WO2014066442A2 - Methods of managing childhood cerebral injury - Google Patents

Description

METHODS OF MANAGING CHILDHOOD CEREBRAL INJURY

GOVERNMENTS SUPPORT

This invention was made with government support under Grant U01NS062676-01 awarded by the National Institutes of Health. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Numbers

61/717,812, 61/717,821, and 61/717,839 all filed October 24, 2012, all hereby incorporated by reference in their entirety.

BACKGROUND

Stroke is a cause of morbidity in childhood. Traditional adult stroke risk factors such as hypertension, diabetes, smoking, and hypercholesterolemia are uncommon in children. Children who have had a stroke typically have long-term neurological problems.

Pediadic patients with stroke are often evaluated by vascular imaging of the head and neck, echocardiogram, and thrombophilia studies. Treatment varies, but often consists of antithrombotic therapy, maintaining normal body temperature with acetaminophen, cooling blankets, and fluids. Clincal guidelines specifically recommend against treatment of neonates with first arterial ischemic stroke without evidence of an ongoing cardioembolic source. See Monagle et al. Antithrombotic therapy in neonates and children: American college of chest physicians evidence-based clinical practice guidelines (8th Edition) Chest. 2008;133(6 Suppl):887S-968S.

The steroid hormone progesterone has been reported to have neuroprotective effects in several stroke models. See Sayeed et al, Restor Neurol Neurosci 2007; 25: 151-159, Ishrat et al, Exp Neurol 2010; 226: 183-190, Ishrat et al, Brain Res 2009; 1257: 94-101.

SUMMARY

Described herein are methods for treating or reducing the risks and/or effects of cerebral injury due to ischemia or stroke. In certain embodiments, this disclosure relates to methods of treating or preventing seizures or brain injury comprising administering an effective amount progesterone or analog to human patient, e.g., a subject less than 18 years old. In certain embodiments, the subject is diagnosed with congenital heart disease, sickle cell disease, hematologic abnormalities, cerebral arteriopathy, or vascular malformations. In certain embodiments, the human subject has had an ischemic or hemorrhagic stroke. In certain embodiments, the subject is less than 6 months, 1 year, or 6 years in age.

In certain embodiments, the human subject is diagnosed with an aneurysm. In certain embodiments, the human subject is diagnosed with sickle cell disease. In certain embodiments, the human subject is diagnosed with a cerebral vascular abnormality. In certain embodiments, the cerebral vascular abnormality is cervicocephalic arterial dissection, moyamoya, vasculitis, sickle cell disease arteriopathy, post varicella angiopathy, and idiopathic focal cerebral arteriopathy. In certain embodiments, the human subject is diagnosed with a genetic syndrome associated with vascular abnormalities. In certain embodiments, the genetic syndrome is neurofibromatosis type 1, Alagille syndrome, Williams Syndrome, PHACES syndrome, and trisomy 21.

In certain embodiments, the disclosure relates to methods disclosed herein wherein the progesterone or analog is administered in combination with another therapeutic agent.

DETAILED DISUSSION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the

publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of immunology, medicine, surgery, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

As used herein, the term "combination with" when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof.

As used herein, "subject" refers to any animal, typically a human patient, livestock, or domestic pet.

As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression. Pediatric Cereberal Vascular Abnormalities

Congenital heart disease, sickle cell disease, hematologic abnormalities, cerebral arteriopathy, and vascular abnormalities are risk factors for stroke in pediatric patients. Vascular abnormalities such as aneurysms are the major cause of hemorrhagic stroke in children. Vascular anomalies are typically present symptoms such as headaches and/or seizures before a stroke has occurred. Sickle cell disease is another common risk factor for pediatric stroke. Many patients have a clinically evident stroke and/or a silent stroke by age 20.

Ischemic stroke typically refers to arterial ischemic stroke and cerebral sinovenous thrombosis resulting in venous infarction. Cerebral arteriopathy is typically diagnosed after imaging vascular abnormalities such as cervicocephalic arterial dissection, moyamoya, vasculitis, sickle cell disease arteriopathy, post varicella angiopathy, and idiopathic focal cerebral arteriopathy.

Several genetic syndromes are associated with cervical and cerebral arteriopathy such as neurofibromatosis type 1, Alagille syndrome, Williams Syndrome, PHACES syndrome, and trisomy 21.

Hemorrhagic stroke typically refers to spontaneous intracerebral hemorrhage and non-traumatic subarachnoid hemorrhage.

In certain embodiments, this disclosure relates to methods of treating or preventing seizures or brain injury comprising administering an effective amount progesterone or analog to human patient, e.g., a subject less than 18 years old. In certain embodiments, the human subject has had an ischemic or hemorrhagic stroke. In certain embodiments, the subject is less than 1 month, 6 months, 1 year, or 6 years in age.

In certain embodiments, the human subject is diagnosed with an aneurysm. In certain embodiments, the human subject is diagnosed with sickle cell disease. In certain embodiments, the human subject is diagnosed with a cerebral vascular abnormality. In certain embodiments, the cerebral vascular abnormality is cervicocephalic arterial dissection, moyamoya, vasculitis, sickle cell disease arteriopathy, post varicella angiopathy, and idiopathic focal cerebral arteriopathy. In certain embodiments, the human subject is diagnosed with a genetic syndrome associated with vascular abnormalities. In certain embodiments, the genetic syndrome is neurofibromatosis type 1, Alagille syndrome, Williams Syndrome, PHACES syndrome, and trisomy 21.

Described herein are methods for treating or reducing the risks and/or effects of cerebral injury due to ischemia or stroke. The methods involve administering a therapeutically effective amount of a progesterone/analog (or a pharmaceutically acceptable salt, ester or prodrug thereof), and optionally therapeutically effective amount of a second therapeutic agent.

In certain embodiments, the disclosure relates to methods of treating cerebral injury due to ischemia comprising administering an effective amount of progesterone or analog to a human subject diagnosed with ischemic or hemorrhagic stroke.

In certain embodiments, the administration is after cerebral arteriography, angiography, computerized tomography scan, and/or magnetic resonance imaging.

In certain embodiments, the progesterone or analog is administered less than 6, 8, or 24 hours after ischemic onset.

In certain embodiments, the progesterone or analog is administered more than 24 hours after ischemic onset.

In certain embodiments, a first injection is provided by less than 24 hours after diagnosis and a second injection is provided more than 24 hours after ischemic onset.

In certain embodiments, the progesterone/analog is administered daily for more than two days.

In certain embodiments, the subject exhibits one or more seizures as a symptom of stroke.

In certain embodiments, the subject is diagnosed with a viral or bacterial infection. In accordance with some embodiments, the methods may comprise administering more than one progesterone/analog. In such embodiments, the progesterone/analog may be administered in separate compositions by the same or different routes of administration (sequentially or substantially simultaneously) or in a single composition comprising two or more progesterone/analog(s). In accordance with some embodiments, the methods may further comprise one or more further administrations of the same or different

progesterone/ analog(s) .

In accordance with any of the embodiments discussed herein, the composition(s) may be administered once or several times a day, and the duration of the treatment may be for a period of about 1, 2, 3, 4, 5, 6, 7 days or more. In specific embodiments, a daily dose can be administered in a single administration or in multiple administrations.

As discussed above, the methods may comprise administering the composition(s) by any route of administration, including, but not limited to, systemic injection, orally, nasally, parenterally, intravenously, intraperitoneally, intramuscularly, transdermally, buccally, subcutaneously, and/or topically. In some embodiments, a subject contemplated to undergo therapy provided herein may be diagnoses with any type of cerebral injury or disease, traumatic brain injury (TBI), ischemic CNS injury, spinal cord injury, ischemic stroke, or anterior optic nerve ischemic injury. Thus, the subject may be a subject suffering from or at risk of developing ischemic injury, including a subject suffering from or at risk of developing any of these or similar conditions.

In some embodiments, the subject is at risk of ischemic injury, such as ischemic stroke, such as a subject suffering from atherosclerosis or with a family history of heart disease, or smoker, or over age 55. In other embodiments, the subject is at risk of another ischemic injury, such as a subject whose work, status or lifestyle places him/her at risk for ischemic injury, such as CNS injury or TBI, including athletes and soldiers.

In accordance with specific embodiments, a patient at risk of ischemic injury is considered to be a subject who will be administered a thrombolytic agent, and is

administered a progesterone/analog in order to reduce the risks of treatment with a thrombolytic agent. In such embodiments, the progesterone/analog may be administered "prophylactically," i.e., before the onset or occurrence of ischemic injury or stroke, such as being administered as part of an ongoing therapeutic regimen.

For example, in specific embodiments, the methods and compositions described herein may decrease edema in affected tissue, such as by at least about 15%, about 15% to 30%, about 30% to 45%, about 45% to 60%, about 60% to 80%, or about 80% to 95%, or greater. Additionally or alternatively, in specific embodiments, the methods and

compositions described herein may reduce neuronal cell death, such as by increasing neuronal survival in treated patients by at least about 10%>, about 10%> to 20%>, 20%> to 30%>, 30%o to 40%o, 40%o to 60%o, 60%> to 80%>, or greater, compared to a patient in a control group. Combination therapies

In certain embodiments, the disclosure relates to methods disclosed herein wherein the progesterone or analog is administered in combination with another therapeutic agent.

In certain embodiments, the disclosure relates to methods disclosed herein wherein the progesterone or analog is administered in combination with an antithrombotic therapy. Antithrombotic therapy includes both antiplatelet (e.g. aspirin) and anticoagulant (e. g. unfractionated heparin, low molecular weight heparin, and warfarin) medications.

In certain embodiments, the disclosure relates to methods disclosed herein wherein the progesterone or analog is administered in combination with an anticoagulant. In certain embodiments, the disclosure relates to methods disclosed herein wherein the progesterone or analog is administered in combination with unfractionated heparin, low molecular weight heparin, or warfarin.

In certain embodiments, the low molecular weight heparin is fractionated, e.g., enoxaparin or salt thereof. In certain embodiments, subcutaneous injection of enoxaparin is about 1 mg/kg to 2 mg/kg every 12h. Dosing adjustments and coagulation monitoring, an anti-Xa level is contemplated for in the following situations: bleeding complications, body weight (less than 50 kg, greater than 150 kg), renal insufficiency (creatinine clearance less than 40 mL/min), pregnancy, acute burns, and recurrent thrombosis despite drug treatment.

In certain embodiments, IV administration of unfractionated heparin is an inital bolus 60 Units/kg with tPA and related fibrinolytics. In certain embodiments, initial infusion may be 15 Units/kg/hr to 1200 units/hr and tPA or GPIIb/IIIa receptor antagonist 12 Units/kg/hr to 1000 units/hr. In certain embodiments, initial infusion may be 18

Units/kg/hr to 1800 units/hr.

In certain embodiments, the disclosure relates to methods disclosed herein wherein the progesterone or analog is administered in combination with an antiplatelet.

Contemplated antiplatelets include aspirin, adenosine diphosphate (ADP) receptor inhibitors, clopidogrel, prasugrel, ticagrelor, ticlopidine, phosphodiesterase inhibitors, cilostazol, glycoprotein IIB/IIIA inhibitors, abciximab, eptifibatide, tirofiban, adenosine reuptake inhibitors, dipyridamole, thromboxane inhibitors, thromboxane synthase inhibitors, thromboxane receptor antagonists, terutroban.

In certain embodiments, the disclosure relates to methods disclosed herein wherein the progesterone or analog is administered in combination with an thrombolytic agent. In certain embodiments, the disclosure relates to methods disclosed herein wherein the progesterone or analog is administered in combination with tissue plasminogen activator (tPA), streptokinase, urokinase, and/or desmoteplase.

Managing adult stroke may involve using intravenous (IV) tissue plasminogen activator (tPA) for patients 18 years and older who meet strict inclusion criteria. See N Engl J Med, 1995, 333(24): 1581-7. Time to presentation is a major limiting factor since IV thrombolysis typically is started within 4 hours of symptom onset. In certain embodiments, the methods disclosed herein may be performed in combination with a thrombolytic agent. As used herein, the term "thrombolytic agent" includes but is not limited to thrombolytic agents such as tissue plasminogen activator (tPA), streptokinase, urokinase and

desmoteplase, which are used (or being developed) to treat ischemic injury. tPA is a serum protease acting specifically on serum plasminogen. tPA also is known as alteplase and is available commercially as Activase® (Genentech). The tPA in Activase® is a recombinant glycoprotein having 527 amino acid residues. Activase® is provided as a lyophilized powder comprising tPA, L-arginine, phosphoric acid, and polysorbate 80, and is reconstituted with sterile water for injection prior to use. tPA can be isolated from animal sources (including human sources) or produced recombinantly.

Streptokinase is a protein that acts to convert plasminogen to plasmin. Streptokinase is commercially available in some countries as Streptase® (Aventis Behring). Streptase® is provided as a lyophilized white powder comprising streptokinase, cross-linked gelatin polypeptides, sodium L-glutamate, sodium hydroxide and albumin, and is reconstituted and diluted for injection prior to use. The streptokinase in Streptase® is derived from streptococci bacteria. Streptokinase also can be produced recombinantly.

Urokinase is a protein that converts plasminogen to plasmin. Urokinase is commercially available in some countries as Abbokinase® (Abbott) and Kinlytic™

(Microbix). Both of these commercial compositions contain the low molecular weight form of urokinase, consisting of an A chain of 2,000 daltons linked by a sulfydryl bond to a B chain of 30,400 daltons. Abbokinase® and Kinlytic™ are provided as a lyophilized white powder containing 250,000 IU urokinase per vial, mannitol, albumin and sodium chloride. Prior to use, both of these commercial compositions are reconstituted with sterile water to produce from 50,000 to 750,000 International Units (IU) of urokinase activity per mL, 0.5% mannitol, 5% albumin and 1% sodium chloride. The urokinase in Abbokinase® and Kinlytic™ is obtained from human neonatal kidney cells grown in tissue culture. Urokinase also can be produced recombinantly.

Desmoteplase is a thrombolytic enzyme that converts plasminogen into plasmin. Desmoteplase is currently undergoing clinical trials as DIAS-3 and DIAS-4 (Lundbeck), which are recombinantly produced.

Progesterone and analogs

Progesterone and analogs for methods disclosed herein may be prodrugs, metabolites and derivatives of progesterone; analogues of progesterone metabolites or derivatives, and other steroid compounds that exhibit in vivo efficacy in the methods described herein. Progesterone is also known as D4-pregnene-3,20-dione; delta-4- pregnene-3,20-dione; or pregn-4-ene-3,20-dione. In certain embodiments, progesterone analogs may be derivative steroids. Exemplary steroids include those described herein and in U.S. publication 2011/0263553 and PCT publication WO 2009/108804. For example, steroids useful in the methods and compositions described herein include those set forth in WO 2010/088409 and stereoisomers thereof.

In some specific embodiments, the analog is allopregnanolone, a principal metabolite of progesterone. Allopregnanolone is a potent, positive modulator of CNS GABAA receptor functions. Orchinik et al, Brain Res, 1994, 646(2): 258-66; Paul et al, Faseb J, 1992, 6(6): 2311-22. Allopregnanolone also is known as 3 -hydroxy-5a-pregnan- 20-one or 3a,5a-tetrahydroprogesterone.

Progesterone analogs include, for example, derivatives of progesterone such as 5-a- dihydroprogesterone, 6-dehydro-retroprogesterone (dydrogesterone), hydroxyprogesterone caproate, levonorgestrel, norethindrone, norethindrone acetate; norethynodrel, norgestrel, medroxyprogesterone, chlormadinone, and megestrol.

Progesterone analogs also includes, but is not limited to, modifications that produce 17a-OH esters of progesterone, as well as, modifications that introduce 6-a-methyl, 6- methyl, 6-ene, and 6-chloro substituents onto progesterone, and/or 19-norprogesterones. Further, non-limiting examples, of synthetic progesterone analogs include, norethindrone (Micronor®), norgestrel (Ovrette®), levonorgestrel (Norplant®; with ethinyl estradiol; Alesse®, Nordette®), gestodene, medroxyprogesterone acetate (Provera®), promegestone, nomegestrol acetate, lynestrenol and dienogest.

In some embodiments, the progesterone analog is selected from the group consisting of norethynodrel, norethidrone acetate, medroxyprogesterone, medroxyprogesteron 17- acetate, levonorgestrel, dydrogesterone, hydroxyprogesterone caproate, norethidrone, gestodene, nomegestrol acetate, promegestone, dienogest, chlormadinion, megestrol, megestrol acetate, and/or mixtures thereof.

In some embodiments the progesterone analog is selected from the group consisting of 5-a-dihydroprogesterone, medroxyprogesterone, dydrogesterone, and progesterone and/or mixtures thereof.

The progesterone or analog for uses herein can be administered as a "prodrug." As used herein, a "prodrug" is an inactive or less active form of the neuroprotective steroid that, once administered, is metabolized in vivo into an active or more active form. In some embodiments, the prodrug is oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, and/or dephosphorylated to produce the active drug form. In other embodiments, the prodrug is a compound that has at least one biologically labile protecting group on a functional moiety of the compound, which protecting group is removed in vivo. A number of prodrug ligands are described in Jones and Bischofberger, Antiviral Research, 1995, 27: 1-17. In specific embodiments, alkylation, acylation or other lipophilic modification of the steroid will increase the stability of the steroid. Examples of substituent groups that can replace one or more hydrogens on the steroid are alkyl, aryl, steroids, carbohydrates, including sugars, 1,2-diacyl glycerol and alcohols.

In certain embodiments, the progesterone analog for uses disclosed herein is one disclosed in WO 2009/108804. One example, of a progesterone analog for uses herein is 1- (3-(hydroxyimino)-10,13-dimethyl-2,3,6,7,8,9,10,l 1,12,13, 14,15, 16,17-tetradecahydro-lH- cyclopenta[a]phenanthren-17-yl)ethanone optionally substituted with one or more substituents or derivative optionally substituted with one or more substituents.

In particular embodiments, the progesterone analog is represented by Formula I:

Formula I

wherein X is O, N or S;

Y is O, N or S;

R¹, R², R⁵ and R⁶ are independently hydrogen, alkyl, halogen, hydroxyl cycloalkyl, cycloalkenyl, alkenyl, alkynyl, aryl, alkylaryl, arylalkyl, heterocyclic, heteroaryl, amino, thiol, alkoxy, sulfide, nitro, cyano, azide, sulfonyl, acyl, carboxyl, an ester, an amide, carbamate, carbonate, an amino acid residue or a carbohydrate;

R⁴ is hydrogen or alkyl; or R⁴ and R⁷ together form a double bond;

R is hydrogen, optionally substituted acyl, a residue of an amino acid, a

carbohydrate, -OR¹¹, -NRⁿR¹² or R³ is absent;

R⁷ is hydrogen or is absent, or R⁷ together with R⁴ forms a double bond;

R is hydrogen, optionally substituted acyl, a residue of an amino acid, a

carbohydrate, -OR¹¹, -NRⁿR¹² or R⁸ absent;

R⁹ is hydrogen or alkyl; or R⁹ and R¹⁰ together form a double bond;

R¹⁰ is hydrogen or is absent, or R¹⁰ together with R⁹ forms a double bond; R is the residue of an amino acid, a carbohydrate or an optionally substituted ester or

a substituted acyl;

R 12 is hydrogen or alkyl; and

the dotted line indicates the presence of either a single bond or a double bond, wherein the valences of a single bond are completed by hydrogens,

provided that at least one of XR³R⁷ or YR⁸R¹⁰ is not =0 or OH, and that if the dotted line between

C4 and C5 or between C5 and C6 represents a double bond then the other dotted line between C4 and C5 or between C5 and C6 represents a single bond.

In certain embodiments, the progesterone analog for uses herein may be those disclosed in U.S. Patent Application Number 2013/021785, hereby incorporated by reference in its entirety.

In certain embodiments, the progesterone analog for uses herein are compounds of Formula II

Formula II

or esters, prodrugs, or salts thereof wherein

X is O, N-OR¹⁰, or N-OCR¹R²OPZ(OR³)₂;

Y is O, N-OR¹⁰, or N-OCR¹R²OPZ(OR³)₂; provided at least one X or Y is N-

OCR^OPOCOR³)^

Z is =0 or =S;

R¹ is hydrogen, alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R¹ is optionally substituted with one or more, the same or different, R⁴;

R is hydrogen, alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R is optionally substituted with one or more, the same or different, R⁴; or R and R form a carbocyclic ring;

each R is independently and individually phosphate ion, hydrogen, - CH₂0(C=0)alkyl, or a group selected from alkyl, alkanoyl, and formyl further substituted with one or more, the same or different, alkyl, amino, hydroxyl, thiol, halogen, aryl, carbocyclyl, or heterocyclyl, wherein R is optionally substituted with one or more, the same or different, R⁴;

R⁴ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁴ is optionally substituted with one or more, the same or different, R⁵;

R⁵ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, tertbutyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl- N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N- dimethylcarbamoyl, Ν,Ν-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfmyl, ethylsulfmyl, mesyl, ethylsulfonyl, methoxycarbonyl,

ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, Ν,Ν-dimethylsulfamoyl, N,N- diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl;

R¹⁰ is hydrogen or a group selected from alkyl and formyl further substituted with one or more, the same or different, alkyl, amino, hydroxyl, thiol, halogen, aryl, carbocyclyl, or heterocyclyl, wherein R¹⁰ is optionally substituted with one or more, the same or different, R²⁰;

20

R is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R 20 is optionally

30

substituted with one or more, the same or different, R ;

30

R is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R 30 is optionally substituted with one or more, the same or different, R⁴⁰; and

R⁴⁰ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, Ν,Ν-dimethylcarbamoyl, N,N- diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfmyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N- ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

Progesterone or analogs can be administered as a pharmaceutically acceptable salt or ester.

As used herein, "alkyl" means a noncyclic straight chain or branched, unsaturated or saturated hydrocarbon such as those containing from 1 to 10 carbon atoms, while the term "lower alkyl" or "Ci_₄alkyl" has the same meaning as alkyl but contains from 1 to 4 carbon atoms. The term "higher alkyl" has the same meaning as alkyl but contains from 7 to 20 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-septyl, n-octyl, n-nonyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an "alkenyl" or "alkynyl", respectively). Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2- pentenyl, 3 -methyl- 1-butenyl, 2-methyl-2-butenyl, 2,3- dimethyl-2-butenyl, and the like; while representative straight chain and branched alkynyls include acetylenyl, propynyl, 1 - butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3- methyl- 1-butynyl, and the like.

Non-aromatic mono or polycyclic alkyls are referred to herein as "carbocycles" or

"carbocyclyl" groups. Representative saturated carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated carbocycles include cyclopentenyl and cyclohexenyl, and the like.

"Heterocarbocycles" or heterocarbocyclyl" groups are carbocycles which contain from 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur which may be saturated or unsaturated (but not aromatic), monocyclic or polycyclic, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen

heteroatom may be optionally quaternized. Heterocarbocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,

tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like. "Aryl" means an aromatic carbocyclic monocyclic or polycyclic ring such as phenyl or naphthyl. Polycyclic ring systems may, but are not required to, contain one or more non- aromatic rings, as long as one of the rings is aromatic.

As used herein, "heteroaryl" refers an aromatic heterocarbocycle having 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including both mono- and polycyclic ring systems. Polycyclic ring systems may, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic. Representative heteroaryls are furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. It is contemplated that the use of the term "heteroaryl" includes N-alkylated derivatives such as a l-methylimidazol-5-yl substituent.

As used herein, "heterocycle" or "heterocyclyl" refers to mono- and polycyclic ring systems having 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom. The mono- and polycyclic ring systems may be aromatic, non-aromatic or mixtures of aromatic and non-aromatic rings. Heterocycle includes heterocarbocycles, heteroaryls, and the like.

"Alkylthio" refers to an alkyl group as defined above attached through a sulfur bridge. An example of an alkylthio is methylthio, (i.e., -S-CH₃).

"Alkoxy" refers to an alkyl group as defined above attached through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i- propoxy, n-butoxy, s-butoxy, t-butoxy, n- pentoxy, and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy, n-propoxy, i- propoxy, n-butoxy, s-butoxy, t-butoxy.

"Alkylamino" refers an alkyl group as defined above attached through an amino bridge. An example of an alkylamino is methylamino, (i.e., -NH-CH₃).

"Alkanoyl" refers to an alkyl as defined above attached through a carbonyl bride (i.e., -(C=0)alkyl).

"Alkylsulfonyl" refers to an alkyl as defined above attached through a sulfonyl bridge (i.e., -S(=0)₂alkyl) such as mesyl and the like, and "Arylsulfonyl" refers to an aryl attached through a sulfonyl bridge (i.e., - S(=0)₂aryl).

"Alkylsulfmyl" refers to an alkyl as defined above attached through a sulfmyl bridge (i.e. -S(=0)alkyl). The term "substituted" refers to a molecule wherein at least one hydrogen atom is replaced with a substituent. When substituted, one or more of the groups are "substituents." The molecule may be multiply substituted. In the case of an oxo substituent ("=0"), two hydrogen atoms are replaced. Example substituents within this context may include halogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, - NRaRb, -NRaC(=0)Rb, -NRaC(=0)NRaNRb, -NRaC(=0)ORb, - NRaS0₂Rb, -C(=0)Ra, - C(=0)ORa, -C(=0)NRaRb, -OC(=0)NRaRb, -ORa, -SRa, -SORa, - S(=0)₂Ra, -OS(=0)₂Ra and -S(=0)₂ORa. Ra and Rb in this context may be the same or different and independently hydrogen, halogen hydroxyl, alkyl, alkoxy, alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl.

The term "optionally substituted," as used herein, means that substitution is optional and therefore it is possible for the designated atom to be unsubstituted.

As used herein, "salts" refer to derivatives of the disclosed compounds where the parent compound is modified making acid or base salts thereof. Examples of salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkylamines, or dialkylamines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. In preferred embodiment the salts are conventional nontoxic

pharmaceutically acceptable salts including the quaternary ammonium salts of the parent compound formed, and non-toxic inorganic or organic acids. Preferred salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; 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, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

As used herein, the term "derivative" refers to a structurally similar compound that retains sufficient functional attributes of the identified analog. The derivative may be structurally similar because it is lacking one or more atoms, substituted, a salt, in different hydration/oxidation states, or because one or more atoms within the molecule are switched, such as, but not limited to, replacing an oxygen atom with a sulfur or nitrogen atom or replacing an amino group with a hydroxyl group or vice versa. The derivative may be a prodrug. Derivatives may be prepare by any variety of synthetic methods or appropriate adaptations presented in synthetic or organic chemistry text books, such as those provide in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007) Michael B. Smith or Domino Reactions in Organic Synthesis, Wiley (2006) Lutz F. Tietze hereby incorporated by reference.

Pharmaceutical Compositions

In certain embodiments, progesterone/analog and optionally a second therapeutic agent can be administered in the same or separate pharmaceutical compositions. The pharmaceutical composition(s) can include a pharmaceutically acceptable carrier or diluent as is known in the art. For example, the pharmaceutically acceptable carrier may be inorganic or organic, solid or liquid, a particulant, a powder, a solution, a suspension, an emulsion, or micronized form. Suitable vehicles and their formulation are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES (16th Ed.) (A. Osol, Ed. Mack Publishing Co., Easton Pa., 1980).

In some embodiments, one or more of the compositions is a solution or suspension for parenteral, intramuscular, intradermal, subcutaneous, or topical application. Such a composition can include excipients known for use in such dosage forms, such as a sterile diluent such as water, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as

ethylenediammetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. In some embodiments, one or more of the compositions is a parental preparation and is optionally provided in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

In specific embodiments, a composition suitable for parental administration comprises a sterile aqueous preparation of a thrombolytic agent and progesterone or analog, which can be isotonic with the blood of the recipient patient.

In some embodiments, one or more of the compositions is in the form of a nasal spray formulation, and may comprise one or more preservative agents and isotonic agents. Such formulations may be adjusted to a pH and isotonic state compatible with the nasal mucous membranes.

In some embodiments, one or more of the compositions is in a form suitable for administration as an aerosol by inhalation. Such compositions may comprise solid particles of the active agent(s) suitable for inhalation. The composition may be provided in a small chamber and nebulized. Nebulization may be accomplished by compressed air or by ultrasonic energy to form a plurality of liquid droplets or solid particles comprising the compounds or salts.

In some embodiments, one or more of the compositions is an oral composition that includes an inert diluent or an edible carrier. In specific embodiments, the composition is enclosed in gelatin capsules or compressed into tablets. For example, the therapeutic agent(s) can be incorporated with excipients and prepared in the form of tablets, troches or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials suitable for use in such dosage forms are known in the art and can be included as part of the composition.

In some embodiments, one or more of the compositions is a controlled release composition. Controlled release preparations may be prepared using polymers to complex or absorb the therapeutic agent(s) to be delivered. The controlled delivery may be exercised by selecting appropriate macromolecules (for example, polyesters, polyamino acids, hydrogels, poly(lactic acid), polyvinyl pyrrolidone, ethylene -vinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate). The rate of drug release may also be controlled by altering the concentration of such macromolecules. Alternatively, it is possible to entrap the therapeutic agents in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethyl cellulose or gelatin-microcapsules or poly(methylmethacrylate)

microcapsules, respectively, or in a colloid drug delivery system, for example, liposomes, albumin, microspheres, microemulsions, nanoparticles, nanocapsules, or in

macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980).

In some embodiments, one or more of the compositions comprises a liposomal formulation. When the compounds or salts thereof are an aqueous-soluble salt, using conventional liposome technology, the same may be incorporated into lipid vesicles. In such an instance, due to the water solubility of the compound or salt, the compound or salt will be substantially entrained within the hydrophilic center or core of the liposomes. The lipid layer employed may be of any conventional composition and may either contain cholesterol or may be cholesterol-free. When the compound or salt of interest is water- insoluble, again employing conventional liposome formation technology, the salt may be substantially entrained within the hydrophobic lipid bilayer that forms the structure of the liposome. In either instance, the liposomes that are produced may be reduced in size, as through the use of standard sonication and homogenization techniques. The liposomal formulations containing the progesterone analogue or salts thereof, may be lyophilized to produce a lyophilizate which may be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.

In some embodiments the progesterone or analog is provided in a composition that includes a therapeutically effective amount. For example, the effective amount may be present at a dose of about 0.1 ng to about 100 g per kg of body weight, about 10 ng to about 50 g per kg of body weight, from about 100 ng to about 1 g per kg of body weight, from about 1 ug to about 100 mg per kg of body weight, from about 1 ug to about 50 mg per kg of body weight, from about 1 mg to about 500 mg per kg of body weight, or from about 1 mg to about 50 mg per kg of body weight. In other embodiments, the effective amount administered to achieve a therapeutic effective dose is about 0.1 ng, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 500 mg per kg of body weight or greater.

In some embodiments, the progesterone is provided in a composition that includes an amount effective to achieve a serum level of progesterone/analog of about 100 ng/ml to about 2000 ng/ml, including about 100 ng/ml to about 1000 ng/ml, including about 200 ng/ml to about 450 ng/ml, such as about 350 ng/ml to about 450 ng/ml, as described, for example, in U.S. Patent 7,473,687.

In some embodiments, the second therapeutic agent is provided in a composition suitable for intravenous administration, or in a composition to be diluted prior to intravenous administration.

In some embodiments, the second therapeutic agent is provided in a composition that includes a therapeutically effective amount of thrombolytic agent, such as a

therapeutically effective amount in accordance with protocols approved by the U.S. Food and Drug Administration for the treatment of acute ischemic stroke or in clinical development for the treatment of ischemic stroke or other ischemic injury. For example, tPA may be provided in a composition formulated for intravenous administration to provide a dose of 0.9 mg/kg body weight of the patient, such as up to a total dose of 90 mg;

streptokinase may be provided in a composition formulated for intravenous administration, such as to provide a dose of from 1,500,000 to 2,400,000 IU; urokinase may be provided in a composition formulated for intravenous administration, such as to provide a dose of from 50,000 IU to 750,000 IU, including 500,000 or 300,000 IU; desmoteplase may be provided in a composition formulated for intravenous administration, such as to provide 90 μg/kg, such as up to 9,000 μg (or 9 mg).

EXAMPLES Progesterone Attenuates Acute Seizure and Brain Infarction by Modulating Pro- Inflammatory Cytokines in a Mouse Model of Neonatal Stroke

Neonatal stroke is among the top ten causes of childhood death and causes long- term or permanent disability and neurological deficits in survivors. In most cases, recurrent clonic seizure is the only clinical manifestation which worsens the stroke outcomes. P12 CD1 (mixed gender) mice underwent permanent unilateral right common carotid ligation (pUCCL) or sham surgery (n=10/group). Pups which showed seizure activity during the lh post-pUCCL were randomly assigned to receive PROG (8 mg/kg) or vehicle injections at 1, 3 and every 24h post-pUCCL for 6 days. Acute behavioral seizures (during the first 4h post- pUCCL), serum pro-inflammatory cytokines (IL-Ιβ, IL-6, TNFa at 6, 24 and 48h) and brain infarction (at day 7 by cresyl -violet staining) were assessed. Cytokine data were analyzed by repeated measures one-way ANOVA followed by LSD. For seizure and infarct data, a two-tailed unpaired t-test was employed.

Acute seizures were observed during the first 4h post-pUCCL in the vehicle group (90.8±11.77). PROG treatment significantly (P<0.05) reduced seizure occurrence

(58.4±8.97) by 35% compared to vehicle. Serum IL-Ιβ, IL-6 and TNFa were significantly higher (P<0.05) at 6 and 24h and remained elevated until 48h post-pUCCL in the vehicle group compared to sham. The PROG-treated group showed a significant (P<0.05) decrease in all pro-inflammatory cytokine levels at all time points. Further, pUCCL resulted in severe hemispheric damage (16.88±1.48) as evidenced by cresyl-violet staining at 7 days post- pUCCL. PROG treatment showed a significant (P<0.05) reduction (4.93±1.1) in infarct volume (~70%>) compared to vehicle. This data indicates that PROG reduces acute seizures and brain infarction following neonatal stroke.

Claims

CLAIMS What we claim:

1. A method of treating or preventing seizures comprising administering an effective amount progesterone or analog to a human subject less than 18 years old.

2. The method of Claim 1, wherein the human subject has had an ischemic or hemorrhagic stroke.

3. The method of Claim 1, wherein the subject is less than 6 months, 1 year, or 6 years in age.

4. The method of Claim 1, wherein the human subject is diagnosed with an aneurysm.

5. The method of Claim 1, wherein the human subject is diagnosed with sickle cell disease.

6. The method of Claim 1, wherein the human subject is diagnosed with a vascular abnormality.

7. The method of Claim 6, wherein the vascular abnormality is cervicocephalic arterial dissection, moyamoya, vasculitis, sickle cell disease arteriopathy, post varicella angiopathy, and idiopathic focal cerebral arteriopathy.

8. The method of Claim 1, wherein the human subject is diagnosed with a genetic syndrome associated with stroke.

9. The method of Claim 8, wherein the genetic syndrome associated with stroke is neurofibromatosis type 1 , Alagille syndrome, Williams Syndrome, PHACES syndrome, and trisomy 21.

10. The method of Claim 1, wherein the progesterone or analog is administered in combination with another therapeutic agent.