetyltransferase containing cells (ChAT) of the Basal forebrain cholinergic system on P14 shows a significant decrease in ChAT in HI in comparison to HI+ALLO and RA groups. Quantification of ChAT cells. N = 5 animals / group & 4 sections / animal. Bars represent the Mean ± SE.

[0028] Fig 12A-12D: RNAseq Data (12A) PCA analysis of RNA-Seq transcriptome on top 1000 variance genes in HI and HI+AL1O. (12B) Differentially Expressed Genes and GSEA enriched pathways that are up and down regulated in HI+ALLO group as compared to HI group. (12C) Top 10 significantly up and downregulated genes ranked by log2 fold change values in HI+ALLO group as compared to HI group. (12D) Molecular network analysis revealed that ALLO induced an increase in genes with the neurogenesis network Genes with increased expression in HI +ALLO versus HI are shown in red, while genes with decreased expression are shown in green. N=5 animals / group.

[0029] FIG. 13 is a graph of a DCFDA cellular ROS assay, measuring superoxide and hydroperoxyl in neonate mouse cortex on P12, showing HI causes significant increase in ROS levels. ALLO decreased those levels to same levels as normoxia. N = 8 animals / group. Bars represent the Mean ± SE.

[0030] 3

[0031] 45803005.1 FIG. 14A-14D are the results of Mitochondrial stress tests. 14Ais a schematic diagram of typical Seahorse Mito Stress Test assay. FIG. 14B shows the effects of FCCP and rotenone+antimycin A. 14C shows the basal and maximal respiration and spare capacity ratio (SCR). 14D shows ATP production represented as a ratio to RA. N=6 animals / group(3 wells per group with 25,000 cells / well). Bars represent the Mean ± SE.

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] Hypoxic ischemic (HI) brain injury in premature infants is associated with high morbidity, mortality and cerebral palsy (CP). HI is most common between 23-30 weeks gestation. During this time there is axon and dendrite formation, differentiation synaptogenesis, myelination and synaptic pruning and development of circuitry. Therefore, any injury in this stage will lead to developmental disturbance. Many factors play a pivotal role in the pathogenesis of HI brain injury in premature infants. These include neuro-inflammation, microglial activation, susceptibility of preoligodendrocytes to reactive oxidative (ROS), nitrative stress (RNS) and excitotoxicity caused by glutamate accumulation. No current therapy can reverse or ameliorate this injury.

[0034] Allopregnanolone (ALLO) is an endogenous neurosteroid, a reduced metabolite of progesterone, 90% of which is produced by the placenta and 10% is produced de novo in the fetal brain. ALLO is essential for brain growth, neuronal and glial cell survival. ALLO peaks to 10 times normal levels in the last part of the third trimester, when the brain grows 4- fold. Complete, acute loss of ALLO occurs with pre-term birth, which represents 10% of US births. The long-term consequences of early ALLO deprivation are associated with increased risk for neurodevelopmental delay and CP. ALLO de novo synthesis in the brain is further compromised by hypoxic ischemic (HI) brain injury, which leads to increase risk of cerebral palsy (CP) even further.

[0035] ALLO promotes neuro-regeneration and cognitive function. ALLO suppresses excitability as it is an allosteric modulator of GABAA receptors, therefore it is used to control seizure activity. ALLO exerts promyelinating and cytoprotective effects on oligodendrocytes against inflammatory stimuli. Therefore, ALLO can be administered as a replacement therapy for the lost placental supply and the compromised de novo brain ALLO production due to brain injury. In addition, ALLO targets many of the mechanisms of brain injury in premature infants. ALLO decreases many downstream harmful effects of (HI) which is the main contributor of HI brain injury. HI brain injury in the preterm is characterized by oligodendroglial loss and arrest of development, neuronal apoptosis and death, occasional seizures, pro-inflammatory cytokine release, glutamate excitotoxicity, microglial activation with consequent oxidative stress and more inflammation and therefore poor neurodevelopmental outcome. ALLO targets most of these events, improves neuronal regeneration and decreases neuronal apoptosis, decreases oligodendroglial loss and

[0036] 4

[0037] 45803005.1 improves myelination. ALLO has potent anti-inflammatory and anti-oxidant properties. ALLO decreases microglial activation thus preserving neuronal integrity and circuitry. ALLO is a positive allosteric modulator of GABA receptors thus decreases seizures and glutamate excitotoxicity. ALLO enhances memory and cognition.

[0038] I. Definitions

[0039] The term “analogue”, as used herein, refers to a chemical compound with a structure similar to that of another (reference compound) but differing from it in respect to a particular component, functional group, atom, etc.

[0040] The term “derivative”, as used herein, refers to compounds which are formed from a parent compound by one or more chemical reaction(s).

[0041] The term “prodrug”, as used herein, refers to an active drug chemically transformed into a per se inactive derivative which, by virtue of chemical or enzymatic attack, is converted to the parent drug within the body before or after reaching the site of action. Prodrugs are frequently (though not necessarily) pharmacologically inactive until converted to the parent drug.

[0042] “Pharmaceutically acceptable salt”, as used herein, refer modification of the parent compound by making the acid or base salts thereof. Example of pharmaceutically acceptable salts include but are not limited to mineral or organic acid salts of basic residues such as amines; and alkali or organic salts of acidic residues such as carboxylic acids. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. Such conventional nontoxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric acids; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, tolunesulfonic, naphthalenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic salts.

[0043] The pharmaceutically acceptable salts of the compounds can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704; and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use," P. Heinrich Stahl and Camille G. Wermuth, Eds., Wiley-VCH, Weinheim, 2002.

[0044] 5

[0045] 45803005.1 As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0046] Modified release dosage form is one for which the drug release characteristics of time, course and / or location are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage forms such as solutions, ointments, or promptly dissolving dosage forms. Delayed release, extended release, and pulsatile release dosage forms and their combinations are types of modified release dosage forms.

[0047] Delayed release dosage form is one that releases a drug (or drugs) at a time other than promptly after administration.

[0048] Extended release dosage form is one that allows at least a twofold reduction in dosing frequency as compared to the drug presented as a conventional dosage form (e.g. as a solution or prompt drug-releasing, conventional solid dosage form).

[0049] Pulsatile release dosage form is one that mimics a multiple dosing profile without repeated dosing and allows at least a twofold reduction in dosing frequency as compared to the drug presented as a conventional dosage form (e.g. as a solution or prompt drug-releasing, conventional solid dosage form). A pulsatile release profile is characterized by a time period of no release (lag time) or reduced release followed by rapid drug release.

[0050] IL Compositions

[0051] A. ALLO

[0052] The compositions contains a naturally occurring metabolite of progesterone, 3a-hydroxy-5a-pregnan-20-one (APa), also known as tetrahydroprogesterone (THP), as well as the pharmaceutically acceptable salts and hydrates thereof. 3a-hydroxy-5a-pregnan-20-one (THP) is generally classified a neurosteriod as it is produced in the central nervous system and previously has been found to be an allosteric modulator of GABA receptors. See, for example, U. S. Patent Nos.

[0053] 5,925,630; 6,143, 736; and 6,277, 838.

[0054] Other suitable analogs and derivatives include variant molecules of 3a-hydroxy-5a-pregnan-20-one or substituted derivatives of 3a-hydroxy-5a-pregnan-20-one, such as 3a-oxy derivatives, 3a-alkyl derivatives, 3a-alkenyl derivatives, 3a-ester derivatives, 3a-ether derivatives; 3ss-phenylethynyl derivatives of 3a-hydroxy-5a-pregnan-20-one, and 3p-phenylethynyl derivatives of 3 a-hydroxy-5a-pregnan- 20-one, as described in Hawkinson, etal. J. Pharmacology &

[0055] 6

[0056] 45803005.1 Experimental Therapeutics 287: 198-207 (1998); as well as steroids derivatives of the 5a pregnan-20-one series such as those described in U. S. Patent Nos. 5,925, 630,6, 143,736 and 6,277, 838.

[0057] Analogs or derivatives of 3a-hydroxy-5a-pregnan-20-one include progesterone-like molecules that arc cither natural precursors or metabolites of progesterone or synthetic variants of progesterone that exhibit substantially equivalent neurogenic activity as 3a-hydroxy-5a-pregnan-20-one. Substantially equivalent neuro-enhancing activity is defined as approximately 30% to approximately 300% of the neuro-enhancing activity of 3a-hydroxy-5a-pregnan-20-one.

[0058] The neuro-enhancing agents are administered at dosages and for periods of time effective to stimulate or induce neural proliferation and / or to protect against the neural loss in an individual. Dosage regimes may be adjusted for purposes of improving the therapeutic response to the particular composition administered. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The dosages of the one or more neuro-enhancing agents is in the range of about 0.1 mg to about 1000 mg, more preferably in the range of about 1 mg to about 500 mg, most preferably in the range from about 10 mg to about 100 mg. However, the particular dose depends on the particular neurological disease or defect being targeted and can be readily determined by the treating physician.

[0059] The compounds described herein may have one or more chiral centers and thus exist as one or more stereoisomers. Such stereoisomers can exist as a single enantiomer, a mixture of diastereomers or a racemic mixture. As used herein, the term "stereoisomers" refers to compounds made up of the same atoms having the same bond order but having different three-dimensional arrangements of atoms which are not interchangeable. The three-dimensional structures are called configurations. As used herein, the term "enantiomers" refers to two stereoisomers which are nonsuperimpos able mirror images of one another. As used herein, the term "optical isomer" is equivalent to the term "enantiomer". As used herein the term "diastereomer" refers to two stereoisomers which are not mirror images but also not superimposable. The terms "racemate", "racemic mixture" or "racemic modification" refer to a mixture of equal parts of enantiomers. The term "chiral center" refers to a carbon atom to which four different groups are attached. Choice of the appropriate chiral column, eluent, and conditions necessary to effect separation of the pair of enantiomers is well known to one of ordinary skill in the art using standard techniques (see e.g. Jacques, J. et al., "Enantiomers, Racemates, and Resolutions", John Wiley and Sons, Inc. 1981).

[0060] B. Additional active agents

[0061] The compositions can further contain one or more additional active agents. In one embodiment, the additional active agent is a steroid. Suitable steroids include biologically active 7

[0062] 45803005.1 forms of vitamin D3 and D2, such as those described in U. S. Patent Nos. 4,897,388 and 5,939,407. The steroids may be co-administered to further aid in neurogenic stimulation or induction and / or prevention of neural loss, particularly for treatments of Alzheimer's disease. Estrogen and estrogen related molecules also may be co-administered with the neuro-enhancing agents to enhance neuroprotection as described in Brinton (2001) Learning and Memory 8 (3): 121-133.

[0063] Other neuroactive steroids, such as various forms of dehydroepi-androsterone (DHEA) as described in U. S. Patent No. 6,552, 010, can also be co-administered to further aid in neurogenic stimulation or induction and / or prevention of neural loss. Other agents that cause neural growth and outgrowth of neural networks, such as Nerve Growth Factor (NGF), Brain- derived Neurotrophic Factor (BDNF), can also be administered either along with or before or after the administration of THP. Additionally, inhibitors of neural apoptosis, such as inhibitors of calpains and capases as described in Haughey et al.(2002) J Neurochemistry 83: 1509-1524, and other cell death mechanisms, such as necrosis, can be co-administered with the neuro-enhancing agents o further prevent neural loss associated with certain neurological diseases and neurological defects.

[0064] C. Formulations

[0065] Depending upon the manner of introduction, the neuro-enhancing agents described herein may be formulated in a variety of ways. Formulations containing allopregnanolone or other substantially equivalent variant molecules can be prepared in various pharmaceutical forms, such as controlled release formulations and suspensions. Preferably, these formulations are employed in solid dosage forms suitable for intrauterine administration of precise dosages.

[0066] III. Methods of Treatment

[0067] Disorders to be Treated

[0068] Hypoxia-Ischemia (HI) in premature infants is characterized by mainly white matter injury and is associated with high morbidity, mortality and cerebral palsy (CP). Brain development during gestational age 24-34 weeks, mainly axon and dendrite formation, differentiation synaptogenesis, myelination and synaptic pruning and development of circuitry, are attenuated and compromised by any injury in this stage. Such injury can lead to significant developmental delay and retardation. Many factors play a pivotal role in the pathogenesis of HI brain injury. They include neuroinflammation, microglial activation, susceptibility of pre-oligodendrocytes to reactive oxidative (ROS), nitrative stress (RNS) and excitotoxicity induced by glutamate accumulation. No current therapy can reverse or ameliorate HI injury in premature infants.

[0069] There remains a serious unmet need for novel targets and neuro-therapeutics to address HI-induced brain injury. Particularly, a natural neuro-steroid which ameliorates brain injury and enhances neuro-regeneration would be highly impactful on the short and long-term

[0070] 8

[0071] 45803005.1 neurodevelopmental outcome of premature infants. The data show that ALLO treatment results in significant neuroprotection in HI model of the immature brain. RNA sequencing data analysis identified the genetic signature of ALLO treatment in vivo, which significantly enhances neurogenesis, increases myelination, and suppress inflammatory response.

[0072] As shown by the examples, an animal model of HI injury was supplemented with ALLO which has promyelinating and cytoprotective effects on oligodendrocytes against inflammatory stimuli induced by HI injury. The data supports the use of ALLO in promoting neurogenesis, neuroregeneration, oligodendrogenesis and reducing neuro-inflammation as well as attenuating glutamate excitotoxicity in utero and in pre-term or hypoxic infants.

[0073] Human studies provided strong evidence of the relation and proportion of prematurity and ALLO serum level. Preterm neonates who are born <32 wk„ of gestational age, showed a very low semm level of ALLO, which explains why premature neonates are more prone to brain injury; mainly delayed myelination, and significant white matter brain injury. Myelin generation is dependent on high levels of cholesterol, and ALLO increases HMG-CoA reductase expression to meet the demand for the cholesterol required for myelin membrane growth. The HI mouse model mimics Hl in premature human infants mainly as they both have ventricular enlargement resulting from periventricular white matter loss as shown by H&E staining spanning the lateral ventricles (Fig 2). In contrast, the HI+ALLO group demonstrated minimal ventriculomegaly and therefore minimal white matter injury.

[0074] Cortical cytokine levels were significantly reduced in animals treated with ALLO compared to non-treated HI group. TLRs family are key components of the innate immune system and are activated after HI in the neonatal brain. Upon activation, a signaling cascade is initiated leading to activation of different transcription factors. Genomic studies of brain tissues confirmed strong dysregulation of inflammatory and apoptotic pathways in Hi-exposed mice, which were rectified in neonate mice treated with ALLO. Dysregulated signaling, suppressing the inflammatory cytokine gene expression through the modulation of TLRs, was restored by ALLO treatment. Macrophages express functional GABA-A receptors, and the activation of the receptors by ALLO leads to reduced production of inflammatory cytokines. ALLO treatment reduced macrophage expression of TNFa, IL- IB, and IDO. Similar to macrophages, microglia have been illustrated to express both GABA-A and GABA-B receptors, and treatment with the GABA-A agonist reduces microglial production of inflammatory mediators following LPS stimulation. ALLO also reduces Blood brain barrier (BBB) dysfunction following focal ischemia, due to suppression of MMP-2 and MMP-9 expression in ischemic brain following ALLO treatment. These findings highlight ALLO’s antiinflammatory properties.

[0075] 9

[0076] 45803005.1 The studies described in the examples specifically assessed neurogenesis and neuroproliferation of neurons after HI. ALLO treatment to the HI mouse model showed significant improvement in neuronal regeneration, a significant decrease in neuronal apoptosis, and improved myelination. ALLO treatment also showed a significant decrease in microglial activation thus preserving neuronal integrity and circuitry. ALLO synthesis occurs primarily in glial cells; astrocytes, oligodendrocytes, and Schwann cells as well as neural progenitors. The mechanism of action for ALLO is mediated by binding to GABAAR to elicit chloride efflux and calcium influx that induces the transcription of mitotic genes and downregulates anti-mitotic genes. ALLO administration activates a signaling cascade to trigger cell proliferation and subsequently neurogenesis. Increasing evidence indicates that altered cholesterol homeostasis is linked to neuropathologies. ALLO regulates cholesterol homeostasis via liver-X-receptor (LXR) and pregnane-X-receptor (PXR). LXR is a nuclear hormone receptor, primarily expressed in glial cells, acts as a molecular sensor of cholesterol levels initiating cholesterol clearance. Loss of LXR has been shown to repress cortical neurogenesis, particularly during late-embryonic stage development of layer Il / m. LXR activation improved cognitive function in multiple mice models of amyloidogenesis.

[0077] RNAseq analysis of neonate brain tissue among Hl+ALLO group vs HI groups, highlights the signature of ALLO treatment in triggering neurogenesis, oligo, glial cell proliferation and differentiation pathways as well as augmenting antioxidative pathways. It also showed significant attenuating of inflammation pathways (FIG. 6). To validate some of the findings, RT-PCR of studied neonate brain tissue was done on selected genes and the findings were lined up with RNAseq data.

[0078] One of the significant validated genes was the EN1 gene which is overexpressed in the HI group treated with ALLO compared to the HI group. Engrailed- 1 (EN1) is a developmental gene involved in regionalization during early embryogenesis and maintenance of normal neurons. After birth, EN 1 exerts a protective effect on midbrain dopaminergic (mDA) neurons, and loss of EN 1 causes mDA neurons in the ventral midbrain to gradually die approximately 6 weeks after birth, resulting in motor and non- motor symptoms, and neurodevelopmental abnormalities. Another crucial gene which was significantly over-expressed in HI+ALLO group and validated by RT-PCR is Pitx3 gene. PitX3 is a transcription factor required for the development and survival of midbrain dopaminergic (mdDA) neurons. Pitx3 is crucial for maintaining normal function and preserving the specific molecular identities of postnatal mDA neurons, is required for proper survival of an mDA neuron subset in prenatal and postnatal mouse brains. The transcriptomic analysis strongly highlights the direct involvement of ALLO in regulating genes highly associated with neurogenesis, myelination, and cell repair.

[0079] 10

[0080] 45803005.1 ALLO promotes efficient mitochondrial respiration through multiple mechanisms involving electron transport chain function and coupling efficiency. The viability and efficiency of the bioenergetic system is a primary determinant of synaptic and brain function. Compromised mitochondrial bioenergetics is among the earliest pathogenic events in neonatal HI insult. HI insult in neonates induces depletion of high energy phosphates. HI causes mitochondrial membrane permeabilization which results in membrane transporter deficits, accumulation of sodium and calcium within the cell, subsequent depolarization, and release of excitotoxic levels of glutamate. In premature infants, mitochondria are even less efficient at buffering influxed calcium, which leads to mitochondrial swelling few hours following the HI insult. Calmodulin-dependent activation of nitric oxide synthase generates nitric oxide which in turn results in the blockage of mitochondrial respiratory complex IV and formation of peroxy nitrites. In addition, calcium overload coincides with upregulated superoxide production and accumulation of hydroxyl and peroxynitrite radicals leads to lipid peroxidation, protein nitrosylation, and DNA damage which is more acute in the immature brain given its limited antioxidant capacity.

[0081] In the studies in the examples, it was observed that the ALLO treated HI group had a significantly lower accumulation of both ROS and lipid peroxids.

[0082] HI has been shown to damage mitochondrial ultrastructure. HI causes damage to the mitochondrial membrane and cristae causing vacuolization. HI induces perturbation of mitochondrial dynamics (fission, fusion) and mitophagy. In HI+ALLO group, there was a significant preservation of mitochondrial ultrastructure. The findings are supported by observations that ALLO suppressed mitochondrial uncoupling, increased mitochondrial respiration, reduced lipid peroxidation, and improved mitochondrial efficiency in Alzheimer’s mouse model.

[0083] Biogenesis is required for the increase in mitochondrial mass. This process is dependent on the fission of existing mitochondria followed by a coordinated program of transcription which is regulated by Peroxisome proliferator-activated receptor gamma coactivator la (PGC-la). PGC-la is often expressed at high levels within neurons to meet the high demand for energy production. PGC-la is the master regulator of mitochondrial biogenesis, respiratory capacity, oxidative phosphorylation, and fatty B oxidation. PGC-la plays a role in the formation and maintenance of synapses during development. PGC-la is significantly suppressed in the HI group, decreasing mitochondrial biogenesis and subsequently decreasing mitochondrial DNA, mitochondria volume, and size as shown in our data (FIG. 8A&B). After ALLO treatment, there was a significant increase in mitochondrial biogenesis as evidenced by the increase in mitochondrial DNA copy number to a level comparable to RA group (FIG. 8A-G). Pyruvate dehydrogenase (PDH) converts pyruvate into acetyl-CoA, thus connecting glycolysis to the TCA cycle. PDH functions as a large protein complex 11

[0084] 45803005.1 (PDHC) and localizes to the mitochondria, and its activity is regulated by phosphorylation of the PDH-Ela subunit. Previous studies showed that PDH-E1 is downregulated under prolonged hypoxic conditions, which is similar to the findings in the Hl group. ALLO treatment to the Hl group showed a significant increase in PDH-E1 level and was comparable to RA group (FIG. 8H). This crucial effect of ALLO treatment offers a new avenue of intervention for immature infants who suffer lifelong disabilities due to HI insult.

[0085] In conclusion, the data shows that ALLO is a unique natural neuro-steroidal therapeutic for HI injury in neonates. ALLO treatment partially reverses the devastating HI effects in the immature brain and has the potential to decrease CP incidence and severity by promoting neurogenesis, neuro-regeneration, and oligodendrogenesis, while reducing neuro-inflammation and glutamate excitotoxicity. ALLO has potent anti-oxidant effects, reversing HI damage to mitochondrial ultrastructure, biogenesis, and bioenergetics targeting many of the molecular mechanisms involved in HI.

[0086] The present invention will be further understood by reference to the following non-limiting example.

[0087] Example 1: Measurement of alloprenanolone in umbilical cord blood of neonates.

[0088] This study measured ALLO in preterm and full term human infant plasma at the time of birth.

[0089] Material & Methods

[0090] Sample Collection

[0091] UA institute IRB was obtained, and parents were consented prior to cord blood collection. A total of 60 neonates, with Gestational age (GA) ranged between 23-38 week of gestation, were enrolled. Umbilical cord plasma was collected and ALLO (ALLO) measurement was done using mass spect.

[0092] Serum Analysis

[0093] Umbilical cord plasma was collected and ALLO (ALLO) measurement was done using mass spect.

[0094] Serum ALLO concentrations were quantified using a sensitive liquid chromatography tandem mass spectrometry assay with minor modifications. Briefly, an aliquot of serum sample or calibration standard was spiked with the internal standard (ALLO -d5) and extracted with tert-butyl methyl ether. The organic layer was dried and underwent a derivatization procedure using 1-amino-4-methylpiperazine. The reactant was reconstituted with 50% methanol and injected on the LC-MS system. The chromatographic separation was achieved by a Cl 8 reverse phase column and an isocratic mobile phase of formic acid and methanol. The mass spectrometer was operated in

[0095] 12

[0096] 45803005.1 positive ion mode utilizing electrospray ionization. Detection was through multiple reaction monitoring, with the transition of m / z 416 to 99 monitored for ALLO and the transition of m / z 421 to 99 for the internal standard. The assay was linear over the concentration range of 0.04 - 100 ng / ml.

[0097] Serum ALLO concentrations were quantified using a sensitive liquid chromatography tandem mass spectrometry assay with minor modifications. Briefly, an aliquot of serum sample or calibration standard was spiked with the internal standard (ALLO -d5) and extracted with tert-butyl methyl ether. The organic layer was dried and underwent a derivatization procedure using 1-amino-4-methylpiperazine. The reactant was reconstituted with 50% methanol and injected on the LC-MS system. The chromatographic separation was achieved by a C18 reverse phase column and an isocratic mobile phase of formic acid and methanol. The mass spectrometer was operated in positive ion mode utilizing electrospray ionization. Detection was through multiple reaction monitoring, with the transition of m / z 416 to 99 monitored for ALLO and the transition of m / z 421 to 99 for the internal standard. The assay was linear over the concentration range of 0.04 - 100 ng / ml.

[0098] Results:

[0099] On a pilot study of 20 neonates, ALLO levels were measured in umbilical cord plasma (reflective of neonate’s plasma level at birth).

[0100] FIG. 1 is a graph of ALLO (ALLO, ng / ml) measured from umbilical cord plasma of preterm and full term human infants (from 23 -38 weeks gestational age) reflecting their plasma ALLO concentration levels at birth. Plasma ALLO concentration were quantified using a sensitive liquid chromatography tandem mass spectrometry assay. N = 20 neonates. Bars represent the Mean ±SE.

[0101] The level of ALLO is very low in 23-28 week premature infants at birth (13.6 ±3.2 ng / ml), and significantly increases in 29-32 weeks premature infants to 49.8±10.1 ng / ml, with another significant increase to 92.3 ± 12.6 ng / ml for neonates with GA bet 33-38 weeks.

[0102] Human ALLO plasma levels at birth were 9 times higher in term infants compared to extreme preterm infants. ALLO-treated HI mice showed significant improvement in neuroregeneration, myelination, motor function, coordination, learning and memory, along with significant attenuation of neuro-inflammation. RNA sequencing analysis showed that ALLO-treated HI group has a significant downregulation of inflammatory responsive genes, significant reduction of glutamate excitotoxicity, improved neurogenesis and neural repair restoring neural integrity and cognitive function. Mitochondrial ultrastructure in hippocampal neurons, using electron microscopy, showed small, fragmented mitochondria, with loss of mitochondrial membrane and cristae in HI group, while ALLO protected mitochondrial ultrastructure after HI exposure. ALLO 13

[0103] 45803005.1 significantly reversed deficits in biogenesis and key mitochondrial enzyme activity and reduced lipid peroxidation in HI.

[0104] ALLO serum level was measured using umbilical cord blood collected at birth from a total of 60 human neonates who were born with gestational age ranged between 23 to 40 weeks of gestational age (GA). Serum ALLO concentration was quantified using a sensitive liquid chromatography tandem mass spectrometry assay. ALLO serum levels were very low in preterm neonates with GA between 23-28 week (13.6 ±3.2 ng / ml), and significantly increased in premature neonates with GA 29-32 weeks (49.8+10.1 ng / ml), and significantly peaked to (72 ±5.2 ng / ml) in neonates with GA bet 33-36 weeks of GA and to value of (92.3 ± 12.6 ng / ml) in full term infants with GA of 38-39 weeks. (FIG. 1)

[0105] Human ALLO plasma levels at birth were 9 times higher in term infants compared to extreme preterm infants. ALLO-treated HI mice showed significant improvement in neuroregeneration, myelination, motor function, coordination, learning and memory, along with significant attenuation of neuro-inflammation. RNA sequencing analysis showed that ALLO-treated HI group has a significant downregulation of inflammatory responsive genes, significant reduction of glutamate excitotoxicity, improved neurogenesis and neural repair restoring neural integrity and cognitive function. Mitochondrial ultrastructure in hippocampal neurons, using electron microscopy, showed small, fragmented mitochondria, with loss of mitochondrial membrane and cristae in HI group, while ALLO protected mitochondrial ultrastructure after HI exposure. ALLO significantly reversed deficits in biogenesis and key mitochondrial enzyme activity and reduced lipid peroxidation in HI.

[0106] Conclusion:

[0107] The data showed that ALLO is a unique neuro-therapeutic natural steroid in HI injury . ALLO treatment can lead to a decrease in CP incidence and severity; by promoting neurogenesis, neuro-regeneration, oligodendrogenesis and reducing neuro-inflammation and glutamate excitotoxicity and preserving mitochondrial structure and function.

[0108] Example 2: Treatment of Neonate Mice with ALLO to reduce Hypoxia Damage Materials and Methods:

[0109] Animal studies:

[0110] A neonate mouse model of HI was treated with 3 doses of ALLO (lOmg / kg / dose IP) on P5, P8 and Pl 1. Another group with HI was treated with saline. Some of the studied animal groups were sacrificed on Pl 2 for molecular, biochemical, histopathological, and transcriptome studies. Others were allowed to survive till P60 for neurobehavioral studies.

[0111] 14

[0112] 45803005.1 To determine efficacy of ALLO ameliorating the neurodegenerative effect of HI and promote generation of neural cells, using the mouse model of HI of the immature brain, mice were treated with ALLO (lOmg / kg) after HI on postnatal day 5, 8 and 11 based on the optimal dose response. To elucidate ALLO’s neuro-regenerative potential in this model, the mice were injected with

[0113] All procedures were performed in accordance with the NIH Guidelines on the care and use of vertebrate animals and approved by the Institutional Animal Care and Use Committee of the University of Arizona. Adult pregnant mice were housed in a 12-h light / dark cycle in a virus / Ag-free facility with controlled temperature and humidity and provided with water and food ad libitum.

[0114] Animal Model

[0115] An animal model of HI injury of the immature brain was use. In brief, male and female swiss webster mice pups were randomly allocated to experimental groups at postnatal day 5 (P5). Under complete aseptic precautions, P5 mouse pups were anaesthetized with isoflurane, midline neck incision was performed, and both carotid arteries were temporary ligated (using 6.0 silk sutures double knot) for 10 min. After that, sutures were removed, and the neck incision closed. Pups were allowed to recover for 30 min on thermal blanket, then were placed in hypoxia chamber (FiCL 8%) for 20 min after which they returned to their dams. During surgery and recovery and in the hypoxic chamber, pups were placed on thermal blanket and their rectal temperature maintained at 36.5 °C measured by an ultrathin rectal probe (BiosebLab, France). Sham controls at P5 were anaesthetized with isoflurane, and midline neck incision was performed. Both carotid arteries were isolated but not ligated. Then, the neck incision was closed. During surgery and recovery, sham mice were placed on thermal mattress and rectal temperature was maintained at 37 °C.

[0116] ALLO injection:

[0117] ALLO was purchased from Steraloids, Inc. (Newport, RI). ALLO was injected at a dose of lOmg / kg / dose IP on P5, P8 and PH. ALLO was injected IP after surgery (HI+ALLO) or sham surgery (RA+ALLO) to half the pups. The other half received saline IP using the same volume and interval (RA) and (HI) groups. Treatment assignment was blinded to the investigator who participated in drug administration and endpoint analyses. Some of studied animal groups were sac’d on day of life pl2 for molecular, biochemical, histopathological and transcriptome studies. Others were allowed to survive till P60 for neurobehavioral studies.

[0118] Measuring neuronal proliferation and regeneration:

[0119] Neonate mice were injected with 5-bromo-2’ -deoxyuridine (BrdU) IP, a proliferation marker, at a dose of lOOmg / kg, with every ALLO injection at P5, P8 and Pl 1. They were sacrificed at P14, and IF staining was performed using Brdu (novus biologicals 500-235) and co-stained with 1

[0120] 45803005.1 NeuN (Millipore MAB377). Hippocampus was examined for Brdu positive neuronal cells to visualize neuronal progenitor proliferation. Brdu -i-ve cells was counted in the hippocampus CAI, CA2, CA3 and dentate gyrus and analyzed with assistance of slideBook stereology software in 5 animals / group and 4 sections / animal. In addition, IF staining for other markers of regeneration and proliferation in the hippocampus doublecortin (neuronal migration marker) and Ki67 (cell proliferation marker) and co-staining with NeuN was performed.

[0121] GABA and glutamate neurotransmitters:

[0122] To gain insight into neuronal neurotransmitter function, quantitative spatial and temporal GABA through IF staining (abcam55051) and glutaminergic receptor expression (abcaml74785) were determined by IF staining at Pl 4. In addition, extracellular glutamate was quantified using a glutamate assay kit (abcam252893). Supernatants were collected and Glutamate concentration measured using Enzy Chrome- Glutamate Assay Kits, a spectrophotometric assay where color is measured at 565nm is proportionate to the Glutamate concentration in the sample.

[0123] MRI T1,T2 , T2 flair and DWI:

[0124] MRI was performed at P14 and P60. MRI data: was obtained using 9.4 / 30 BioSpect Spectrometer (Bruker BioSpin Corp., Germany) equipped with 72 mm volume coil as a transmitter and 4-channel mouse brain coil. High resolution RARE T2-weighted images in axial and coronal plain, T2 flair and DWI will be acquired using specific parameters. MRI will be obtained at 2 time points P14 and P60) 8 animals per group at both time points.

[0125] Image post processing:

[0126] The axially acquired T2-weighted images (0.5 mm slice thickness) were used for data analysis. Masks of the mouse brain and ventricle will be obtained by manually tracing area of interest. Coronally acquired images were used for conformation purposes. Images were then imported into Analyze 7.5 software (Biomedical Imaging Resource, Mayo Clinic, Rochester, MN) for manual and semi-automated volume rendering. Total brain tissue volume is defined as the total intracranial volume minus all CSF spaces. Hippocampal volume was traced manually and volumes measured.

[0127] Additional Neuro dev elopmental testing at P60:

[0128] The automated CatWalk System (Noldus Inc., The Netherlands) was used to further quantify gait and motor deficits. This has the advantages of providing and automated assessment of the mouse motor function by taking high-resolution videos of the mice, with specialized software to measure several aspects of the animal’ s gait. In brief, at P60 mice walk on a green- illuminated glass platform contrasted by a red- illuminated ceiling, to allow for momentarily highlight of the footprints. A high- speed camera under the platform records movement and transfer the data to a 16

[0129] 45803005.1 computer, where paw prints will be analyzed with the software CatWalk XT 10.5 (Noldus Inc., The Netherlands).

[0130] Barnes Maze test: to test spatial memory ability, mice were placed in an opaque box at the central of platform with 20 holes, only one of which allows the mouse to exit the platform into the safety cage. Analysis of the number of incorrect hole and time finding the correct hole were recorded.

[0131] Histopatholgy was performed at P65 after completion of MRI and neurobehavioral testing. This includes H&E and IF staining for mylein basic protein (MBP), NeuN and caspase 3.

[0132] Animal experiments

[0133] In vivo studies:

[0134] Inflammatory cytokines assay

[0135] Assay of proinflammatory cytokines IL- 1 P, IL-6, and TNF-a was done on the periventricular brain area on postnatal day 12 using Quantikine ELISA kits (R&DSystems), which was used according to the manufacturer’s instructions. N = 8 animals / group.

[0136] Western Blot of ChAT and P65 in cortical tissue homogenates

[0137] Western blot was used to determine quantity of ChA T, phosphorylated p65 (marker of NF-KB activation) and P65. After protein extraction, protein concentration was estimated using the Modified Lowry Protein Assay (Thermo Fisher Scientific, Rockford, IL, USA). Standard SDS-PAGE techniques were followed. After electrophoresis, proteins were transferred to a PVDF membrane using a Wet / Tank Blotting System (Bio-Rad, Hercules, CA, USA). Membranes were briefly washed, incubated with respective primary antibody in 5% BSA with PBST overnight. After washing, the membranes were incubated with HRP-conjugatcd secondary antibodies for 60 min, washed, processed using Amersham ECL detection systems (GE healthcare, Piscataway, NJ USA) and exposed to 8x10 Fuji x-ray Film. Density of ChAT band was presented as a ratio to Actin band density. Density of phosphoP65 was presented as a ratio to P65. The following primary antibodies were used: ChAT antibody (Millipore, { 1 : 1000} Billerica, MA, USA); phospho p65 (Cell Signaling Technology {1:500}, Danvers, MA, USA); P65 (Cell Signaling Technology {1:500}, Danvers, MA, USA); and anti-Beta-Actin protein (as an internal control) (Cell Signaling Technology { 1 : 1000}, Danvers, MA, USA). Horseradish Peroxidase (HRP)-Conjugated Goat Anti-Rabbit and goat anti-mouse IgG conjugate were used for detection of rabbit and mouse primary antibodies respectively (Bio-Rad { 1:5,000}, Hercules, CA, USA). N = 6 mice / group.

[0138] DCFDA cellular ROS assay

[0139] The DCFDA Intracellular ROS Assay (Abeam, Cambridge, MA, USA) was done per the manufacturer’s instractions. Clear tissue lysates are placed in a 96- well cell culture plate and then 17

[0140] 45803005.1 pre-incubated with DCFH-DA, a standard substrate. The sample lysates were then added to the DCFH-DA. After an incubation period of 30 minutes, the samples were read on a standard fluorescence plate reader at 480 nm excitation / 538 nm emission. The ROS levels in the samples were determined by comparison with the predetermined DCF standard curve.

[0141] H2O2 assay

[0142] A colorimetric hydrogen peroxide detection kit (Enzo Life Sciences (ADI-907-015) was used for the assay according to the manufacturers instructions. Standard concentrations of H2O2 were run along with the sample lysates. After 3 min. incubation, the absorbance was measured between 540 and 570 . The samples’ H2O2 content was determined by comparison with the predetermined H2O2 standard curve.

[0143] Lipid Peroxidation

[0144] Lipid peroxides in hippocampal lysates were accessed using the leucomethylene blue assay, using tert-butyl hydroperoxide as a standard, by monitoring the 650 nm absorbance after 1 h incubation at RT. The aldehyde product or termination production of lipid peroxidation in brain mitochondria was determined by measuring thiobarbituric acid reactive substances (TBARS). Samples were mixed with 0.15 M phosphoric acid. After the addition of thiobarbituric acid, the reaction mixture was heated to 100 °C for 1 h. After cooling and centrifugation, the formation of TBARS was determined by the absorbance of chromophore (pink dye) at 531 nm using 600 nm as reference wavelength.

[0145] Glutamate concentration assay

[0146] Glutamate concentration was measured using EnzyChrome-Glutamate Assay Kits, a spectrophotometric assay where color is measured at 565nm is proportionate to the Glutamate concentration in the sample.

[0147] Histo-pathological & immunological studies

[0148] Brain tissue was fixed on Pl 5 in 4% paraformaldehyde for 24 h, processed, embedded in paraffin, and subsequently cut into 6-pm-thick sections. Following deparaffinization, hematoxylin and eosin (H&E) staining was performed according to standard protocols. Typical sections of hippocampus and cerebrum were made in each group of animals.

[0149] Immunohistochemistry

[0150] Animals were deeply anesthetized on P12 with a lethal dose of xylazene / ketamine and perfused transcardially with normal saline, then 4% paraformaldehyde. Brains were sectioned coronally or sagittally at 6-pm-thick using a microtome. Sections were incubated for 2 h at room temperature in TBS+ 1% Triton-X + 10% donkey serum. Samples were incubated for 24 h at 4 °C with primary antibodies, followed by 2-h incubation at RT with secondary antibodies. All images were captured 18

[0151] 45803005.1 on a Zeiss confocal microscope (Carl Zeiss, Thornwood, NY, USA). The following primary antibodies were used to detect the following markers: CD 68 (AbS Serotec { 1:100}, Raleigh, NC, USA), cleaved caspase 3 (Cell Signaling Technology { 1:50}, Danvers, MA, USA), Ibal (Wako { 1:400}, Richmond, VA, USA), CNPase (Abeam {1:200} Cambridge, MA, USA), NeuN (EDM Millipore {1:250} Billerica, MA, USA), and secondary antibodies (Species specific Cy3 and FITC 1:150 Jackson Immunoresearch, (Westgroove, PA, USA). N= 5 animals / group.

[0152] I mnuinos tabling analysis

[0153] Digital images were obtained using confocal software and then exported to Image J.

[0154] Excitation and acquisition parameters were adjusted to fully eliminate pixel saturation and all images were collected under identical settings. Each section corresponds to 750 x 750 pm. The fluorescence intensity of each pixel was performed in 4 sections per mouse and 5 mice per group using image J. Cell counting was performed using image J using plugins AnalyzeSkeleton (2D / 3D). Skeletonized images are assessed for accuracy by creating an overlay of the skeleton and the original image. Cell counting was performed on 4 sections per animal (750 x 750 pm each) and 5 animals per group. Even though analysis was automated, all analysis was performed by one investigator for consistency who was blinded to the study group to eliminate bias.

[0155] Transmission Electron Microscopy to visualize mitochondria

[0156] Pl 2 neonate mice brain hippocampal and cortex region samples were fixed with 2.5% glutaraldehyde in 0.1 M PIPES buffer, pH 7.4 overnight at 4 °C. The samples were then washed with 0.1 M PIPES, pH 7.4 three times for 10 min each. The samples were then post-fixed with 1% osmium tetroxide in PIPES, pH 7.4 for 1 h, washed with deionized water two times for 10 min, followed by 20 min in aqueous 2% uranyl acetate, and washed again with deionized water for 10 min. The samples were then dehydrated with a graded series of increasing concentrations of ethanol (50%, 70%, 90%, and 100%) in a Pelco Biowave Pro microwave set at 250 W, 20 °C, and vacuum for 40 s. The samples were then infiltrated (microwave, 1 : 1 Spurr’s resin ethanol, 250 W, 20 °C, vacuum 3 min, and Spun’ s resin, 25 W, 20 °C vacuum twice, 3 min each) and embedded in Spurr’s resin overnight at 60 °C. Ultrathin (60 nm) sections were cut onto uncoated copper mesh grids and stained with 2% lead acetate for 2 min. The samples were examined using FEI CM12 TEM operated at 80 kV. Digital images were obtained in 8-bit TIFF format using a 4 x 4 digital camera at different objective and mitochondrial length and shape in hippocampus regions were visualize and measured using IMAGE J software provided by NIH.

[0157] 19

[0158] 45803005.1 Mitochondrial DNA Copy Number Measurement

[0159] Total DNA was isolated from hippocampal tissues with QIAamp DNA mini kit (Qiagen, Valencia, CA) and analyzed by quantitative PCR. Relative mtDNA / nDNA ratio was calculated as the relative fold change of mt-NDl(mtDNA) content to HK2 (nDNA) content.

[0160] Measuring brain neuronal proliferation and regeneration:

[0161] Neonate mice were injected intra-peritoneally with 5-bromo-2-deoxyuridine (BrdU), a proliferation marker, at a cone, of lOOmg / kg, on P5, P8 and Pl 1. On Pl 2, mice were sacrificed and IF staining was performed on the hippocampus for Brdu and DAPI. Brdu positive cells allocated in CAI area of the hippocampus, were analyzed with assistance of slideBook stereology software in 5 animals / group and 4 sections / animal.

[0162] Results:

[0163] A HI brain injury model of the immature brain, which was developed in our laboratory, validated and published, was used for this study. Male and female swiss webster mice were randomly allocated to experimental groups at postnatal day 5 (P5). Under aseptic conditions, P5 pups were anaesthetized, a vertical midline neck incision was performed and both carotid arteries were temporarily ligated for 10-minutes using 6.0 silk sutures. Following ischemic insult, the sutures were removed and neck incision closed. All pups were allowed to recover for 30 min., after that pups were placed in a hypoxia chamber (8% O2) for 20 minutes. Half of HI neonate mice were treated with ALLO, (at a dose of lOmg / kg / dose IP on P5, P8 and Pl 1) (HI+ALLO group). The other half were treated with saline IP (same volume & interval) (HI group). ALLO was purchased from Steraloids,Inc.(Newport,RI). Sham room air (RA) controls at P5 were anaesthetized with isoflurane, midline neck incision was performed. Both carotid arteries were isolated but not ligated. Then neck incision was closed. Control groups were treated with ALLO or saline at the same schedule as HI group. All groups were placed during surgery and recovery on a thermal blanket and their rectal temperature maintained at 36.5°C measured by ultrathin rectal probe (BiosebLab, France). In total four groups were studied: HI group, HI+ ALLO group, Sham RA control group, Sham RA+ALLO group. Treatment assignment was blinded to the investigator who participated in drug administration and endpoint analyses.

[0164] Animals were sacrificed at P12, P14 and P60. P5 was chosen as it corresponds to 28 wks of gestation in human, an equivalent stage of oligodendroglial development, where preoligodendrocytes are most abundant and the most sensitive to oxidative and pro-inflammatory stress (< 28wk premature infant is at high risk of HI brain injury). These HI mice had more white matter injury with enlarged lateral ventricles than grey matter injury but still exhibited neuronal apoptosis

[0165] 20

[0166] 45803005.1 and necrosis. HI neonate mice mimicked human HI brain injury of the preterm in paraplegia, incoordination, neurocognitive deficits and Cerebral palsy (CP).

[0167] ALLO treated Hl pups showed minimal paresis and coordination deficits as compared to saline treated HI group, which had severe deficits. Histopathological studies showed significant ventriculomegaly (both lateral ventricles and third ventricle) in HI, in comparison to HI+ALLO group and sham controls (FIG.2).To provide evidence that the ventriculomegally is due to white matter loss and not due to hydrocephalus (increased cerebrospinal fluid volume and pressure) we performed immuno-florescent (IF) staining for oligodendroglia cells using CNPase marker (FIG.3).

[0168] There was a significant reduction in CNPase in HI group compared to HI+ALLO group indicating that ALLO preserves oligodendroglia and myelination.

[0169] Neuronal apoptosis shown by immunoflorescent (IF) co-localization of activated caspase 3 (apoptosis marker) and NeuN (general neuronal marker) was induced by HI but ameliorated by ALLO treatment (FIG.4).

[0170] To demonstrate neuronal proliferation, we performed western blot on hippocampus for proliferating cell nuclear antigen (PCNA) (Novus biologicals) which is a known proliferation marker and doublecortin, a marker of neurogenesis and neuronal migration. There is an increase in PCNA and doublecortin band density in HI ALLO group as compared to HI group, indicating that ALLO increases cell proliferation with at least a subset of this cell proliferation in neuronal proliferation (FIG.5).

[0171] Neurodevelopmental evaluation of mice at P60 including analysis of gait, grip strength, coordination, spatial learning and memory with 15 animals / group are studied. After training and habituation, Grip strength measurements for forelimb and hind limb were performed using a grip strength meter (Columbus Instruments). There is a significant reduction in grip strength especially of the hind limbs in HI group, while HI ALLO group did not have such a reduction in grip strength. HI +ALLO group had significantly higher grip strength than HI group (FIG.6A). Testing of coordination, we used rotarod device (Columbus Instruments, Columbus, OH). HI causes incoordination. HI+ALLO group had improved coordination in comparison to HI group (FIG.6B) Novel object recognition is conducted to assess declarative memory function. Retention is tested by presenting mice with one novel and one familiar object. Mice who remember the familiar object will spend more time exploring the novel object. Preference index is outlined in this formula:

[0172] (exploring novel object time- exploring familiar object time) / (total exploration time for novel and familiar objects).29Preference index for the novel object in the novel object recognition test was significantly increased in HI+ALLO group indicating improved memory function in ALLO treated

[0173] 21

[0174] 45803005.1 than non treated HI group. (FIG.6C). FIG.7 shows MRI done on Pl 4 on RA and HI groups only as a proof of feasibility.

[0175] Summary and Conclusions

[0176] Based on the preliminary data, it was hypothesized that ALLO decreases pro-inflammatory cytokines, microglial activation, and neuronal apoptosis while increasing oligodendroglial number, myelination and neuroregeneration. Data from the brain transcriptome analysis shows that myelin metabolism related genes, neurogenesis and neural repair genes are upregulated while inflammatory responsive genes are downregulated with ALLO treatment of HI mice. Single cell RNA sequencing transcriptome profiling was performed on the different cell types and subtypes in the brain.

[0177] Transcriptome profiling will provide mechanistic insight into ALLO’s neuroprotective potential.

[0178] Mitochondria are major cellular sources of reactive oxygen species (ROS). HI causes excess ROS production further exacerbating neuroinflammation. ALLO has been shown to reverse bioenergetic deficits in Alzheimer’ s mouse model. ALLO decreases intracellular ROS levels in the HI model. The effect of ALLO on mitochondrial ultrastructure was assessed by electron microscopy. The effect of ALLO on mitochondrial respiration and ATP production was measured by using Seahorse XL. The effect of ALLO in frozen brain homogenates was analyzed to determine the activity of glycolytic, tricarboxylic and electron transport chain complexes, markers of mitochondrial biogenesis, dynamics and autophagy.

[0179] ALLO, an endogenous neurosteroid, is proposed as a treatment for premature infants suffering from HI brain injury as a therapy to mitigate the harmful effects of the HL ALLO has not been previously investigated in HI brain injury of the preterm infant to date. De novo synthesis of ALLO in fetal pluripotent progenitor brain cells is critical for cortical and hippocampal development and glial differentiation. Therefore, ALLO deficit due to prematurity could lead to neurodevelopmental impairment even in the absence of HI. When adding HI brain injury to the picture, the brain’s ability to de novo produce ALLO is significantly compromised and interferes with the recovery process after HI injury. Using single nuclear RNA sequencing to elucidate mechanism for ALLO’s neuro regenerative potential is a technical innovation in our study.

[0180] The results demonstrate that ALLO improves myelination in our HI model. ALLO-induced neurogenesis, survival of newly generated neurons and behavioral outcomes are linked to the unique properties of immature neurons in the dentate gyrus to associative learning across time and with the memory functions of the entire hippocampal circuit. Newly generated neurons have increased synaptic plasticity which is critical for neural network formation. ALLO improves myelination, thus decreases white matter brain injury, in addition myelinated axons have increased conduction velocity. Myelin generation is dependent on high levels of cholesterol, and synthesis of 22

[0181] 45803005.1 cholesterol requires expression of HMG-CoA reductase, a rate-limiting step in cholesterol synthesis. ALLO increases expression of HMG-CoA reductase to meet the demand for the cholesterol required for myelin membrane growth.

[0182] ALLO administration after hypoxia ischemia brain injury in the immature brain will ameliorate HI injury. ALLO leads to new neuron formation and improves myelination which in turn improves neurodevelopmental outcome.

[0183] The animal model of HI is well established and mimics HI in human premature neonates. ALLO was given at a dose of lOmg / kg / dose IP (total of 3 doses). This dose was selected based on experiments using adult animal model with Alzheimer’s disease. This same dose was used and replicated in many other neurological disorders both in animals and humans, although given at different intervals and different age groups. In PVL there is increased neuronal excitotoxicity and neuronal apoptosis and a great need for neuronal regeneration. The interval of giving a dose every 3 days was chosen to allow the combination of decreasing neuronal excitotoxicity (once daily dose) and neuro-regeneration (once weekly dose).

[0184] ALLO has few side effects. One major one is that continuous infusions in humans has led to excess sedation and unresponsiveness. Different dosing (5, 15, 20 mg / kg / dose) and different intervals, starting with once weekly, once every 2 weeks, once monthly or once every other day may be used. Optimal dosing is based on neuro-regeneration assessed by PCNA western blot and glutamate levels. The optimal dose and interval ideally should have the highest PCNA and lowest glutamate levels. The study design was built on starting the intervention post-insult (HI) which could have a direct impact on the outcome (based on brain damage extent, the long-term outcome could be variable). In this case, a combined prophylactic / therapeutic approach, can be used, starting with ALLO, prior to the HI injury, and continuing post-injury as planned. This approach could strengthen the long-term outcome.

[0185] Example 3: Determination of the neuro-regenerative and neuroprotective mechanisms of ALLO in HI mouse model of the immature brain.

[0186] ALLO plays a neuroprotective role following different brain injuries such as ischemic damage, seizures and oxygen-glucose deprivation. ALLO reduces microglial activation and proinflammatory cytokine gene expression in preclinical models of traumatic brain, spinal cord injury, and multiple sclerosis.

[0187] ALLO promotes neuronal regeneration and oligodendroglial regeneration. Cytokines, chemokines, stress proteins, cell adhesion molecules and immune molecules are extensively up- or down-regulated in response to hypoxia ischemia. Wnt signaling pathway plays a role in promoting the physical change of microglia post-ischemic insult and also correlates with pro-inflammatory 23

[0188] 45803005.1 microglia signaling. Excessive pro-inflammatory signaling promotes lipid oxidation which subsequently, increase damage due to the lipid-rich nature of the brain. Neuron stress results from specific changes in the microenvironment, which include insufficient metabolic supplies, excess extracellular glutamate, calcium overload, acidosis and oxidative damage. Extent of exposure to such factors will ultimately influence the fate of the neuron. Transcriptome analysis can be used to examine neuronal, glial (astrocyte and microglia) and oligodendroglial transcripts, analyzing cellular response profile. Transcriptome profiling will provide mechanistic insight on ALLO’s neuroprotection potential through pathway analysis.

[0189] Materials and Methods

[0190] The same experimental groups were used as described in Example 2.

[0191] Choline-acetyltransferase containing neurons ( ChAT) Assay

[0192] To define ALLO’s anti-inflammatory potential, choline-acetyltransferase containing neurons (ChAT) synthesizing acetylcholine in the Basal forebrain cholinergic system (BFCS) were examined. Basal forebrain cholinergic neurons innervate the cortex (neocortex), hippocampus, and other forebrain regions.

[0193] RNA isolation.

[0194] RNA was isolated from both cortex and hippocampus on P14 as previously described. Staining for activated microglia

[0195] IF staining for activated microglia using CD68 marker was conducted in activated microglia in ALLO treated HI group and compared to the saline HI group.

[0196] RNAseq transcriptome of neonate brain in all studied groups

[0197] RNA isolation. Total RNA was isolated from the cortical and hippocampal tissues using the RNeasy Kit (Qiagen, Valencia, CA) following the manufacturer’s instruction. The quality and quantity of RNA samples were determined using the Experion RNA analysis kit (Bio-Rad, Hercules, CA). RNA samples were reverse-transcribed to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) following the manufacturer’ s instructions and stored at -80°C for gene array analysis.

[0198] RNA Sequencing (RNA-Seq) Transcriptomics

[0199] RNA-Seq was conducted on cerebral hemispheres at Vanderbilt Technologies for Advanced Genomics (VANTAGE) at P14. Only RNA samples with an acceptable RNA quality indicator score (RQI > 7) were used for sequencing. Enrichment of mRNA and library preparation of cDNA were done using a stranded mRNA (poly(A) - selected) sample preparation kit. Sequencing was performed at 100 bp paired-end on NovaSeq. 600, targeting 30 million reads per sample.

[0200] Transcripts were mapped to mouse genome (ensemble release 90) using Kallisto 0.4.351. Tximport 24

[0201] 45803005.1 VI.6.052 was used to generate a counts table from Kallisto output, and DESeq. 2 VI.18.153 was used to calculate normalized read counts for each gene and / or transcript and to perform expression analysis.

[0202] Principle component analysis (PC A)

[0203] The gene counts were transformed in the “DESeq. 2” package in R by variance stabilizing transformation. Then, PCAs were computed based on the top 1000 variable genes. Projections over the first and second principal components were used to present the separations.

[0204] Single nucleus RNA seq (snRNAseq):

[0205] Samples were processed with 10X Genomics 5’ Gene Expression kits on the 10X Genomics Chromium controller. After generating sequencing libraries for 10.000 cells / sample, the libraries were pooled and sequenced at VANTAGE. 10,000 reads were sequenced per cell. Sequencing data for each sample was processed through the 10X Genomics Cell Ranger pipeline which was read into the Seurat single-cell analysis package in R, where we used the ‘sctransform’ method to integrate data from all samples. The integrated data sets were then be visualized with the Uniform Manifold Approximation and Projection (UMAP) method and clustered with Louvain clustering as implemented in Seurat. Cell type annotation was conducted using the Garnett semi-automated cell type assignment package.

[0206] Neurobehavior al testing and long-term outcome (N = 25 mice / group)

[0207] Forelimb and hindlimb grip strength were measured using a grip strength meter (Columbus Instruments) at 60 days of age. Each session consisted of the average of three tests per animal. Rotarod device (Columbus Instruments, Columbus, OH, USA) was used to measure motor function and balance at P60. Each session consisted of the average of three trials on the elevated accelerating rotarod beginning at 5 RPM, measuring the time the mouse was able to remain on the rod. Novel object recognition test was performed on P60 to evaluate learning and memory. Mice were adapted to empty box once a day for 3 days, then adapted to a box containing 2 identical objects once a day for 3 days. One of the objects in the box was replaced by a new one for which they were adapted also once a day for 3 days. Then testing began. When mice sniffed or touched the objects, but not climbed over the objects, it was considered an effective exploration. The exploration time was recorded by two blinded observers to treatment / exposure allocation. The “recognition index” was figured out according to a formula: (exploring novel object time- exploring familiar object time) / ( total exploration time for novel and familiar objects).

[0208] Mitochondrial ultrastructure, emitochondrial respiration and ATP production

[0209] This is defined in HI and effects of ALLO assessed by electron microscopy. The effect of ALLO on by using Seahorse XF is performed on primary neurons (treated with 1 mL papain) from 25

[0210] 45803005.1 RA, HI and HI ALLO groups at Pl 2 which were cultured on poly-D-lysine coated 24 well plate at 25,000 cells per well, respectively.

[0211] Neurons were grown in Neurobasal Plus Medium + B27 Plus supplement for one week prior to experiment. On the day of analysis, culture medium is replaced with unbuffered DMEM and incubated at 37°C in a CO2-free incubator for 1 h. Seahorse XF assay will be done per manufacture instruction.

[0212] Basal respiration, maximum respiration (respiration after mitochondrial uncoupling) and spare capacity ratio (SCR, the ratio of maximal to basal respiration, an indicator for utilization of oxidative respiratory capacity) as well as ATP production is reduced in primary neurons obtained from HI mice versus RA mice. This will be confirmed on primary neurons obtained from HI+ALLO mice at P12 (N=6 animals / group with 2 animals per well, 3 wells per group with 25,000 cells / well).

[0213] The effect of ALLO in frozen brain homogenates was assessed by determining a) the activity of glycolytic, tricarboxylic and electron transport chain complexes; b) mitochondrial biogenesis using Peroxisome proliferator-activated receptor gamma coactivator 1 -alpha (PGC-la) western blot; c) mitochondrial dynamics (fission and fusion) assessed by electron microscopy; and d) Mitophagy evaluated by florescent microscopy with colocalization of microtubule-associated protein 1 light chain 3 (LC3) puncta and mitotracker, a mitochondrial proteins. Targeted mitochondrial transcriptome analysis analyzing RNAseq was used to explore the mechanisms by which ALLO regulates brain metabolism.

[0214] The effect of ALLO in frozen brain homogenates was assessed by determining a) the activity of glycolytic, tricarboxylic and electron transport chain complexes; b) mitochondrial biogenesis using Peroxisome proliferator-activated receptor gamma coactivator 1 -alpha (PGC-la) western blot; c) mitochondrial dynamics (fission and fusion) assessed by electron microscopy; and d) Mitophagy evaluated by florescent microscopy with colocalization of microtubule-associated protein 1 light chain 3 (LC3) puncta and mitotracker, a mitochondrial proteins. Targeted mitochondrial transcriptome analysis analyzing RNAseq was used to explore the mechanisms by which ALLO regulates brain metabolism.

[0215] All statistical tests are performed with Graph Pad Prism 8 software (La Jolla, CA).

[0216] Statistical analysis of mean differences between groups will be performed by one way-Anova followed by bonferroni post hoc. All P values and N values are indicated in figure legends. No sex difference was found in the preliminary results.

[0217] 26

[0218] 45803005.1 Results

[0219] ALLO decreases intracellular ROS levels mainly superoxide and hydrogen peroxide in our Hl model.. RNAseq analysis shows that mitochondrial energy / redox metabolism related genes (oxidative phosphorylation) are significantly upregulated in HI +ALLO as compared to HI (FIG.

[0220] 12).

[0221] FIG. 13 is a graph of a DCFDA cellular ROS assay, measuring superoxide and hydroperoxyl in neonate mouse cortex on Pl 2, showing HI causes significant increase in ROS levels. ALLO decreased those levels to same levels as normoxia.

[0222] FIG. 14A-14D are the results of Mitochondrial stress tests. 14Ais a schematic diagram of typical Seahorse Mito Stress Test assay. FIG. 14B shows the effects of FCCP and rotenone+antimycin A. 14C shows the basal and maximal respiration and spare capacity ratio (SCR). 14D shows ATP production represented as a ratio to RA.

[0223] Basal respiration, maximum respiration (respiration after mitochondrial uncoupling) and spare capacity ratio (SCR, the ratio of maximal to basal respiration, an indicator for utilization of oxidative respiratory capacity) as well as ATP production is reduced in primary neurons obtained from Hl mice versus RA mice. This was confirmed on primary neurons obtained from Hl+ALLO mice at Pl 2 (N=6 animals / group with 2 animals per well, 3 wells per group with 25,000 cells / well).

[0224] HI damages mitochondrial ultrastructure, alters dynamics, decreases mitochondrial respiration, ATP production, mitochondrial enzymes and complexes, mitochondrial biogenesis , while increasing ROS levels and mitophagy. ALLO administration after HI ameliorates most of these deleterious effects, preserving ATP and reducing ROS levels.

[0225] Summary

[0226] Hypoxia-Ischemia (HI) in premature infants is characterized by mainly white matter injury and is associated with high morbidity, mortality and cerebral palsy (CP). Brain development during gestational age 24-34 weeks, mainly axon and dendrite formation, differentiation synaptogenesis, myelination and synaptic pruning and development of circuitry, are attenuated and compromised by any injury in this stage. Such injury can lead to significant developmental delay and retardation. Many factors play a pivotal role in the pathogenesis of HI brain injury. They include neuroinflammation, microglial activation, susceptibility of pre-oligodendrocytes to reactive oxidative (ROS), nitrative stress (RNS) and excitotoxicity induced by glutamate accumulation. No current therapy can reverse or ameliorate HI injury in premature infants.

[0227] ALLO is an endogenous neuro-steroid that promotes neuro-regeneration and enhances memory and cognitive function. ALLO suppresses excitability as it is an allosteric modulator of GABAA receptors, therefore it is used to control seizure activity. ALLO exerts promyelinating and 27

[0228] 45803005.1 cytoprotective effects on oligodendrocytes against inflammatory stimuli. The mechanism of ALLO neuroprotection and neuro-regeneration has been studied extensively in Alzheimer’s disease. ALLO has shown success in many clinical trials, Alzheimer’s disease, traumatic brain injury, major depression, fragile X-associated tremor ataxia syndrome, refractory seizures and status epilepticus in children. ALLO is already FDA-approved in the treatment of postpartum depression. However, ALLO has NOT been investigated in neonates with HI brain injury.

[0229] ALLO improves myelination, thus decreasing white matter brain injury. Myelin generation is dependent on high levels of cholesterol, and synthesis of cholesterol requires the expression of HMG-CoA reductase. ALLO increases HMG-CoA reductase expression to meet the demand for the cholesterol required for myelin membrane growth. ALLO-induced neurogenesis and survival of newly generated neurons. Behavioral outcomes are linked to the unique properties of immature neurons in the dentate gyrus to associative learning across time and memory functions of the entire hippocampal circuit. Newly generated neurons have increased synaptic plasticity which is critical for neural network formation;

[0230] During gestation, ALLO expression peaks up to 10-fold normal levels in the last part of the third trimester. Premature infants born at the end of the 2ndtrimester or early part of the third trimester, have low ALLO brain levels compared to full-term infants. In addition, their de novo synthesis of ALLO in the brain is reduced due to the loss of the placental supply of progesterone that would have been converted to ALLO in the brain. Postnatal hypoxia-ischemia insult (HI) will further reduce premature infants’ brain capacity to produce ALLO.

[0231] Administering ALLO can replenish the stores and target many of the mechanisms that lead to the development of brain injury in premature infants. In the studies described above, ALLO administration in neonate mice with HI injury showed a significant decrease in many downstream harmful effects of (HI). ALLO treatment to the neonate mouse model showed significant improvement in neuronal regeneration, a significant decrease in neuronal apoptosis, and improved myelination. ALLO treatment also showed a significant decrease in microglial activation thus preserving neuronal integrity and circuitry.

[0232] Materials

[0233] Serum ALLO in human neonates at birth, a proof of concept.

[0234] ALLO serum level was measured using umbilical cord blood collected at birth from a total of 60 human neonates who were born with gestational age ranged between 23 to 40 weeks of gestational age (GA). Serum ALLO concentration was quantified using a sensitive liquid chromatography tandem mass spectrometry assay. ALLO serum levels were very low in preterm neonates with GA between 23-28 week (13.6 ±3.2 ng / ml), and significantly increased in premature 28

[0235] 45803005.1 neonates with GA 29-32 weeks (49.8±10.1 ng / ml), and significantly peaked to (72 ±5.2 ng / ml) in neonates with GA bet 33-36 weeks of GA and to value of (92.3 ± 12.6 ng / ml) in full term infants with GA of 38-39 weeks. (FIG. 1)

[0236] Amelioration of injury by ALLO treatment to HI mice pups

[0237] ALLO treated HI pups showed minimal paresis and coordination deficits as compared to saline treated HI group, which had severe deficits. HI pups displayed ventricular enlargement resulting from periventricular white matter loss as shown by H&E staining of serial coronal section spanning the lateral ventricles. HI ALLO groups have minimal ventriculomegaly and therefore minimal white matter injury (FIG.2A),

[0238] To provide further evidence that the ventriculomegaly is due to white matter loss and not due to hydrocephalus (increased cerebrospinal fluid volume and pressure), we performed immuno-florescent (IF) staining for oligodendroglial cells in the corpus callosum using CNPase marker (FIG.2B). There was a significant reduction in CNPase in HI group compared to HI+ALLO group indicating that ALLO preserves oligodendroglia and myelination, which is direct evidence of neuroprotective properties of ALLO. Neuronal apoptosis was also shown by immunoflorescent (IF) co-localization of activated caspase-3 and NeuN was induced by HI but ameliorated by ALLO treatment (FIG.2C). Western blot assessment for cleaved Caspase-3 showed similar result in the studied groups (FIG. 2D).

[0239] ALLO is a neuro-regenerative therapeutic in HI.

[0240] ALLO has been shown to be a neuro-regenerative therapeutic in Alzheimer’s. Western blot was performed on hippocampus for proliferating cell nuclear antigen (PCNA) which is a known proliferation marker and doublecortin, which is a marker of neurogenesis and neuronal migration. A significant increase in PCNA and doublecortin band density in HI ALLO group as compared to HI saline group, indicating that ALLO increases cell proliferation (FIG. 3A). IF staining for doublecortin is significantly increased in the dentate gyms of the hippocampus of the ALLO treated HI group as compared to HI saline group. This indicates that ALLO neuronal cell proliferation (FIG. 3B). Examining hippocampus region for Brdu incorporation, as an indicator of neuronal progenitor proliferation showed that Brdu +ve cells were significantly increased in the hippocampus particularly the CAI area in ALLO treated versus non treated HI group (FIG.3C). This finding supports and highlights the pivotal role of ALLO in neuronal proliferation and regeneration.

[0241] ALLO has anti-inflammatory properties in HI model.

[0242] To define ALLO’s anti-inflammatory potential, we examined choline-acetyltransferase containing neurons (ChAT) synthesizing acetylcholine in the Basal forebrain cholinergic system (BFCS). Basal forebrain cholinergic neurons innervate the cortex (neocortex), hippocampus, and 29

[0243] 45803005.1 other forebrain regions. Basal forebrain cholinergic signaling has an important role in cognition and inhibition of neuro-inflammation. A significant reduction in ChAT expression was observed in HI group compared to Hl+ALLO group indicating that ALLO preserved ChAT neurons and acetylcholine levels (FIG. 4A).

[0244] Microglia is activated in response to HI injury which is initially beneficial but prolonged activation can release pro-inflammatory cytokines and free radicals that can result in oligdendroglial injury as well as neuronal injury. IF staining for activated microglia using CD68 marker showed a significant reduction of activated microglia in HI+ALLO group compared to HI group. (FIG.4B). ALLO decreased microglial activation but did not have an effect on microgliosis. Looking at downstream effect of microglial activation, we conducted analyses for inflammatory cytokines. ALLO induced a significant reduction of Hi-induced TNF-a, IL-ip, IL-4, IL-6 (FIG. 4C) and phosphorylated NF-KB p65 compared to saline HI group (FIG.4D).

[0245] HI is known to increase glutamate concentration to very high levels causing neurotoxicity and neuronal injury. We found that HI+ALLO group has significantly lower glutamate concentration than HI group, thus preventing neuronal injury. (FIG.4E). All these findings provide direct evidence of ALLO’s anti-inflammatory properties, which partially explains ALLO’s enhancement of cognitive function.

[0246] ALLO improved cognition and the long-term neurodevelopment outcomes of HI neonate mice.

[0247] To investigate if the improved histopathological findings, along with neuro-regenerative and anti-inflammatory properties of ALLO translate to improved neurobehavioral outcomes, mice at P60 were evaluated for gait analysis, grip strength, coordination, spatial learning and memory with 25 animals / group. After training and habituation, Grip strength measurements for forelimb and hind limb were performed using a grip strength meter (Columbus Instruments). A significant reduction in grip strength was observed, especially of the hind limbs in HI group, while HI+ALLO group did not have such a reduction in grip strength. HI +ALLO group had significantly higher grip strength than HI group (FIG.5A). For coordination testing, we used rotarod device (Columbus Instruments, Columbus, OH). HI mice have significant in-coordination. HI+ALLO group had significantly improved coordination in comparison to HI group (FIG. 5B). Novel object recognition was conducted to assess declarative memory function. Retention was tested by presenting mice with one novel and one familiar object. Mice who remember the familiar object spent more time exploring the novel object. Preference index for the novel object was calculated using this formula: (exploring novel object time- exploring familiar object time) / (total exploration time for novel and familiar objects).29Preference index for the novel object in the novel object recognition test was 30

[0248] 45803005.1 significantly increased in HI+ALLO group indicating improved memory function in ALLO treated than non- treated HI group. (FIG. 5C).

[0249] Effect of ALLO treatment on transcriptome expression in Hi-exposed neonates.

[0250] Principle component analysis plot and a heat map of Pearson’ s coefficient correlation across two studied groups: HI+ALLO and HI group. Bulk RNA seq data were analyzed and provided the assessment of the variability in the transcriptomic dataset. Both the PCA plot and Pearson’s correlation heat map were generated using normalized reads per kilobases of transcript per 1 million mapped reads (RPKM) counts. The PCA demonstrated expected grouping among replicates within samples and sample groups spread across the three PCs (FIG. 6A). PCI accounts for 31.36% of the variance, PC2 accounts for an additional 18.35%, and finally PC3 accounts for 17.45%. The scree plot (FIG. 5A) confirmed that most of the variance within the dataset was described by the first two PCs. Although the PCA plot emphasizes intergroup variability, Heat map depicting the top upregulated and downregulated DEGs selected genes from comparisons of RNA sequencing data of HI+ALLP and HI groups. Gene expression values are represented by the z-score (FIG. 6B).

[0251] Differential gene expressions (DGEs) were interpreted for two groups: HI+ALLO and HI groups; were illustrated in volcano plots to show log 2 (fold changes) on the x-axis and significant -loglO(p- value) on y-axis (FIG. 6C). Total DGEs are calculated are 346 genes of which 170 are upregulated and 176 genes are downregulated for P value <0.05, FDR<0.5 and fold change< 1.5 (FIG. 6D). Overall, the RNA seq data set reveals that ALLO treatment can robustly and consistently regulate neuronal cell gene expression.

[0252] Enrichment and pathway analysis was performed on the selected genes using String protein- protein interactome network and Shiny GO analysis platform which provided a list of pathways in GO Biological processes, that are highly enriched in upregulating central nervous system myelination, oligo proliferation and differentiation and neurogenesis pathways (FIG.

[0253] 6D). OG biological processes also showed downregulation of inflammatory responses and cytokines production pathways (FIG. 6E). FIG. 6F, showed list of top 10 upregulated genes and top 10 down regulated genes ranked by log2 fold change values in HI+ALLO group as compared to HI group. RT-PCT validation of significantly up-regulated genes was done. EN1 gene, important in neurogenesis and neural repair restoring neural integrity and cognitive function; Pcp2 gene (Purkinje cell protein 2); Pitx3 gene most important in dopaminergic signaling); S1PR5 gene (Sphingosine 1 -phosphate receptor 5 mediates the immune quiescence of the brain endothelial barrier by lowering NF-KB activation); & Dnml gene (localizes to the presynaptic terminal and mediates the uptake of synaptic vesicles), all of these genes were significantly highly expressed in HI+ALLO in comparison to HI group (P<0.05) (FIG. 6G). CXCL13 genes (inflammation

[0254] 31

[0255] 45803005.1 pathway); was significantly down regulated in HI+ALLO group in comparison to HI group (P<0.05) (FIG.6G).

[0256] ALLO reversed the increase in mitochondrial ROS production seen in HL Mitochondria are major cellular sources of reactive oxygen species (ROS)12. HI causes excess ROS production, that is of mitochondrial origin as well as extra-mitochondrial, further exacerbating neuroinflammation.

[0257] ALLO reversed Hi-induced elevation of intracellular levels of ROS, mainly superoxide and hydrogen peroxide (FIG.7A). The impact of ALLO on lipid peroxides as another indicator of oxidative damage. HI induced a significant increase in lipid peroxidation which was reversed by both ALLO to levels comparable to RA groups (FIG.7B).

[0258] ALLO reversed the decline in mitochondrial biogenesis and function following HL Mitochondrial ultrastructure in hippocampal analyzed by electron microscopy showed small fragmented mitochondria., loss of mitochondrial membrane, and cristae in the HI group In the HI ALLO-treated group, mitochondria have intact membranes and retain their structure and cristae in both hippocampal and cortical neurons (FIG.8D).

[0259] Peroxisome proliferator- activated receptor gamma coactivator 1 -alpha (PGC-la) was analyzed by western blot to evaluate mitochondrial biogenesis. PGC-la is significantly increased in HI ALLO versus HI saline group (FIG.8B) Mitochondrial DNA copy number was measured in hippocampal tissue as further evidence of mitochondrial biogenesis. Mitochondrial DNA I Nuclear DNA (mtDNA / nDNA) ratio was significantly decreased in HI as compared to RA groups. ALLO restored the Hi-induced decrease in mitochondrial DNA to levels comparable to RA group (FIG.

[0260] 8A). This suggests that ALLO enhances cellular mitochondrial function in HI mice by regulating mitochondrial biogenesis. For definitive unbiased assessment, using image J to assess mitochondrial number and mitochondrial size, we assessed total mitochondrial area / per neuron. HI + ALLO group had significantly increased mitochondrial area as compared to HI group indicating an increase in the number and size of mitochondria (FIG.8E).

[0261] To investigate the direct impact of ALLO on key mitochondrial enzymes, we assessed the protein levels of the pyruvate dehydrogenase subunit El a (PDHEla, a primary regulator linking glycolysis to the TCA cycle and lipogenesis), oxaloglutarate dehydrogenase (OGDH, the determiner of metabolic flux through the TCA cycle). HI induced significant decrease in expression in PDHEla. ALLO treatment reversed the Hi-induced decrease in the key mitochondrial enzyme PDHEla expression (FIG.8F, 8G, 8H). OGDH showed a similar trend but was not statistically significant.

[0262] 32

[0263] 45803005.1 Material & Methods

[0264] Human Experiments

[0265] University of Arizona Institute internal review board (1RB) was obtained, and parents were consented prior to cord blood collection. A total of 60 neonates, with Gestational age (GA) ranged between 23-38 week of gestation, were enrolled. Umbilical cord plasma was collected and AUUO (AUUO) measurement was done using mass spect.

[0266] Serum ALLO measurement in human cord blood serum

[0267] Serum AULO concentrations were quantified using a sensitive liquid chromatography tandem mass spectrometry assay with minor modifications. Briefly, an aliquot of serum sample or calibration standard was spiked with the internal standard (AULO -d5) and extracted with tert-butyl methyl ether. The organic layer was dried and underwent a derivatization procedure using 1-amino-4-methylpiperazine. The reactant was reconstituted with 50% methanol and injected on the LC-MS system. The chromatographic separation was achieved by a Cl 8 reverse phase column and an isocratic mobile phase of formic acid and methanol. The mass spectrometer was operated in positive ion mode utilizing electrospray ionization. Detection was through multiple reaction monitoring, with the transition of m / z 416 to 99 monitored for ALLO and the transition of m / z 421 to 99 for the internal standard. The assay was linear over the concentration range of 0.04 - 100 ng / ml.

[0268] Animal experiments

[0269] Hypoxia ischemia insult - Animal model

[0270] All procedures were performed in accordance with the NIH Guidelines on the care and use of vertebrate animals and approved by the Institutional Animal Care and Use Committee of the University of Arizona. Adult pregnant mice were housed in a 12-h light / dark cycle in a virus / Ag-free facility with controlled temperature and humidity and provided with water and food ad libitum.

[0271] An animal model of HI injury of the immature brain was used. In brief, male and female Swiss Webster mice pups were randomly allocated to experimental groups at postnatal day 5 (P5). Under complete aseptic precautions, P5 mouse pups were anaesthetized with isoflurane, midline neck incision was performed, and both carotid arteries were temporary ligated (using 6.0 silk sutures double knot) for 10 min. After that, sutures were removed, and the neck incision closed. Pups were allowed to recover for 30 min on thermal blanket, then were placed in hypoxia chamber (FiO2 8%) for 20 min after which they returned to their dams. During surgery and recovery and in the hypoxic chamber, pups were placed on thermal blanket and their rectal temperature maintained at 36.5 °C measured by an ultrathin rectal probe (BiosebLab, France). Sham controls at P5 were anaesthetized with isoflurane, and midline neck incision was performed. Both carotid arteries were 33

[0272] 45803005.1 isolated but not ligated. Then, the neck incision was closed. During surgery and recovery, sham mice were placed on thermal mattress and rectal temperature was maintained at 37 °C.

[0273] ALLO injection:

[0274] ALLO was purchased from Steraloids, Inc. (Newport, RI). ALLO was injected at a dose of lOmg / kg / dose IP on P5, P8 and Pll. ALLO was injected IP after surgery (HI+ALLO) or sham surgery (RA+ALLO) to half the pups. The other half received saline IP using the same volume and interval (RA) and (HI) groups. Treatment assignment was blinded to the investigator who participated in drug administration and endpoint analyses. Some of studied animal groups were sac’d on day of life pl2 for molecular, biochemical, histopathological and transcriptome studies. Others were allowed to survive till P60 for neurobehavioral studies.

[0275] In vivo studies:

[0276] Inflammatory cytokines assay

[0277] Assay of proinflammatory cytokines IL-ip, IL-6, and TNF-a was done on the periventricular brain area on postnatal day 12 using Quantikine ELISA kits (R&DSystems), which was used according to the manufacturer’s instructions. N = 8 animals / group.

[0278] Western Blot of ChAT and P65 in cortical tissue homogenates

[0279] Western blot was used to determine quantity of ChAT, phosphorylated p65 (marker of NF-KB activation) and P65. After protein extraction, protein concentration was estimated using the Modified Lowry Protein Assay (Thermo Fisher Scientific, Rockford, IL, USA). Standard SDS-PAGE techniques were followed. After electrophoresis, proteins were transferred to a PVDF membrane using a Wet / Tank Blotting System (Bio-Rad, Hercules, CA, USA). Membranes were briefly washed, incubated with respective primary antibody in 5% BSA with PBST overnight. After washing, the membranes were incubated with HRP-conjugated secondary antibodies for 60 min, washed, processed using Amersham ECL detection systems (GE healthcare, Piscataway, NJ USA) and exposed to 8x10 Fuji x-ray Film. Density of ChAT band was presented as a ratio to Actin band density. Density of phosphoP65 was presented as a ratio to P65. The following primary antibodies were used: ChAT antibody (Millipore, {1:1000} Billerica, MA, USA): phospho p65 (Cell Signaling Technology { 1:500}, Danvers, MA, USA); P65 (Cell Signaling Technology { 1:500}, Danvers, MA, USA); and anti-Beta-Actin protein (as an internal control) (Cell Signaling Technology { 1:1000}, Danvers, MA, USA). Horseradish Peroxidase (HRP)-Conjugated Goat Anti-Rabbit and goat anti-mouse IgG conjugate were used for detection of rabbit and mouse primary antibodies respectively (Bio-Rad { 1:5,000}, Hercules, CA, USA). N = 6 mice / group.

[0280] 34

[0281] 45803005.1 DCFDA cellular ROS assay

[0282] The DCFDA Intracellular ROS Assay (Abeam, Cambridge, MA, USA) was done per the manufacturer’s instructions. Clear tissue lysates are placed in a 96- well cell culture plate and then pre-incubated with DCFH-DA, a standard substrate. The sample lysates were then added to the DCFH-DA. After an incubation period of 30 minutes, the samples were read on a standard fluorescence plate reader at 480 nm excitation / 538 nm emission. The ROS levels in the samples were determined by comparison with the predetermined DCF standard curve.

[0283] H2O2 assay

[0284] A colorimetric hydrogen peroxide detection kit (Enzo Life Sciences (ADI-907-015) was used for the assay according to the manufacturer’s instructions. Standard concentrations of H2O2 were run along with the sample lysates. After 3 min. incubation, the absorbance was measured between 540 and 570 . The samples’ H2O2 content was determined by comparison with the predetermined H2O2 standard curve.

[0285] Lipid Peroxidation

[0286] Lipid peroxides in hippocampal lysates were accessed using the leucomethylene blue assay, using tert-butyl hydroperoxide as a standard, by monitoring the 650 nm absorbance after 1 h incubation at RT. The aldehyde product or termination production of lipid peroxidation in brain mitochondria was determined by measuring thiobarbituric acid reactive substances (TBARS). Samples were mixed with 0.15 M phosphoric acid. After the addition of thiobarbituric acid, the reaction mixture was heated to 100 °C for 1 h. After cooling and centrifugation, the formation of TBARS was determined by the absorbance of chromophore (pink dye) at 531 nm using 600 nm as reference wavelength.

[0287] Glutamate concentration assay

[0288] Glutamate concentration was measured using Enzy Chrome- Glutamate Assay Kits, a spectrophotometric assay where color is measured at 565nm is proportionate to the Glutamate concentration in the sample.

[0289] Histo-pathological & immunological studies

[0290] Brain tissue was fixed on Pl 5 in 4% paraformaldehyde for 24 h, processed, embedded in paraffin, and subsequently cut into 6-pm-thick sections. Following deparaffinization, hematoxylin and eosin (H&E) staining was performed according to standard protocols. Typical sections of hippocampus and cerebrum were made in each group of animals.

[0291] Immunohistochemistry

[0292] Animals were deeply anesthetized on Pl 2 with a lethal dose of xylazene / ketamine and perfused transcardially with normal saline, then 4% paraformaldehyde. Brains were sectioned 35

[0293] 45803005.1 coronally or sagittally at 6-pm-thick using a microtome. Sections were incubated for 2 h at room temperature in TBS+ 1% TRITON™-X + 10% donkey serum. Samples were incubated for 24 h at 4 °C with primary antibodies, followed by 2-h incubation at RT with secondary antibodies. All images were captured on a Zeiss confocal microscope (Carl Zeiss, Thornwood, NY, USA). The following primary antibodies were used to detect the following markers: CD 68 (AbS Serotec { 1:100}, Raleigh, NC, USA), cleaved caspase 3 (Cell Signaling Technology { 1:50}, Danvers, MA, USA), Ibal (Wako { 1:400}, Richmond, VA, USA), CNPase (Abeam { 1:200} Cambridge, MA, USA), NeuN (EDM Millipore {1:250} Billerica, MA, USA), , and secondary antibodies (Species specific Cy3) and FITC 1:150 Jackson Immunoresearch, (Westgroove, PA, USA). N - 5 animals / group.

[0294] Immunostaining analysis

[0295] Digital images were obtained using confocal software and then exported to Image J.

[0296] Excitation and acquisition parameters were adjusted to fully eliminate pixel saturation and all images were collected under identical settings. Each section corresponds to 750 x 750 pm. The fluorescence intensity of each pixel was performed in 4 sections per mouse and 5 mice per group using image J. Cell counting was performed using image J using plugins ANALYZES KELETON (2D / 3D). Skeletonized images are assessed for accuracy by creating an overlay of the skeleton and the original image. Cell counting was performed on 4 sections per animal (750 x 750 pm each) and 5 animals per group. Even though analysis was automated, all analysis was performed by one investigator for consistency who was blinded to the study group to eliminate bias.

[0297] Transmission Electron Microscopy to visualize mitochondria

[0298] Pl 2 neonate mice brain hippocampal and cortex region samples were fixed with 2.5% glutaraldehyde in 0.1 M PIPES buffer, pH 7.4 overnight at 4 °C. The samples were then washed with 0.1 M PIPES, pH 7.4 three times for 10 min each. The samples were then post-fixed with 1% osmium tetroxide in PIPES, pH 7.4 for 1 h, washed with deionized water two times for 10 min, followed by 20 min in aqueous 2% uranyl acetate, and washed again with deionized water for 10 min. The samples were then dehydrated with a graded series of increasing concentrations of ethanol (50%, 70%, 90%, and 100%) in a PELCO BIOWAVE PRO microwave set at 250 W, 20 °C, and vacuum for 40 s. The samples were then infiltrated (microwave, 1:1 Spurr’s resin ethanol, 250 W, 20 °C, vacuum 3 min, and Spurr’s resin, 25 W, 20 °C vacuum twice, 3 min each) and embedded in Spurr’s resin overnight at 60 °C. Ultrathin (60 nm) sections were cut onto uncoated copper mesh grids and stained with 2% lead acetate for 2 min. The samples were examined using FEI CM12 TEM operated at 80 kV. Digital images were obtained in 8-bit TIFF format using a 4 x 4

[0299] 36

[0300] 45803005.1 digital camera at different objective and mitochondrial length and shape in hippocampus regions were visualize and measured using IMAGE J software provided by NIH.

[0301] Mitochondrial DNA Copy Number Measurement

[0302] Total DNA was isolated from hippocampal tissues with QIAamp DNA mini kit (Qiagen, Valencia, CA) and analyzed by quantitative PCR. Relative mtDNA / nDNA ratio was calculated as the relative fold change of mt-NDl(mtDNA) content to HK2 (nDNA) content.

[0303] Measuring brain neuronal proliferation and regeneration:

[0304] Neonate mice were injected intra-peritoneally with 5-bromo-2-deoxyuridine (BrdU), a proliferation marker, at a cone, of lOOmg / kg, on P5, P8 and Pl 1. On P12, mice were sacrificed and IF staining was performed on the hippocampus for Brdu and DAPI. Brdu positive cells allocated in CAI area of the hippocampus, were analyzed with assistance of slideBook stereology software in 5 animals / group and 4 sections / animal.

[0305] RNAseq transcriptome of neonate brain in all studied groups

[0306] RNA isolation. Total RNA was isolated from the cortical and hippocampal tissues using the RNeasy Kit (Qiagen, Valencia, CA) following the manufacturer’s instruction. The quality and quantity of RNA samples were determined using the Experion RNA analysis kit (Bio-Rad, Hercules, CA). RNA samples were reverse-transcribed to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) following the manufacturer’ s instructions and stored at -80°C for gene array analysis.

[0307] RNA Sequencing (RNA-Seq) Transcriptomics

[0308] RNA-Seq was conducted on cerebral hemispheres at Vanderbilt Technologies for Advanced Genomics (VANTAGE) at P14. Only RNA samples with an acceptable RNA quality indicator score (RQI > 7) were used for sequencing. Enrichment of mRNA and library preparation of cDNA were done using a stranded mRNA (poly(A) - selected) sample preparation kit. Sequencing was performed at 100 bp paired-end on NovaSeq. 600, targeting 30 million reads per sample.

[0309] Transcripts were mapped to mouse genome (ensemble release 90) using Kallisto 0.4.351. Tximport VI.6.052 was used to generate a counts table from Kallisto output, and DESeq. 2 VI.18.153 was used to calculate normalized read counts for each gene and / or transcript and to perform expression analysis.

[0310] Principle component analysis (PCA)

[0311] The gene counts were transformed in the “DESeq. 2” package in R by variance stabilizing transformation. Then, PCAs were computed based on the top 1000 variable genes. Projections over the first and second principal components were used to present the separations.

[0312] 37

[0313] 45803005.1 Single nucleus RNA seq (snRNAseq):

[0314] Samples were processed with 10X Genomics 5’ Gene Expression kits on the 10X Genomics Chromium controller. After generating sequencing libraries for 10,000 cells I sample, the libraries were pooled and sequenced at VANTAGE. 10,000 reads per cell were sequenced. Sequencing data for each sample was processed through the 10X Genomics Cell Ranger pipeline which was read into the Seurat single-cell analysis package in R, where the ‘sctransform’ method was used to integrate data from all samples. The integrated data sets were then be visualized with the Uniform Manifold Approximation and Projection (UMAP) method and clustered with Louvain clustering as implemented in Seurat. Cell type annotation was conducted using the Garnett semi-automated cell type assignment package.

[0315] Neurobehavioral testing and long-term outcome N = 25 mice / group)

[0316] Forelimb and hindlimb grip strength were measured using a grip strength meter (Columbus Instruments) at 60 days of age. Each session consisted of the average of three tests per animal. Rotarod device (Columbus Instruments, Columbus, OH, USA) was used to measure motor function and balance at P60. Each session consisted of the average of three trials on the elevated accelerating rotarod beginning at 5 RPM, measuring the time the mouse was able to remain on the rod. Novel object recognition test was performed on P60 to evaluate learning and memory. Mice were adapted to empty box once a day for three days, then adapted to a box containing two identical objects once a day for three days. One of the objects in the box was replaced by a new one for which they were adapted also once a day for three days. Then testing began. When mice sniffed or touched the objects, but not climbed over the objects, it was considered an effective exploration. The exploration time was recorded by two blinded observers to treatment / exposure allocation. The “recognition index” was figured out according to a formula: (exploring novel object time- exploring familiar object time) / ( total exploration time for novel and familiar objects).

[0317] Statistical analyses

[0318] All statistical tests were performed with Graph Pad Prism 8 software (La Jolla, CA).

[0319] Statistical analysis of mean differences between groups was performed by using one-way ANOVA, followed by a Bonferroni post-hoc analysis. All p values and n values are indicated in figure legends. No sex difference was found in the studies.

[0320] Results

[0321] Serum ALLO in human neonates at birth, a proof of concept.

[0322] ALLO serum level was measured using umbilical cord blood collected at birth from a total of 60 human neonates who were born with gestational age ranged between 23 to 40 weeks of gestational age (GA). Serum ALLO concentration was quantified using a sensitive liquid

[0323] 38

[0324] 45803005.1 chromatography tandem mass spectrometry assay. ALLO serum levels were very low in preterm neonates with GA between 23-28 week (13.6 ±3.2 ng / ml), and significantly increased in premature neonates with GA 29-32 weeks (49.8+10.1 ng / ml). and significantly peaked to (72 +5.2 ng / ml) in neonates with GA bet 33-36 weeks of GA and to value of (92.3 + 12.6 ng / ml) in full term infants with GA of 38-39 weeks. (FIG. 1)

[0325] Amelioration of injury by ALLO treatment to HI mice pups

[0326] ALLO treated HI pups showed minimal paresis and coordination deficits as compared to saline treated HI group, which had severe deficits. HI pups displayed ventricular enlargement resulting from periventricular white matter loss as shown by H&E staining of serial coronal section spanning the lateral ventricles. HI ALLO groups have minimal ventriculomegaly and therefore minimal white matter injury (FIG.2A),

[0327] To provide further evidence that the ventriculomegaly is due to white matter loss and not due to hydrocephalus (increased cerebrospinal fluid volume and pressure), we performed immuno-florescent (IF) staining for oligodendroglial cells in the corpus callosum using CNPase marker (FIG.2B). There was a significant reduction in CNPase in HI group compared to HI+ALLO group indicating that ALLO preserves oligodendroglia and myelination, which is direct evidence of neuroprotective properties of ALLO. Neuronal apoptosis was also shown by immunoflorescent (IF) co-localization of activated caspase-3 and NeuN was induced by HI but ameliorated by ALLO treatment (FIG.2C). Western blot assessment for cleaved Caspase-3 showed similar result in the studied groups (FIG. 2D).

[0328] ALLO is a neuro-regenerative therapeutic in HL

[0329] ALLO has been shown to be a neuro-regenerative therapeutic in Alzheimer’s. The aim was to determine if this is true after HL Therefore, western blot performed on hippocampus for proliferating cell nuclear antigen (PCNA) which is a known proliferation marker and doublecortin, which is a marker of neurogenesis and neuronal migration. A significant increase in PCNA and doublecortin band density in HI ALLO group as compared to HI saline group, indicating that ALLO increases cell proliferation (FIG. 3A). IF staining for doublecortin is significantly increased in the dentate gyrus of the hippocampus of the ALLO treated HI group as compared to HI saline group. This indicates that ALLO neuronal cell proliferation (FIG. 3B). Examining hippocampus region for Brdu incorporation, as an indicator of neuronal progenitor proliferation showed that Brdu +ve cells were significantly increased in the hippocampus particularly the CAI area in ALLO treated versus non treated HI group (FIG. 3C). This finding supports and highlights the pivotal role of ALLO in neuronal proliferation and regeneration.

[0330] 39

[0331] 45803005.1 ALLO has anti-inflammatory properties in HI model.

[0332] To define ALLO’s anti-inflammatory potential, choline-acetyltransferase containing neurons (ChA T) synthesizing acetylcholine in the Basal forebrain cholinergic system (BFCS) were examined. Basal forebrain cholinergic neurons innervate the cortex (neocortex), hippocampus, and other forebrain regions. Basal forebrain cholinergic signaling has an important role in cognition and inhibition of neuro-inflammation. It was found that a significant reduction in ChAT expression in HI group compared to HI+ALLO group indicating that ALLO preserved ChAT neurons and acetylcholine levels (FIG. 4A).

[0333] Microglia is activated in response to HI injury which is initially beneficial but prolonged activation can release pro-inflammatory cytokines and free radicals that can result in oligdendroglial injury as well as neuronal injury. IF staining for activated microglia using CD68 marker showed a significant reduction of activated microglia in HI+ALLO group compared to HI group. (FIG.4B). ALLO decreased microglial activation but did not have an effect on microgliosis. Looking at downstream effect of microglial activation, we conducted analyses for inflammatory cytokines. ALLO induced a significant reduction of Hi-induced TNF-a, IL-ip, IL-4, IL-6 (FIG. 4C) and phosphorylated NF-KB p65 compared to saline HI group (FIG.4D).

[0334] HI is known to increase glutamate concentration to very high levels causing neurotoxicity and neuronal injury. The HI+ALLO group has significantly lower glutamate concentration than HI group, thus preventing neuronal injury. (FIG.4E). All these findings provide direct evidence of ALLO’s anti-inflammatory properties, which partially explains ALLO’s enhancement of cognitive function.

[0335] ALLO improved cognition and the long-term neurodevelopment outcomes of HI neonate mice.

[0336] To investigate if the improved histopathological findings, along with neuro-regenerative and anti-inflammatory properties of ALLO translate to improved neurobehavioral outcomes, mice at P60 were evaluated for gait analysis, grip strength, coordination, spatial learning and memory with 25 animals / group. After training and habituation, Grip strength measurements for forelimb and hind limb were performed using a grip strength meter (Columbus Instruments). A significant reduction in grip strength especially of the hind limbs in HI group was found, while HI+ALLO group did not have such a reduction in grip strength. HI +ALLO group had significantly higher grip strength than HI group (FIG. 5A). For coordination testing, a rotarod device (Columbus Instruments, Columbus, OH) was used. HI mice have significant in-coordination. HI+ALLO group had significantly improved coordination in comparison to HI group (FIG. 5B) Novel object recognition was conducted to assess declarative memory function. Retention was tested by

[0337] 40

[0338] 45803005.1 presenting mice with one novel and one familiar object. Mice who remember the familiar object spent more time exploring the novel object. Preference index for the novel object was calculated using this formula: (exploring novel object time- exploring familiar object time) / (total exploration time for novel and familiar objects). Preference index for the novel object in the novel object recognition test was significantly increased in HI+ALLO group indicating improved memory function in ALLO treated than non- treated HI group. (FIG.5C).

[0339] Effect of ALLO treatment on transcriptome expression in Hi-exposed neonates.

[0340] Principle component analysis plot and a heat map of Pearson’s coefficient correlation across two studied groups: HI+ALLO and HI group. Bulk RNA seq data were analyzed and provided the assessment of the variability in the transcriptomic dataset. Both the PCA plot and Pearson’s correlation heat map were generated using normalized reads per kilobases of transcript per 1 million mapped reads (RPKM) counts. The PCA demonstrated expected grouping among replicates within samples and sample groups spread across the three PCs (FIG.6A). PCI accounts for 31.36% of the variance, PC2 accounts for an additional 18.35%, and finally PC3 accounts for 17.45%. The scree plot (FIG. 5A) confirmed that most of the variance within the dataset was described by the first two PCs. Although the PCA plot emphasizes intergroup variability, Heat map depicting the top upregulated and downregulated DEGs selected genes from comparisons of RNA sequencing data of HI+ALLP and HI groups. Gene expression values are represented by the z-score (FIG.6B).

[0341] Differential gene expressions (DGEs) were interpreted for two groups; HI+ALLO and HI groups; were illustrated in volcano plots to show log 2 (fold changes) on the x-axis and significant -loglO(p- value) on y-axis (FIG.6C). Total DGEs are calculated are 346 genes of which 170 are upregulated and 176 genes are downregulated for P value <0.05, FDR<0.5 and fold change< 1.5 (FIG.6D). Overall, the RNA seq data set reveals that ALLO treatment can robustly and consistently regulate neuronal cell gene expression.

[0342] Enrichment and pathway analysis was performed on the selected genes using String protein- protein interactome network and Shiny GO analysis platform which provided a list of pathways in GO Biological processes, that are highly enriched in upregulating central nervous system myelination, oligo proliferation and differentiation and neurogenesis pathways (FIG.

[0343] 6D). OG biological processes also showed downregulation of inflammatory responses and cytokine production pathways (FIG.6E). FIG. 6F shows a list of top 10 upregulated genes and top 10 down regulated genes ranked by log2 fold change values in HI+ALLO group as compared to HI group. RT-PCT validation of significantly up-regulated genes was done. EN1 gene, important in neurogenesis and neural repair restoring neural integrity and cognitive function; Pcp2 gene (Purkinje cell protein 2); Pitx3 gene most important in dopaminergic signaling); S1PR5 gene 41

[0344] 45803005.1 (Sphingosine 1 -phosphate receptor 5 mediates the immune quiescence of the brain endothelial barrier by lowering NF-KB activation); & Dnml gene (localizes to the presynaptic terminal and mediates the uptake of synaptic vesicles), all of these genes were significantly highly expressed in HI+ALLO in comparison to HI group (P<0.05) (FIG.6G). CXCL13 genes (inflammation pathway); was significantly down regulated in HI+ALLO group in comparison to HI group (P<0.05) (FIG.6G).

[0345] ALLO reversed the increase in mitochondrial ROS production seen in HL Mitochondria are major cellular sources of reactive oxygen species (ROS). HI causes excess ROS production, that is of mitochondrial origin as well as extra-mitochondrial, further exacerbating neuroinflammation.

[0346] ALLO reversed Hi-induced elevation of intracellular levels of ROS, mainly superoxide and hydrogen peroxide (FIG.7A). The impact of ALLO was assessed on lipid peroxides as another indicator of oxidative damage. HI induced a significant increase in lipid peroxidation which was reversed by both ALLO to levels comparable to RA groups (FIG.7B).

[0347] ALLO reversed the decline in mitochondrial biogenesis and function following HL Mitochondrial ultrastructure in hippocampas analyzed by electron microscopy, showed small fragmented mitochondria., loss of mitochondrial membrane, and cristae in the HI group. In the HI ALLO-treated group, mitochondria have intact membranes and retain their structure and cristae in both hippocampal and cortical neurons (FIG.8D)

[0348] Peroxisome proliferator- activated receptor gamma coactivator 1 -alpha (PGC-la) western blots were analyzed to evaluate mitochondrial biogenesis. PGC-la is significantly increased in HI ALLO versus HI saline group (FIG.8B) Mitochondrial DNA copy number was measured in hippocampal tissue as further evidence of mitochondrial biogenesis. Mitochondrial DNA I Nuclear DNA (mtDNA / nDNA) ratio was significantly decreased in HI as compared to RA groups. ALLO restored the Hi-induced decrease in mitochondrial DNA to levels comparable to RA group (FIG.

[0349] 8A). This shows that ALLO enhances cellular mitochondrial function in HI mice by regulating mitochondrial biogenesis. For definitive unbiased assessment, using image I to assess mitochondrial number and mitochondrial size, total mitochondrial area / per neuron. HI + ALLO group had significantly increased mitochondrial area as compared to HI group indicating an increase in the number and size of mitochondria (FIG.8E)

[0350] To investigate the direct impact of ALLO on key mitochondrial enzymes, the protein levels of the pyruvate dehydrogenase subunit Ela (PDHEla, a primary regulator linking glycolysis to the TCA cycle and lipogenesis), oxaloglutarate dehydrogenase (OGDH, the determiner of metabolic flux through the TCA cycle) were assessed. HI induced significant decrease in expression in 42

[0351] 45803005.1 PDHEla. ALLO treatment reversed the Hi-induced decrease in the key mitochondrial enzyme PDHE1 a expression (Fig 8F, 8G, 8H). OGDH showed a similar trend but was not statistically significant.

[0352] Discussion

[0353] The data show that ALLO treatment results in significant neuroprotection in hypoxia induced (HI) model of the immature brain. RNA sequencing data analysis identified the genetic signature of ALLO treatment in vivo, which significantly enhances neurogenesis, increases myelination, and suppress inflammatory response.

[0354] There remains a serious unmet need for neuro-therapeutics to address Hi-induced brain injury. A natural neuro-steroid which ameliorates brain injury and enhances neuro-regeneration would be highly impactful on the short and long-term neurodevelopmental outcome of premature infants.

[0355] These studies targeted Hi-induced brain injury in neonates. The animal model of HI injury was treated with ALLO which has promyelinating and cytoprotective effects on oligodendrocytes against inflammatory stimuli induced by HI injury. The study demonstrated a highly compelling support for ALLO in promoting neurogenesis, neuro-regeneration, oligodendrogenesis and reducing neuro-inflammation as well as attenuating glutamate excitotoxicity in a preclinical neonate murine model of Hi-induced brain injury.

[0356] Human studies provided strong evidence of the relation and proportion of prematurity and ALLO serum level. Preterm neonates who are born <32 wk„ of gestational age, showed a very low semm level of ALLO, which explain why premature neonates are more prone to brain injury; mainly delayed myelination, and significant white matter brain injury. Myelin generation is dependent on high levels of cholesterol, and ALLO increases HMG-CoA reductase expression to meet the demand for the cholesterol required for myelin membrane growth. The HI mouse model mimics HI in premature human infants mainly as they both have ventricular enlargement resulting from periventricular white matter loss as shown by H&E staining spanning the lateral ventricles (FIG 2). In contrast, HI+ALLO group, demonstrated minimal ventriculomegaly and therefore minimal white matter injury.

[0357] Cortical cytokine levels were significantly reduced in animals treated with ALLO compared to non-treated HI group. TLRs family are key components of the innate immune system and are activated after HI in the neonatal brain. Upon activation, a signaling cascade is initiated leading to activation of different transcription factors. Genomic studies of brain tissues confirmed strong dysregulation of inflammatory and apoptotic pathways in Hi-exposed mice, which were rectified in neonate mice treated with ALLO. Dysregulated signaling, suppressing the inflammatory cytokine 43

[0358] 45803005.1 gene expression through the modulation of TLRs, was restored by ALLO treatment. Macrophages express functional GABA-A receptors, and the activation of the receptors by ALLO leads to reduced production of inflammatory cytokines. ALLO treatment reduced macrophage expression of TNFa, IL- IB, and IDO. Similar to macrophages, microglia have been illustrated to express both GABA-A and GABA-B receptors, and treatment with the GABA-A agonist reduces microglial production of inflammatory mediators following LPS stimulation. ALLO also reduces Blood brain barrier (BBB) dysfunction following focal ischemia, due to suppression of MMP-2 and MMP-9 expression in ischemic brain following ALLO treatment. These findings highlight ALLO’s antiinflammatory properties.

[0359] Neurogenesis and neuro-proliferative of neurons after HI was specifically assessed. ALLO treatment to the HI mouse model showed significant improvement in neuronal regeneration, a significant decrease in neuronal apoptosis, and improved myelination. ALLO treatment also showed a significant decrease in microglial activation thus preserving neuronal integrity and circuitry. ALLO synthesis occurs primarily in glial cells; astrocytes, oligodendrocytes, and Schwann cells as well as neural progenitors. The mechanism of action for ALLO is mediated by binding to GABAAR to elicit chloride efflux and calcium influx that induces the transcription of mitotic genes and downregulates anti-mitotic genes. ALLO administration activates a signaling cascade to trigger cell proliferation and subsequently neurogenesis. Increasing evidence indicates that altered cholesterol homeostasis is linked to neuro-pathologies. ALLO regulates cholesterol homeostasis via liver-X-receptor (LXR) and pregnane-X-receptor (PXR). LXR is a nuclear hormone receptor, primarily expressed in glial cells, acts as a molecular sensor of cholesterol levels initiating cholesterol clearance. Loss of LXR has been shown to repress cortical neurogenesis, particularly during late-embryonic stage development of layer H / III. LXR activation improved cognitive function in multiple mice models of amyloidogenesis.

[0360] RNAseq analysis of neonate brain tissue among HI+ALLO group vs HI groups, highlights the signature of ALLO treatment in triggering neurogenesis, oligo, glial cell proliferation and differentiation pathways as well as augmenting antioxidative pathways. It also showed significant attenuation of inflammation pathways (FIG. 6). To validate some of the findings, RT-PCR of studied neonate brain tissue was done on selected genes and the findings were lined up with RNAseq data.

[0361] One of the significant validated genes was the EN1 gene which was overexpressed in HI group treated with ALLO compared to HI group. Engrailed-1 (EN1) is a developmental gene involved in regionalization during early embryogenesis and maintenance of normal neurons. After birth, EN 1 exerts a protective effect on midbrain dopaminergic (mDA) neurons, and loss of EN 1

[0362] 44

[0363] 45803005.1 causes mDA neurons in the ventral midbrain to gradually die approximately 6 weeks after birth, resulting in motor and non- motor symptoms, and neurodevelopmental abnormalities. Another crucial gene which was significantly over-expressed in Hl+ALLO group and validated by RT-PCR is Pitx3 gene. PitX3 is a transcription factor required for the development and survival of midbrain dopaminergic (mdDA) neurons. Pitx3 is crucial for maintaining normal function and preserving the specific molecular identities of postnatal mDA neurons, is required for proper survival of an mDA neuron subset in prenatal and postnatal mouse brains. The transcriptomic analysis strongly highlights the direct involvement of ALLO in regulating genes highly associated with neurogenesis, myelination, and cell repair.

[0364] ALLO promotes efficient mitochondrial respiration through multiple mechanisms involving electron transport chain function and coupling efficiency. The viability and efficiency of the bioenergetic system is a primary determinant of synaptic and brain function. Compromised mitochondrial bioenergetics is among the earliest pathogenic events in neonatal HI insult. HI insult in neonates induces depletion of high energy phosphates. HI causes mitochondrial membrane permeabilisation which results in membrane transporter deficits, accumulation of sodium and calcium within the cell, subsequent depolarization, and release of excitotoxic levels of glutamate. In premature infants, mitochondria are even less efficient at buffering influxed calcium, which leads to mitochondrial swelling few hours following the HI insult. Calmodulin-dependent activation of nitric oxide synthase generates nitric oxide which in turn results in the blockage of mitochondrial respiratory complex IV and formation of peroxy nitrites. In addition, calcium overload coincides with upregulated superoxide production and accumulation of hydroxyl and peroxynitrite radicals leads to lipid peroxidation, protein nitrosylation, and DNA damage which is more acute in the immature brain given its limited antioxidant capacity. The ALLO treated HI group, had a significantly lower accumulation of both ROS and lipid peroxids.

[0365] HI has been shown to damage mitochondrial ultrastructure. HI causes damage to the mitochondrial membrane and cristae causing vacuolization. HI induces perturbation of mitochondrial dynamics (fission, fusion) and mitophagy. In HI+ALLO group, there was a significant preservation of mitochondrial ultrastructure. Our findings are in agreement with previous studies which reported that ALLO suppressed mitochondrial uncoupling, increased mitochondrial respiration, reduced lipid peroxidation, and improved mitochondrial efficiency in Alzheimer’s mouse model.

[0366] Biogenesis is required for the increase in mitochondrial mass. This process is dependent on the fission of existing mitochondria followed by a coordinated program of transcription which is regulated by Peroxisome proliferator-activated receptor gamma coactivator la (PGC-la). PGC-la 45

[0367] 45803005.1 is often expressed at high levels within neurons to meet the high demand for energy production. PGC-la is the master regulator of mitochondrial biogenesis, respiratory capacity, oxidative phosphorylation, and fatty B oxidation. PGC-la plays a role in the formation and maintenance of synapses during development. PGC-la is significantly suppressed in the HI group, decreasing mitochondrial biogenesis and subsequently decreasing mitochondrial DNA, mitochondria volume, and size as shown in our data (FIG.8A and 8B). After ALLO treatment, there was a significant increase in mitochondrial biogenesis as evidenced by the increase in mitochondrial DNA copy number to a level comparable to RA group (FIG.8A-G). Pymvate dehydrogenase (PDH) converts pyruvate into acetyl-CoA, thus connecting glycolysis to the TCA cycle. PDH functions as a large protein complex (PDHC) and localizes to the mitochondria, and its activity is regulated by phosphorylation of the PDH-Ela subunit. Previous studies showed that PDH-E1 is downregulated under prolonged hypoxic conditions, which is similar to the findings in HI group. ALLO treatment to the HI group showed a significant increase in PDH-E1 level and was comparable to RA group (FIG.8H). This crucial effect of ALLO treatment offers a new avenue of intervention for immature infants who suffer lifelong disabilities due to HI insult.

[0368] Summary of Results:

[0369] Human ALLO plasma levels at birth were 9 times higher in term infants compared to extreme preterm infants. ALLO-treated HI mice showed significant improvement in neuroregeneration, myelination, motor function, coordination, learning and memory, along with significant attenuation of neuro-inflammation. RNA sequencing analysis showed that ALLO-treated HI group has a significant downregulation of inflammatory responsive genes, significant reduction of glutamate excitotoxicity, improved neurogenesis and neural repair restoring neural integrity and cognitive function. Mitochondrial ultrastructure in hippocampal neurons, using electron microscopy, showed small, fragmented mitochondria, with loss of mitochondrial membrane and cristae in HI group, while ALLO protected mitochondrial ultrastructure after HI exposure. ALLO significantly reversed deficits in biogenesis and key mitochondrial enzyme activity and reduced lipid peroxidation in HI.

[0370] The data shows that ALLO is a unique neuro-therapeutic natural steroid in HI injury. ALLO treatment can lead to a decrease in CP incidence and severity; by promoting neurogenesis, neuroregeneration, oligodendrogenesis and reducing neuro-inflammation and glutamate excitotoxicity and preserving mitochondrial structure and function

[0371] During gestation, ALLO expression peaks up to 10-fold normal levels in the last part of the third trimester. Premature infants born at the end of the 2ndtrimester or early part of the third trimester, have low ALLO brain levels compared to full-term infants. In addition, their de novo 46

[0372] 45803005.1 synthesis of ALLO in the brain is reduced due to the loss of the placental supply of progesterone that would have been converted to ALLO in the brain. Postnatal hypoxia-ischemia insult (HI) will further reduce premature infants’ brain capacity to produce ALLO.

[0373] ALLO improves myelination, thus decreasing white matter brain injury. Myelin generation is dependent on high levels of cholesterol, and synthesis of cholesterol requires the expression of HMG-CoA reductase. ALLO increases HMG-CoA reductase expression to meet the demand for the cholesterol required for myelin membrane growth. ALLO-induced neurogenesis and survival of newly generated neurons. Behavioral outcomes are linked to the unique properties of immature neurons in the dentate gyrus to associative learning across time and memory functions of the entire hippocampal circuit. Newly generated neurons have increased synaptic plasticity which is critical for neural network formation;

[0374] In these examples, administering of ALLO can replenish the stores and target many of the mechanisms that lead to the development of brain injury in premature infants. As shown in the examples, ALLO administration in neonate mice with HI injury showed a significant decrease in many downstream harmful effects of (HI). ALLO treatment in the neonate mouse model showed significant improvement in neuronal regeneration, a significant decrease in neuronal apoptosis, and improved myelination. ALLO treatment also showed a significant decrease in microglial activation thus preserving neuronal integrity and circuitry. ALLO might influence leukocyte biology and associated neuroinflammatory mechanisms independent of its neuro-regenerative properties.

[0375] In conclusion, the data showed that ALLO is a unique natural neuro-steroidal therapeutic for HI injury in neonates. ALLO treatment partially reverses the devastating HI effects in the immature brain and has the potential to decrease CP incidence and severity by promoting neurogenesis, neuro-regeneration, and oligodendrogenesis, while reducing neuro-inflammation and glutamate excitotoxicity. ALLO has potent anti-oxidant effects, reversing HI damage to mitochondrial ultrastructure, biogenesis, and bioenergetics targeting many of the molecular mechanisms involved in HI.

[0376] 47

[0377] 45803005.1

Claims

1. CLAIMS2.We claim:

1. A method of reducing hypoxia induced damage comprising administering to the individual in need thereof an effective amount of alloprcgnanalonc or analogue or derivative thereof having equivalent activity.

2. The method of claim 1 comprising administering allopregnanalone.

3. The method of claim 1 wherein the individual is a pre-term or oxygen deprived infant.

4. The method of claim 1 wherein the individual has had a stroke, cardiac event, or oxygen deprivation.

5. The method of any one of claims 1-4 wherein the allopregnanalone is administered by injection, infusion, or orally.

6. The method of claim 1 wherein the allopregnanalone is administered multiple times.

7. A dosage formulation for use in the method of any of claims 1-6.10.4845803005.1

Images