WO2007011926A2

WO2007011926A2 - Methods for treating and diagnosing complications of premature birth

Info

Publication number: WO2007011926A2
Application number: PCT/US2006/027808
Authority: WO
Inventors: Judah Folkman; Anne Hansen; Carmen Barnes
Original assignee: Children's Medical Center Corporation
Priority date: 2005-07-15
Filing date: 2006-07-17
Publication date: 2007-01-25
Also published as: AU2006269994A1; CA2614677A1; EP1906969A4; EP1906969A2; JP2009501729A; WO2007011926A3

Abstract

Methods and compositions for treating and/or preventing complications of preterm birth are provided. The methods involve administration of 2-methoxyestradiol, or an analog thereof, to a subject. Also provided are methods for diagnosing or predicting complications of preterm birth. The methods involve detection of 2- methoxyestradiol, or a precursor or metabolite thereof.

Description

METHODS FOR TREATING AND DIAGNOSING COMPLICATIONS OF

PREMA TURE BIRTH

BACKGROUND

[0001] While there has been a steady decrease in the mortality rate for profoundly preterm infants, their rate of survival with considerable morbidity is of great concern. A cornerstone of the care of the preterm infant is to try to mimic the in-utero environment regarding as many details as possible: thermoregulation, nutrition, acid/base balance and oxygenation to name a few.

[0002] Infants born at term are exposed to the anti-angiogenic effects of rising maternal 2ME2 levels, balanced by a host of other pro- and anti-angiogenic factors produced both by the mother, transmitted across the placenta, and produced by the placenta itself. Once the fetus is delivered, it no longer receives either maternal or placental pro- and anti-angiogenic factors, and its ability to produce and regulate its own factors is likely immature. Some common complications of prematurity such as intraventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP) and hemangioma formation suggest that angio genesis and vascular leak are occurring out of proportion to factors that oppose these processes.

[0003] This likely imbalance of pro- and anti-angiogenic factors is occurring during the ongoing development of the premature infant's immature vasculature, giving the balance more far reaching implications than in adults. Understanding the normal angiogenesis balance in utero offers the possibility of an eventual therapeutic intervention, supplementing anti- or pro- angiogenic factors to mimic the normal balance by gestational age.

SUMMARY

[0004] In one aspect, featured herein are novel prophylactic and therapeutic methods and compositions that permit safer and more effective treatment of premature infants for various disorders related to an imbalance of pro- and anti-angiogenic factors, such as intraventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP) and hemangioma formation. In one embodiment, the method comprises administering to the infant an effective amount of a 2-methoxyestradiol containing composition. [0005] In another aspect, the invention features methods for identifying pre-term infants at risk of developing complications due to their premature birth by assaying the infant or their mother's 2-methoxylestradiol levels, alone or in addition to assaying the levels of other biomarkers.

[0006] Formulations of 2-methoxyestradiol and other therapeutic agents, as well as kits, for the practice of the novel diagnostic and therapeutic methods are also described herein.

[0007] These embodiments of the present invention, other embodiments, and their features and characteristics will be apparent from the description, drawings, and claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIGURE 1 depicts maternal serum levels of 2ME2 during pregnancy and cord blood values for term babies (black font). Extrapolated maternal and cord blood levels for a 25-week old premature infant (in red font, parenthesis). Predicted change in cord blood levels of 2ME2, corresponding to fetal serum levels in utero, (blue line), versus predicted decrease in serum levels of 2ME2 for a 25-week old premature infant (red line), based on half-life measurements of 2ME2 in adults. N.B. numbers and lines in red are extrapolations.

[0009] FIGURE 2 depicts restoration of physiological levels of 2ME2 in the extremely premature infant to levels found in serum in gestationally age matched controls.

DETAILED DESCRIPTION

[0010] A. Definitions

[0011] For convenience, certain terms employed in the specification, examples, and appended claims are collected here. 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 invention belongs.

[0012] The articles "a" and "an" are used herein to refer to one or to more than one

(i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. [0013] The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included.

[0014] The term "including" is used to mean "including but not limited to".

"Including" and "including but not limited to" are used interchangeably. [0015] The term "control", as used herein with reference to the level of 2- methoxyestradiol (or a precursor or metabolite thereof), refers to the level of 2- methoxyestradiol (or a precursor or metabolite thereof) in at least one term infant or at least one preterm infant not suffering from a complication of premature birth. The control may reflect the average or mean value of the level of 2-methoxyestradiol (or a precursor or metabolite thereof) from two or more term infants or preterm infants not suffering from a complication of premature birth. The control may be a predetermined level of 2- methoxyestradiol (or a precursor or metabolite thereof) at term or at a given timepoint preterm. In one embodiment, the control is a graph or chart representing control levels of 2-methoxyestradiol (or a precursor or metabolite thereof) at a variety of gestational ages (preterm, term, etc.). In an exemplary embodiment, the control is a graph or chart representing control levels of 2-methoxyestradiol (or a precursor or metabolite thereof) throughout all stages of gestation (e.g., on a trimester by trimester, month by month, week by week, or day by day basis).

[0016] The term "complication of premature birth", as used herein, refers to any disease or disorder in an infant born prior to 40 weeks gestational age that is associated with an abnormal level of 2-methoxyestradiol in a subject. Diseases or disorders "associated with an abnormal level of 2-methoxyestradiol" is meant to encompass diseases or disorders that are directly or indirectly caused by an abnormal level of 2-methoxyestradiol or diseases or disorders having an independent underlying cause but that are associated with an abnormal level of 2-methoxyestradiol in a subject. A complication of premature birth may refer to a disease or disorder of associated with beingbom prior to 40 weeks gestational age wherein a subject has a higher level of 2-methoxyestradiol than a control subject. Alternatively, a complication of premature birth may refer to a disease or disorder of associated with being born prior to 40 weeks gestational age wherein a subject has a lower level of 2-methoxyestradiol than a control subject. A complication of premature birth may refer to a disease or disorder associated with abnormal angio genesis of the mother, fetus, placenta, uterus, etc. Examples of complications of premature birth include one or more of the following: intraventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP) and hemangioma.

[0017] "Diagnosis" or "diagnosing" as used herein includes diagnosis, prognosis, monitoring, characterizing, selecting or screening patients, including participants in clinical trials, and identifying patients at risk for or having a particular disorder or those most likely to respond to a particular therapeutic treatment, or for assessing or monitoring a patient's response to a particular therapeutic treatment.

[0018] The term "gestational age" means fetal age of a newborn, calculated from the number of completed weeks since the first day of the mother's last menstrual period to the date of birth.

[0019] The term "2-methoxyestradiol analog" refers to a derivative or analog of 2- methoxyestradiol that exhibits a therapeutic effect similar to that of 2-methoxyestradiol when administered to a subject suffering from a "complication of premature birth". In an exemplary embodiment, a 2-methoxyestradiol analog exhibits a therapeutic effect when administered to a subject suffering from a complication of premature birth. In one embodiment, a 2-methoxyestradiol analog exhibits greater stability when administered to a subject than 2-methoxyestradiol itself. In an exemplary embodiment, a 2-methoxyestradiol analog is a compound of Formula I. Examples of 2-methoxyestradiol analogs suitable for use in association with the methods described herein are also described in various U.S. Patents and published applications, including, for example, U.S. Patent No. 6,528,676, and U.S. Patent Application Publication Nos. 2003/0236408, 2002/0147183, 2003/0187076, and 2003/0236439. In an exemplary embodiment, analogs of 2-methoxyestradiol include colchicine and combretastatin A-4.

[0020] The term "precursor", as used herein with reference to 2-methoxyestradiol, or a 2-methoxyestradiol analog, refers to a compound that forms 2-methoxyestradiol, or a 2-methoxyestradiol analog, as at least one of the metabolites formed during a metabolic process. In an exemplary embodiment, precursors of 2-methoxyestradiol include, for example, estradiol, 2-hydroxyestradiol, etc.

[0021] The term "metabolite", as used herein with reference to 2-methoxyestradiol, or a 2-methoxyestradiol analog, refers to a compound that is formed by metabolism of 2- methoxyestradiol, or a 2-methoxyestradiol analog. Metabolite is meant to encompass both anabolites and catabolites. In an exemplary embodiment, metabolites of 2- methoxyestradiol include, for example, 2-methoxyestrone, etc. [0022] The term "biological sample" refers to a sample of biological material obtained from a subject, or present within a subject, including a tissue, tissue sample, or cell sample (e.g., a chorionic villus sample or a tissue biopsy, for example, an aspiration biopsy, a brush biopsy, a surface biopsy, a needle biopsy, a punch biopsy, an excision biopsy, an open biopsy, an incision biopsy, or an endoscopic biopsy), tumor, tumor sample, or biological fluid (e.g., blood, serum, plasma, amniotic fluid, urine, lymph, or spinal fluid). In an exemplary embodiment, a subject may be a human subject.

[0023] The term "about a control level", as used herein with reference to the level of 2-methoxyestradiol and/or an analog thereof in a subject, refers to a level of 2- methoxyestradiol that is less than 50%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, or 1% different from the control level of 2-methoxyestradiol.

[0024] The term "cis" is art-recognized and refers to the arrangement of two atoms or groups around a double bond such that the atoms or groups are on the same side of the double bond. Cis configurations are often labeled as (Z) configurations. [0025] The term "trans" is art-recognized and refers to the arrangement of two atoms or groups around a double bond such that the atoms or groups are on the opposite sides of a double bond. Trans configurations are often labeled as (E) configurations. [0026] The term "covalent bond" is art-recognized and refers to a bond between two atoms where electrons are attracted electrostatically to both nuclei of the two atoms, and the net effect of increased electron density between the nuclei counterbalances the internuclear repulsion. The term covalent bond includes coordinate bonds when the bond is with a metal ion.

[0027] The term "therapeutic agent" is art-recognized and refers to any chemical moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Examples of therapeutic agents, also referred to as "drugs", are described in well-known literature references such as the Merck Index, the Physicians Desk Reference, and The Pharmacological Basis of Therapeutics, and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment. [0028] The term "therapeutic effect" is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance. The term "pharmacologically active substance" includes any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and/or conditions in an animal or human. The phrase "therapeutically-effective amount" means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. The therapeutically effective amount of such substance will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. For example, certain compositions described herein may be administered in a sufficient amount to produce a at a reasonable benefit/risk ratio applicable to such treatment.

[0029] The term "synthetic" is art-recognized and refers to production by in vitro chemical or enzymatic synthesis.

[0030] The term "meso compound" is art-recognized and refers to a chemical compound which has at least two chiral centers but is achiral due to a plane or point of symmetry.

[0031] The term "chiral" is art-recognized and refers to molecules which have the property of non-superimposability of the mirror image partner, while the term "achiral" refers to molecules which are superimposable on their mirror image partner. A "prochiral molecule" is a molecule which has the potential to be converted to a chiral molecule in a particular process.

[0032] The term "stereoisomers" is art-recognized and refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space, hi particular, "enantiomers" refer to two stereoisomers of a compound which are non-superimposable mirror images of one another. "Diastereomers", on the other hand, refers to stereoisomers with two or more centers of dissymmetry and whose molecules are not mirror images of one another.

[0033] Furthermore, a "stereoselective process" is one which produces a particular stereoisomer of a reaction product in preference to other possible stereoisomers of that product. An "enantioselective process" is one which favors production of one of the two possible enantiomers of a reaction product.

[0034] The term "regioisomers" is art-recognized and refers to compounds which have the same molecular formula but differ in the connectivity of the atoms. Accordingly, a "regioselective process" is one which favors the production of a particular regioisomer over others, e.g., the reaction produces a statistically significant increase in the yield of a certain regioisomer.

[0035] The term "epimers" is art-recognized and refers to molecules with identical chemical constitution and containing more than one stereocenter, but which differ in configuration at only one of these stereocenters.

[0036] The term "ED₅₀" is art-recognized, hi certain embodiments, ED₅₀ means the dose of a drug which produces 50% of its maximum response or effect, or alternatively, the dose which produces a pre-deteπnined response in 50% of test subjects or preparations. The term "LD₅o" is art-recognized, hi certain embodiments, LD₅₀ means the dose of a drug which is lethal in 50% of test subjects. The term "therapeutic index" is an art-recognized term which refers to the therapeutic index of a drug, defined as LD₅o/ED_5O. [0037] The term "prodrug" is art-recognized and is intended to encompass

, compounds which, under physiological conditions, are converted into a drug, such as, for example, a 2-methoxyestradiol compound as described herein. A common method for making a prodrug is to select and attach moieties which are hydro lyzed under physiological conditions to provide the desired compound. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal.

[0038] The term "structure-activity relationship" or "(SAR)" is art-recognized and refers to the way in which altering the molecular structure of a drug or other compound alters its interaction with a receptor, enzyme, nucleic acid or other target and the like. [0039] The term "aliphatic" is art-recognized and refers to a linear, branched, cyclic alkane, alkene, or alkyne. hi certain embodiments, aliphatic groups may be linear or branched and have from 1 to about 20 carbon atoms.

[0040] The term "alkyl" is art-recognized, and includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has about 30 or fewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branched chain), and alternatively, about 20 or fewer. Likewise, cycloalkyls have from about 3 to about 10 carbon atoms in their ring structure, and alternatively about 5, 6 or 7 carbons in the ring structure. The term "alkyl" is also defined to include halosubstituted alkyls.

[0041] The term "aralkyl" is art-recognized and refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

[0042] The terms "alkenyl" and "alkynyl" are art-recognized and refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

[0043] Unless the number of carbons is otherwise specified, "lower alkyl" refers to an alkyl group, as defined above, but having from one to about ten carbons, alternatively from one to about six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths.

[0044] The term "heteroatom" is art-recognized and refers to an atom of any element other than carbon or hydrogen. Illustrative heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur and selenium.

[0045] The term "aryl" is art-recognized and refers to 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "heteroaryl." The aromatic ring may be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF₃, -CN, or the like. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

[0046] The terms ortho, rneta and para are art-recognized and refer to 1,2-, 1,3- and

1,4-disubstituted benzenes, respectively. For example, the names 1,2-dimethylbenzene and ort/rø-dimethylbenzene are synonymous. [0047] The terms "heterocyclyl" or "heterocyclic group" are art-recognized and refer to 3- to about 10-membered ring structures, alternatively 3- to about 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles may also be polycycles. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxanthene, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenantliroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, moφholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring may be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF₃, -CN, or the like.

[0048] The terms "polycyclyl" or "polycyclic group" are art-recognized and refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycycle may be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF₃, -CN, or the like.

[0049] The term "carbocycle" is art-recognized and refers to an aromatic or non- aromatic ring in which each atom of the ring is carbon.

[0050] The term "nitro" is art-recognized and refers to -NO₂; the term "halogen" is art-recognized and refers to -F, -Cl, -Br or -I; the term "sulfhydryl" is art-recognized and refers to -SH; the term "hydroxyl" means -OH; and the term "sulfonyl" is art-recognized and refers to -SO₂ ^". "Halide" designates the corresponding anion of the halogens, and "pseudohalide" has the definition set forth on page 560 of "Advanced Inorganic Chemistry" by Cotton and Wilkinson. [0051] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that may be represented by the general formulas:

R50 R50 I

/ I + N N R53

R51 R52

[0052] wherein R50, R51 and R52 each independently represent a hydrogen, an alkyl, an alkenyl, -(CH₂)_m-R61, or R50 and R51, taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R61 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In certain embodiments, only one of R50 or R51 may be a carbonyl, e.g., R50, R51 and the nitrogen together do not form an imide. In other embodiments, R50 and R51 (and optionally R52) each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH₂)_m-R61. Thus, the term "alkylamine" includes an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R50 and R51 is an alkyl group.

[0053] The term "acylamino" is art-recognized and refers to a moiety that may be represented by the general formula:

O

N ^u R54

R50

[0054] wherein R50 is as defined above, and R54 represents a hydrogen, an alkyl, an alkenyl or -(CH₂)_m-R61, where m and R61 are as defined above. [0055] The term "amido" is art recognized as an amino-substituted carbonyl and includes a moiety that may be represented by the general formula:

[0056] wherein R50 and R51 are as defined above. Certain embodiments of the amide described herein will not include imides which may be unstable.

[0057] The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In certain embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH₂)_m-R61, wherein m and

R61 are defined above. Representative alkylthio groups include methylthio, ethyl thio, and the like.

[0058] The term "carbonyl" is art recognized and includes such moieties as may be represented by the general formulas:

[0059] wherein X50 is a bond or represents an oxygen or a sulfur, and R55 and R56 represents a hydrogen, an alkyl, an alkenyl, -(CH₂)_m-R61 or a pharmaceutically acceptable salt, R56 represents a hydrogen, an alkyl, an alkenyl or -(CH₂)_m-R61, where m and R61 are defined above. Where X50 is an oxygen and R55 or R56 is not hydrogen, the formula represents an "ester". Where X50 is an oxygen, and R55 is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when R55 is a hydrogen, the formula represents a "carboxylic acid". Where X50 is an oxygen, and R56 is hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiolcarbonyl" group. Where X50 is a sulfur and R55 or R56 is not hydrogen, the formula represents a "thiolester." Where X50 is a sulfur and R55 is hydrogen, the formula represents a "thiolcarboxylic acid." Where X50 is a sulfur and R56 is hydrogen, the formula represents a "thiolformate." On the other hand, where X50 is a bond, and R55 is not hydrogen, the above formula represents a "ketone" group. Where X50 is a bond, and R55 is hydrogen, the above formula represents an "aldehyde" group.

[0060] The terms "alkoxyl" or "alkoxy" are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O--(CH₂)_m-R61, where m and R61 are described above. [0061] The term "sulfonate" is art recognized and refers to a moiety that may be represented by the general formula:

O

S OR57

O in which R57 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

[0062] The term "sulfate" is art recognized and includes a moiety that may be represented by the general formula:

in which R57 is as defined above.

[0063] The term "sulfonamido" is art recognized and includes a moiety that may be represented by the general formula:

in which R50 and R56 are as defined above.

[0064] The term "sulfamoyl" is art-recognized and refers to a moiety that may be represented by the general formula:

in which R50 and R51 are as defined above.

[0065] The term "sulfonyl" is art-recognized and refers to a moiety that may be represented by the general formula:

in which R58 is one of the following: hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl.

[0066] The term "sulfoxido" is art-recognized and refers to a moiety that may be represented by the general formula:

in which R58 is defined above.

[0067] The term "phosphoryl" is art-recognized and may in general be represented by the formula:

Q50

OR59

[0068] wherein Q50 represents S or O, and R59 represents hydrogen, a lower alkyl or an aryl. When used to substitute, e.g., an alkyl, the phosphoryl group of the phosphorylalkyl may be represented by the general formulas:

[0069] wherein Q50 and R59, each independently, are defined above, and Q51 represents O, S or N. When Q50 is S, the phosphoryl moiety is a "phosphorothioate". [0070] The term "phosphoramidite" is art-recognized and may be represented in the general formulas:

π _

[0071] wherein Q51, R50, R51 and R59 are as defined above.

[0072] T he term "phosphonamidite" is art-recognized and may be represented in the general formulas:

[0073] wherein Q51, R50, R51 and R59 are as defined above, and R60 represents a lower alkyl or an aryl.

[0074] Analogous substitutions may be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.

[0075] The definition of each expression, e.g. alkyl, m, n, and the like, when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

[0076] The term "selenoalkyl" is art-recognized and refers to an alkyl group having a substituted seleno group attached thereto. Exemplary "seleno ethers" which may be substituted on the alkyl are selected from one of -Se-alkyl, -Se-alkenyl, -Se-alkynyl, and -

Se-(CH₂)_m-R61, m and R61 being defined above.

[0077] The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, ^-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, jσ-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively. [0078] The abbreviations Me₅ Et, Ph, Tf, Nf, Ts, and Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, jo-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations.

[0079] Certain compounds contained in compositions described herein may exist in particular geometric or stereoisomeric forms. In addition, polymers described herein may also be optically active. Contemplated herein are all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in the compositions described herein. [0080] If, for instance, a particular enantiomer of a compound described herein is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are fonned with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

[0081] It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.

[0082] The term "substituted" is also contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents may be one or more and the same or different for appropriate organic compounds. In certain embodiments, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Permissible substituents of organic compounds are not intended to be limited in any manner by the instant description.

[0083] Chemical elements may be identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Additionally, the term "hydrocarbon" is contemplated to include all permissible compounds having at least one hydrogen and one carbon atom. In a broad aspect, the permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compounds that may be substituted or unsubstituted.

[0084] The term "protecting group" is art-recognized and refers to temporary substituents that protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed by Greene and Wuts in Protective Groups in Organic Synthesis (2^nd ed., Wiley: New York, 1991).

[0085] The term "hydroxyl-protecting group" is art-recognized and refers to those groups intended to protect a hydroxyl group against undesirable reactions during synthetic procedures and includes, for example, benzyl or other suitable esters or ethers groups known in the art.

[0086] The term "carboxyl-protecting group" is art-recognized and refers to those groups intended to protect a carboxylic acid group, such as the C-termmus of an amino acid or peptide or an acidic or hydroxyl azepine ring substituent, against undesirable reactions during synthetic procedures and includes. Examples for protecting groups for carboxyl groups involve, for example, benzyl ester, cyclohexyl ester, 4-nitrobenzyl ester, t-butyl ester, 4-pyridylmethyl ester, and the like.

[0087] The term "amino-blocking group" is art-recognized and refers to a group which will prevent an amino group from participating in a reaction carried out on some other functional group, but which can be removed from the amine when desired. Such groups are discussed by in Ch. 7 of Greene and Wuts, cited above, and by Barton, Protective Groups in Organic Chemistry ch. 2 (McOmie, ed., Plenum Press, New York, 1973). Examples of suitable groups include acyl protecting groups such as, to illustrate, formyl, dansyl, acetyl, benzoyl, trifluoroacetyl, succinyl, methoxysuccinyl, benzyl and substituted benzyl such as 3,4-dimethoxybenzyl, o-nitrobenzyl, and triphenylmethyl; those of the formula -COOR where R includes such groups as methyl, ethyl, propyl, isopropyl, 2,2,2-trichloroethyl, 1 -methyl- 1-phenylethyl, isobutyl, t-butyl, t-amyl, vinyl, allyl, phenyl, benzyl, p-nitrobenzyl, o-nitrobenzyl, and 2,4-dichlorobenzyl; acyl groups and substituted acyl such as formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl, benzoyl, and p-methoxybenzoyl; and other groups such as methanesulfonyl, p- toluenesulfonyl, p-bromobenzenesulfonyl, p-nitrophenylethyl, and p-toluenesulfonyl- aminocarbonyl. Preferred amino-blocking groups are benzyl (-CH₂C₆Hs), acyl [C(O)Rl] or SiRl₃ where Rl is C₁-C₄ alkyl, halomethyl, or 2-halo-substituted-(C₂-C₄ alkoxy), aromatic urethane protecting groups as, for example, carbonylbenzyloxy (Cbz); and aliphatic urethane protecting groups such as t-butyloxycarbonyl (Boc) or 9- fluorenylmethoxycarbonyl (FMOC).

[0088] The definition of each expression, e.g. lower alkyl, m, n, p and the like, when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

[0089] The term "electron-withdrawing group" is art-recognized, and refers to the tendency of a substituent to attract valence electrons from neighboring atoms, i.e., the substituent is electronegative with respect to neighboring atoms. A quantification of the level of electron-withdrawing capability is given by the Hammett sigma (σ) constant. This well known constant is described in many references, for instance, March, Advanced Organic Chemistry 251-59 (McGraw Hill Book Company: New York, 1977). The Hammett constant values are generally negative for electron donating groups (σ(P) = - 0.66 for NH₂) and positive for electron withdrawing groups (σ(P) = 0.78 for a nitro group), σ(P) indicating para substitution. Exemplary electron-withdrawing groups include nitro, acyl, formyl, sulfonyl, trifluoromethyl, cyano, chloride, and the like. Exemplary electron- donating groups include amino, methoxy, and the like.

[0090] The term "modulation", when used in reference to a functional property or biological activity or process (e.g., enzyme activity or receptor binding), refers to the capacity to either up regulate (e.g., activate or stimulate), down regulate (e.g., inhibit or suppress) or otherwise change a quality of such property, activity or process, hi certain instances, such regulation may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and/or may be manifest only in particular cell types.

[0091] The term "treating" is art-recognized and refers to curing as well as ameliorating at least one symptom of any condition or disease. In an exemplary embodiment, treating a complication of premature birth, includes, for example, inhibiting the disease or disorder, e.g., arresting its development, or ameliorating or relieving at least one symptom of the disease or disorder, e.g., causing regression of the complication. [0092] The term "prophylactic" or "therapeutic" treatment of a complication of premature birth refers to administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects therefrom).

[0093] A "patient," "subject" or "host" to be treated by the subject method may mean either a human or non-human animal.

[0094] The term "mammal" is known in the art, and exemplary mammals include humans, primates, bovines, porcines, canines, felines, and rodents (e.g., mice and rats). [0095] The term "bioavailable" in the context of a compound is art-recognized and refers to a form of the compound that allows for it, or a portion of the amount administered, to be absorbed by, incorporated to, or otherwise physiologically available to a subject or patient to whom it is administered.

[0096] The term "pharmaceutically-acceptable salts" is art-recognized and refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds, including, for example, those contained in compositions described herein.

[0097] The term "pharmaceutically acceptable carrier" is art-recognized and refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with a subject composition and its components and not injurious to the patient. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0098] The terms "systemic administration," "administered systemically,"

"peripheral administration" and "administered peripherally" are art-recognized and refer to the administration of a subject composition, therapeutic or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. [0099] The terms "parenteral administration" and "administered parenterally" are art-recognized and refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articulare, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. [00100] Contemplated equivalents of the compositions described herein include compositions which otherwise correspond thereto, and which have the same general properties thereof, wherein one or more simple variations of substituents or components are made which do not adversely affect the characteristics of the compositions of interest. In general, the components of the compositions described herein may be prepared using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here.

[00101] B. Methods of Treating, Preventing and Diagnosing Complications of

Premature Birth [00102] Provided herein are methods and compositions for treating, preventing and/or diagnosing complications of premature birth. In one embodiment, methods and compositions for treating or preventing a complication of premature birth using 2- methoxyestradiol, or an analog thereof, are provided. In another embodiment, methods for diagnosing a complication of premature birth by determining the level of 2- methoxyestradiol (or a precursor or metabolite thereof) in a subject are provided. In an exemplary embodiment, the methods and compositions described herein may be used for treating or preventing complication of premature birth by administering 2- methoxyestradiol, or an analog of 2-methoxyestradiol, to a subject in need thereof. In another exemplary embodiment, the methods and compositions described herein may be used for diagnosing a complication of premature birth, or the likelihood of developing a complication of premature birth, comprising detecting the level of 2-methoxyestradiol (or a precursor or metabolite thereof) in a subject.

[00103] In certain embodiments, the methods described herein may involve first identifying a subject susceptible to, or suffering from, a complication of premature birth. Subjects susceptible to, or suffering from, a complication of premature birth may be identified by recognizing symptoms of a complication of premature birth that are being exhibited by the patient. Alternatively, subjects susceptible to, or suffering from, a complication of premature birth may be identified by determining the level of 2- methoxyestradiol (or a precursor or metabolite thereof) in a subject and comparing that level to a control. Any deviation from the control level of 2-methoxyestradiol (or a precursor or metabolite thereof) may be indicative of a subject suffering from a complication of premature birth. In an exemplary embodiment, subjects may be screened for levels of 2-methoxyestradiol (or a precursor or metabolite thereof) on a regular basis (or at regular intervals) for purposes of diagnosis of a complication of premature birth or to monitor the stage or development of a complication of premature birth. In one embodiment, screening for levels of 2-methoxyestradiol (or a precursor or metabolite thereof) may be carried out on a regular basis, for example, about once every month, once every 3 weeks, once every 2 weeks, once every 10 days, once every week, or about once every 144, 120, 96, 72, 48, 24, or 12 hours. In an exemplary embodiment, screening for levels of 2-methoxyestradiol (or precursors or metabolites thereof) may be used to identify subjects that may be candidates for therapeutic treatment with 2-methoxyestradiol, or an analog thereof. In another embodiment, screening for levels of 2-methoxyestradiol (or precursors or metabolites thereof) may be used to calculate an effective dose for administering to a subject.

[00104] Subjects identified as having a lower level or a higher level of 2- methoxyestradiol as compared to a control may be susceptible to, or suffering from, a complication of premature birth. In one embodiment, subjects having a low level of 2- methoxyestradiol as compared to a control may be susceptible to, or suffering from, one or more of the following complications of premature birth: intraventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP) and hemangioma formation.

[00105] The degree or severity of a complication of premature birth may be determined based on the degree of deviation in the level of 2-methoxyestradiol in a subject as compared to a control. For example, a subject exhibiting a greater deviation in the level of 2-methoxyestradiol as compared to a control may indicate that the subject is more susceptible to, or suffering from a more severe case of, complication of premature birth. [00106] Screening for levels of 2-methoxyestradiol (or precursors or metabolites thereof) may be used to monitor the course of treatment with 2-methoxyestradiol, or an analog thereof. When treating a subject with an analog of 2-methoxyestradiol it may be useful for monitoring purposes to determine the level of 2-methoxyestradiol (or a precursor or metabolite thereof) and/or the level of the 2-methoxyestradiol analog (or a precursor or analog thereof). In an exemplary embodiment, it may be useful to determine the level of 2- methoxyestradiol plus the level of a 2-methoxyestradiol analog being administered to a subject. In another embodiment, it may be useful to determine the level of a precursor of 2- methoxyestradiol plus the level of a precursor a 2-methoxyestradiol analog. In yet another embodiment, it maybe useful to determine the level of a metabolite of 2-methoxyestradiol plus the level of a metabolite of a 2-methoxyestradiol analog.

[00107] The level of 2-methoxyestradiol in a subject may be determined directly by measuring the level of 2-methoxyestradiol itself. Alternatively, the level of 2- methoxyestradiol may be determined indirectly by measuring the level of a precursor or a metabolite of 2-methoxyestradiol. Similarly, the level of an analog of 2-methoxyestradiol may be determined by directly measuring the level of the analog in a subject or indirectly by measuring the level of a precursor or metabolite of the analog. In yet another embodiment, the level of a precursor or a metabolite of 2-methoxyestradiol, or an analog thereof, may be measured directly for diagnostic or monitoring purposes. In this instance, the precursor or analyte is not being used as a proxy for the level of 2-methoxyestradiol, or an analog thereof, but rather is being used directly for diagnostic or monitoring purposes. [00108] The level of 2-methoxyestradiol, or an analog thereof, may be determined in a biologic sample of a subject or a control subject. In an exemplary embodiment, the level of 2-methoxyestradiol may be determined in a urine, blood or plasma sample from a subject.

[00109] The level of 2-methoxyestradiol, or analog thereof, in a biological sample of a subject may be compared directly to a control. In another embodiment, the level of 2- methoxyestradiol, or analog thereof, in a biological sample of a subject may be used to calculate the physiological concentration of 2-methoxyestradiol, or analog thereof, found in a subject. The physiological concentration of the 2-methoxyestradiol, or analog thereof, in a subject may then optionally be compared to a control.

[00110] The level of 2-methoxyestradiol (or a precursor or metabolite thereof) or an analog of 2-methoxyestradiol (or a precursor or metabolite thereof) in a sample, e.g., in cord blood, amniotic fluid, plasma, breast milk, and the like, may be determined using any method known in the art. For example, the level of 2-methoxyestradiol and/or analogs thereof may be measured using gas chromatography- mass spectrometry (GC-MS) may be used to measure the levels of 2ME2 or analogs thereof in cord blood, amniotic fluid, plasma, breast milk, and the like. GC-MS is the gold standard for detection and quantitation of low molecular weight steroids, such as 2-methoxyestradiol and/or its analogs. In another example, the level of 2-methoxyestradiol and/or analogs thereof (and precursors or metabolites thereof) in a sample may be determined using other chromatographic methods, such as thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and the like. Alternatively, the level of 2-methoxyestradiol and/or analogs thereof (and precursors or metabolites thereof) may be determined using immunoassays, such as ELISA, RIA, and the like.

[00111 ] Further, methods for detecting precursors of 2-methoxyestradiol such as estrogen, estradiol, and the like (see B.T. Zhu and A.H. Conney, Cancer Research (1998) 58:2269 for a review of the pathways to 2-methoxyestradiol) may be used. For example, serum estrogen assays may be used, for example, hemagglutination-inhibition assays such as the rapid, sensitive, and simple non-radioactive serum estrogen assay developed by Kaseki, et al. modifying a commercially available hemagglutination-inhibition kit (Hi- Estrotec) originally designed to measure total urinary estrogens such that it has a minimum sensitivity of 100 pg of 17-beta-estradiol and takes less than 2.5 hours to complete. Another example of an estrogen assay that may be used in the methods of the invention is Applied Biosystems' HitHunter™ Enzyme Fragment Complementation (EFC) Assay. This assay is based on an engineered β-galactosidase enzyme that consists of two fragments - the Enzyme Acceptor (EA) and the Enzyme Donor(ED). When the fragments are separated, there is no β-galactosidase activity, but when the fragments are together they associate (complement) to form active enzyme. The EFC assay utilizes an ED-analyte conjugate in which the analyte may be recognized by a specific binding protein, such as an antibody or receptor. In the absence of the specific binding protein, the ED-analyte conjugate is capable of complementing EA to form active β-galactosidase, producing a positive luminescent signal. If the ED-analyte conjugate is bound by a specific binding protein, complementation with EA is prevented, and there is no signal. If free analyte is provided (in a sample), it will compete with the ED-analyte conjugate for binding to the specific binding protein; free analyte will release ED-analyte conjugate for complementation with EA, producing a signal dependent upon the amount of free analyte present in the sample. [00112] The level of 2-methoxyestradiol, or a precursor or an analog thereof, in a subject may be compared to a control either quantitatively or qualitatively. For example, a qualitative (or unitless) comparison may be carried out by determining whether the level of 2-methoxyestradiol, or an analog thereof, in a subject is higher, lower, or about the same as a control. Optionally, a qualitative comparison may be used to estimate the magnitude of difference in the level of 2-methoxyestradiol, or an analog thereof, in a subject as compared to a control, such as, for example, a 2-fold change, a 50% change, etc. A quantitative comparison may be carried out by determining the quantity of 2-methoxyestradiol, or an analog thereof, in a subject as compared to the quantity in a control, wherein the quantity has some form of units attached (such as, for example, mg of protein, volume of a spot/band in a gel, intensity of a spot on a phosphoimager or autoradiogram exposure, volume of a spot on a chromatography plate, etc.).

[00113] It may be desirable to monitor symptoms of a complication of premature birth in addition to, or instead of, monitoring the level of 2-methoxyestradiol (or a precursor or metabolite thereof) in a subject. For example, in a subject suffering from or susceptible to retinopathy of prematurity, it may be desirable to conduct an ophthalmic examination of the subject before, during and/or after treatment with the subject 2-methoxyestradiol, or an analog thereof. After instilling a series of dilating drops in each eye, the retina is examined with an ophthalmoscope. For example, in a subject suffering from or susceptible to intraventricular hemorrhage, it may be desirable to conduct an cranial ultrasound on the subject before, during and/or after treatment with the subject 2-methoxyestradiol, or an analog thereof. This test uses sound waves to create a picture of internal structures. A cranial ultrasound can view the inside of the baby's brain through the fontanelles, the spaces between the bones of the baby's head. With the ultrasound, the amount of bleeding can be graded. In a subject suffering from or susceptible to necrotizing enterocolitis, it may be desirable to conduct abdominal x-rays and/or blood tests on the subject before, during and/or after treatment with the subject 2-methoxyestradiol, or an analog thereof. An x-ray of the abdomen of a subject having necrotizing enterocolitis may show a bubbly appearance in the intestine and signs of air or gas in the large veins of the liver. Air may also be outside the intestines in the abdomen. A needle may be inserted into the abdominal cavity. Withdrawing intestinal fluid from the abdomen is often a sign of a hole in the intestines, hi a subject suffering from or susceptible to hemangioma, it may be desirable to conduct a physical examination, CT scan and/or MRI scan on the subject before, during and/or after treatment with the subject 2-methoxyestradiol, or an analog thereof. [00114] In one embodiment, the methods described herein involve administering 2- methoxyestradiol to a subject. Alternatively, it may be desirable to administer a mixture of 2-methoxyestradiol and one or more analogs of 2-methoxyestradiol, or a mixture of two or more analogs of 2-methoxyestradiol, to a subject, hi other embodiments, it may be desirable to administer 2-methoxyestradiol and/or an analog(s) thereof to a subject in combination with another therapeutic agent effective for treating or ameliorating at least one symptom of a complication of premature birth. In an exemplary embodiment, 2- methoxyestradiol is administered with another therapeutic agent effective for treating or ameliorating a complication of premature birth, such as, for example, lung surfactant to help the lungs to function normally. Exemplary lung surfactants include, but are not limited to, colfosceril (Exosurf®), Survanta®, and Surfaxin®. In embodiments directed to combination therapies, the drugs may be formulated and administered together as a mixture or may be separately formulated and administered.

[00115] In an exemplary embodiment, a subject susceptible to, or suffering from, a complication of premature birth associated with a low level of 2-methoxyestradiol is administered an amount of 2-methoxyestradiol, or an analog thereof, sufficient to raise the level of 2-methoxyestradiol (or the level of 2-methoxyestradiol plus the level of a 2- methoxyestradiol analog) up to about the level of 2-methoxyestradiol in a control subject. In a further embodiment, a subject may be administered on a regular basis an amount of 2- methoxyestradiol, or an analog thereof, sufficient to maintain the level of 2- methoxyestradiol (or the level of 2-methoxyestradiol plus the level of a 2-methoxyestradiol analog) in the subject at about a control level. For example, in certain embodiments, it may be desirable to administer 2-methoxyestradiol and/or an analog thereof to a subject on a monthly, weekly, or daily basis. In an exemplary embodiment, the level of 2- methoxyestradiol (or the level of 2-methoxyestradiol plus the level of a 2-methoxyestradiol analog) is maintained in a subject at about a control level throughout pregnancy. In order to maintain an appropriate level of 2-methoxyestradiol (or the level of 2-methoxyestradiol plus the level of a 2-methoxyestradiol analog) in a subject it may be desirable to regularly monitor the subject for levels of 2-methoxyestradiol (or the level of 2-methoxyestradiol plus the level of a 2-methoxyestradiol analog) and adjust the amount of therapeutic agent administered to the subject accordingly.

[00116] In one embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I:

[00117] wherein, independently for each occurrence:

[00118] R is H, C_1-6 alkyl, aryl, aralkyl, or carbonyl;

[00119] Ri and R₂ are -OR, -SR, or -N(R)₂;

[00120] R₃ is H, halide, Ci_-6 alkyl, aryl, aralkyl, or carbonyl;

[00121] R₄, R₅, and R₆ is are H, halide, Ci_-6 alkyl, aryl, aralkyl, or carbonyl;

[00122] m is an integer from 1-5 inclusive; and

[00123] n and p are an integer from 1-6 inclusive.

[00124] In a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R is methyl. [00125] In a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R₁ is OH.

[00126] In a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R₂ is OH.

[00127] In a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R₃ is methyl.

[00128] In a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R₄ is H.

[00129] In a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R₅ is H.

[00130] In a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R₆ is H.

[00131] In a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R is methyl and Ri is OH.

[00132] In a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R is methyl and R₂ is OH.

[00133] In a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R is methyl and R₃ is methyl.

[00134] In a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R is methyl, R₁ is OH, and R₂ is OH.

[00135] In a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R is methyl, R₁ is OH, R₂ is OH, and R₃ is methyl.

[00136] In a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R₄, R₅, and R₆ are H.

[00137] In a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R is methyl, and R₄, R₅, and R₆ are H.

[00138] hi a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R is methyl, R₁ is OH, and R₄, R₅, and R₆ are H.

[00139] hi a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R is methyl, R₁ is OH, R₂ is OH, and R₄,

R₅, and R₆ are H.

[00140] hi a further embodiment, a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I and the attendant definitions, wherein R is methyl, R₁ is OH, R₂ is OH, R₃ is methyl, and R₄, R₅, and R₆ are H.

[00141] Also included in the compositions and methods described herein are pharmaceutically acceptable addition salts and complexes of formula I. In cases wherein the compounds may have one or more chiral centers, unless specified, the compositions described herein comprise each unique racemic compound, as well as each unique nonracemic compound.

[00142] hi cases in which the compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are intended to be included in association with the compounds described herein, hi cases wherein the compounds may exist in tautomeric

O OR¹ forms, such as keto-enol tautomers, such as --^^-« and --""^==- , each tautomeric form is intended to be included in association with the compounds described herein, whether existing in equilibrium or locked in one form by appropriate substitution with R'. The meaning of any substituent at any one occurrence is independent of its meaning, or any other substituent's meaning, at any other occurrence. [00143] Also included in the methods and compositions described herein are prodrugs of 2-methoxyestradiol and the compounds of formula I. [00144] The compounds of formula I may be prepared by any conventional method useful for the preparation of analogous compounds. Starting materials for the processes are known or can be prepared by known processes from commercially available materials. A compound used in the methods described herein can be converted to another compound used in the methods described herein using conventional methods. The products of the reactions are isolated by conventional means such as extraction, crystallization, distillation, chromatography, and the like.

[00145] Known compounds that are used in accordance with the invention and precursors to novel compounds according to the invention can be purchased, e.g., from Sigma Chemical Co., St. Louis, Steraloids and Research Plus. Other compounds according to the invention can be synthesized according to known methods from publicly available precursors.

[00146] The chemical synthesis of estradiol has been described (Eder, V. et al., Ber

109, 2948 (1976); Oppolzer, D. A. and Roberts, D A. HeIv. Chim. Acta. 63, 1703, (1980)). The synthetic pathways used to prepare some of the 2-methoxyestradiol analogs of the present invention are based on modified published literature procedures for estradiol derivatives and dimethylhydrazone (Trembley et al., Bioorganic & Med. Chem. 1995 3, 505-523; Fevig et al., J. Org. Chem., 1987 52, 247-251; Gonzalez et al., Steroids 1982, 40, 171-187; Trembley et al., Synthetic Communications 1995, 25, 2483-2495; Newkome et al., J. Org. Chem. 1966, 31, 677-681; Corey et al Tetrahedron Lett 1976, 3-6; Corey et al., Tetrahedron Lett, 1976, 3667-3668) and German Patent No. 2757157 (1977). [00147] In exemplary embodiments, 2-methoxyestradiol analogs suitable for use with the compositions and methods described herein are described in U.S. Patent No. 6,528,676, and U.S. Patent Application Publication Nos. 2003/0236408, 2002/0147183, 2003/0187076, 2002/0082433, and 2003/0236439. 2-methoxyestradiol, or analogs thereof, may be purchased from commercially available sources or may be prepared as described in, for example, U.S. Patent No. 6,528,676, and U.S. Patent Application Publication Nos. 2003/0236408, 2002/0147183, 2003/0187076, 2002/0082433, and 2003/0236439. Examples of 2-methoxyestradiol analogs suitable for use in accordance with the methods and compositions described herein include one or more of the following: 17 substituted 2- methoxyestradiol derivatives: estra-l,3,5(10)-triene-3,17α-diol, 2-methoxyestra-l,3,5(10)- triene-3-ol, 17β-aminoestra-l,3₃5,(10)-triene-3,17β-diol, 2-methoxy-17-oxime-3- hydroxyestra- 1,3,5(10)-triene- 17-one, 2-methoxy-3 , 17β-bis(acetyloxy)estra- 1,3,5,(10)- triene, 2-methoxy-17β-propaneestra-l,3,5(10)-triene-3-ol, 2-methoxy-17β-methylestra- l,3,5(10)-triene-3-ol, 2-methoxy-17(20)-Z-propylideneestra-l,3,5,(10)-triene-3-ol, 17(20)- methyleneestra- 1,3,5(10)-triene-3 -ol, 2-methoxy- 17β-(N-n-(l ) propylamino)estra- l,3,5(10)-triene-3-ol, 2-methoxy-19-norρregna-l,3,5(10)17(20)-tetraen-3-ol, 2-methoxy- 17β-ethylestra-l,3,5(10)-triene-3-ol, 2-methoxy-17-(4-tosylhydrazone)estra-l,3,5(10)- triene-3-ol, 2-methoxy-17(20)-Z-butylideneestra-l,3,5(10)-triene-3-ol, 2-methoxy-17β- butylestra-l,3,5(10)-triene-3-ol; 2 substituted estradiol derivatives: 2-acetylestra-l,3,5(10)- triene-3, 17β-diol, 2-formylestra- 1 ,3 ,5(10)-triene-3 , 17β-diol, 2-(hydroxymethyl)estra- l,3,5(10)-triene-3,17β-diol, 2-ethylestra-l,3,5(10)-triene-3,17β-diol, 2-methylestra- l,3,5(10)-triene-3,17β-diol, 2-nitroestra-l,3,5(10)-triene-3,17β-diol, 2-(N₅N- dimethylamino)estra-l,3,5(10)-triene-3,17β-diol₃ 2-aminoestra-l,3,5(10)-triene-3-ol, 2- formaimdeestra-l,3,5(10)-1riene-3-ol₅ 2-(N-methylammo)estra-l,3,5(10)-triene-3-ol-HCl, 2-(N,N-dimethylamino)estra-l,3,5(10)-triene-3-ol, 2-(N,N-dimethylamino)estra-l,3,5(10)- triene-3-ol-HCl, 2-aminoestra- 1 ,3,5(10)-triene-3, 17β-diol, 2-(N,N-dimethylamino)-l 7(20)- methyleneestra-l,3,5(10)-triene-3-ol-HCL, 2-(r-ρropynyl)estra-l,3,5(10)-triene-3,17β-diol, 2-Azidoestra l,3,5(10)-triene-3,17β-diol, 2-ethoxyestra-l,3,5(10)-triene3,17β-diol, estra- 1 ,3,5(10)-triene-3-ol; 16 substituted 2-methoxyestradiol derivatives: 2-methoxy- 16α- methylestra- l,3,5(10)-triene-3,17β-diol, 2-methoxy-16β-methylestra-l,3,5(10)-triene- 3 , 17β-diol, 2-methoxy- 16-ethylestra- 1,3,5,(10)-triene-3 , 17 β-diol, 16α-(hydroxymethy- l)estra-l, 3,5(10)-triene-3,17β-diol, 16β-(hydroxymethyl)estra-l,3,5(10)-triene-3,17β-diol, 2-methoxy- 16α-propaneestra- 1,3,5(10)-triene-3, 17β-diol, 2-methoxy-l 6β-propaneestra- l,3,5(10)-triene-3,17β-diol, 2-methoxy-16β-butaneestra-l,3,5(10)-triene-3,17β-diol, 2- methoxy-16α-butaneestra-l,3,5(10)-triene-3,17β-diol, 2-methoxy- 16β-iso-butaneestra- l,3,5(10)-triene-3,17β-diol, 2-methoxy-16α-(N,N-dimethylaminomethyl)estra-l,3,5(10)- triene-3,17β-diol, 2-methoxy-16α-ethylestra-l,3,5(10)-triene-3,17β-diol; dehydrogenated/substituted 2-methoxyestradiol derivatives: 2-methoxy-estra- 1,3,5(10)9(1 l)-tetraene-3,17β-diol; other 2-methoxyestradiol derivatives: 2-ethoxy-19- norpregnane- 1 ,3,5(10) 17(20)-tetraen-3-ol, 2-(l -propynyl)- 19-norpregnane- 1 ,3,5(10) 17(20)- tetraen-3-ol, 2-formyl- 19-norpregnane- 1 ,3 ,5 ( 10) 17(20)-tetraen-3-ol, 2-formamide- 19- norpregnane- 1 ,3 ,5( 10) 17(20)-tetraen-3 -ol, 2-methyenehydroxy- 19-norpregnane- l,3,5(10)17(20)-tetraen-3-ol, 2-ethyl-19-norρregnane-l, 3,5(10)17(20)-tetraen-3-ol, 2- methyi- 19-norpregnane- 1,3,5(10)17(20)-tetraen-3 -ol, 2-( 1 -propenyl)- 19-norpregnane- l,3,5(10)17(20)-tetraen-3-ol, 2-ethoxy-17(20)-methyleneestra-l,3₅5(10)-triene-3-ol, 2-(l- propynyl)-17(20)-methyleneestra-l,3,5(10)-triene~3~ol, 2-formyl-17(20)-methyleneestra- l,3,5(10)-triene-3-ol, 2-forniamide-17(20)-methyleneestra-l,3,5(10)-triene-3-ol, 2- methylenehydroxy- 17(20)-methyleneestra-l ,3,5(10)-triene-3-ol₃ 2-ethyl- 17(20)- methyleneestra-l,3,5(10)-triene-3-ol, 2-methyl-17(20)-methyleneestra-l,3,5(10)-triene-3- ol, 2-(l-propenyl)-17(20)-methyleneestra-l,3₃5(10)-triene-3-ol, 2-ethoxyestra-l,3,5(10)- triene-3-ol, 2-(l-propynyl)estra-l,3,5(10)-triene-3-ol, 2-formylestra-l,3,5(10)-triene-3-ol, 2-fomiamideestra-l,3,5(10)-triene-3-ol, 2-(methylenehydroxy)estra-l,3_?5(10)-triene-3-ol, 2-ethylestra-l,3,5(10)-triene-3-ol, 2-methylestra-l,3,5(10)-triene-3-ol, 2-(l-proρenyl)estra- l,3,5(10)-triene-3-ol, 2-ethoxy-17β-methylestra-l,3,5(10)-triene-3-ol, 2-(l-ρropynyl)-17β- methylestra-l,3,5(10)-triene-3-ol, 2-formyl-17β-methylestra-l,3,5(10)-triene-3-ol, 2- formamide- 17β-methylestra- 1,3,5(10)-triene-3 -ol, 2-methylenehydroxy- 17β-methylestra- 1, 3,5(10)-triene-3-ol, 2-ethyl-17β-methylestra-l,3,5(10)-triene-3-ol, 2-methyl-17β- methylestra- 1,3,5(10)-triene-3-ol, 2-(l -propenyl)- 17β-methylestra- 1,3,5(10)-triene-3-ol, 2- ethoxy-17β-ethylestra-l,3,5(10)-triene-3-ol, 2-(l-ρroρynyl)-17β-ethylestra-l,3,5(10)-triene- 3-ol, 2-formyl-17β-ethylestra-l,3,5(10)-triene-3-ol, 2-fortnamide-17β-ethylestra-l,3,5(10)- triene-3-ol, 2-methylenehydroxy- 17β-ethylestra- 1,3,5(10)-triene-3-ol, 2-ethyl-l 7β- ethylestra-l_;,3,5(10)-triene-3-ol, 2-methyl-17β-ethylestra-l,3,5(10)-triene-3-ol, 2-(l- propenyl)-17β-ethylestra-l,3,5(10)-triene-3-ol, 2-ethoxy-17(20)-ρropyleneestra-l,3,5(10)- triene-3-ol, 2-(l-propynyl)-17(20)- ρroρyleneestxa-l,3,5(10)-triene-3-ol, 2-formyl- 17(20)- propyleneestra-l,3,5(10)-triene-3-ol, 2-formamide-17(20)-proρyleneestra-l,3,5(10)-triene- 3-ol, 2-methylenehydroxy-17(20)-proρyleneestra-l,3,5(10)-triene-3-ol, 2-ethyl- 17(20)- propyleneestra- 1,3,5(10)-triene-3 -ol, 2-methyl- 17(20)-propyleneestra- 1,3,5(10)^'-triene-3-ol, 2-( 1 -propenyl)- 17(20)-propyleneestra- 1,3,5(10)-triene-3-ol, 2-methoxy- 17 β- methylenehydroxyestra- 1 ,3 ,5(10)-triene-3-ol, 2-methoxy- 17β-(carboxyliacid)-estra- 1,3,5(10)-triene-3-ol, colchicine, combretastatin A-4, diethylstilbestrol, 2-bromoestradiol, 2-methoxyestrone, 2-hydroxyestradiol, 4-hydroxyestradiol, 17-ethynylestradiol, 2- fluoroestradiol, estradiol, estrone, 2-methoxy-17-ethynylestradiol, estriol, 2-methoxyestriol, estradiol-3-O-methyl ether, 2-methoxyestradiol-3-O-methyl ether, 4-methoxyestradiol, A- methoxestradiol-3-O-methyl ether, podophyllotoxin, dihydrocombretastatin A-4, 3-benzyl- 2-methoxyestradiol, 3-benzyl-2-methoxyestrone, 16α-alkyl-3-benzyl-2-methoxyestrone, 16β-alkyl-3-benzyl-2-methoxyestrone, 16-alkyl-16-carbomethoxy-3-benzyl-2- methoxyestrone, 16-methane-dimethylenamine-3-benzyl-2-methoxyestrone, 16- carbomethoxy-3-benzyl-2-methoxyestrone, l6-alkyl-3-benzyl-2-methoxyestra-17β-diol, 16-methanol-3-benzyl-2-methoxyestradiol, 16-alkyl-3-benzyl-2-methoxyestradiol, 16β- raethyl-2-methoxyestradiol, 16α-methyl-2-methoxyestradiol, 16-ethyl-2-methoxyestradiol, 16α-n-propyl-2-methoxyestradiol, 16β-n-propyl-2-methoxyestradiol, 16β-n-butyl-2- methoxy estradiol, 16β-isobutyl-2-methoxyestradiol, 16β-methyl(dimethyl amine)-2- methoxyestradiol, 16β-methanol-2-methoxyestradiol, 2-Methoxy-17-deoxyestrone, 17- ethyl-2-methoxyestrone, 17-methyl-2-methoxyestrone, 2-N,N-dimethylamino- 17- deoxyestrone, 2-Azido-estradiol, and 16α-methanol-2-methoxyestradiol (see e.g., U.S. Patent No. 6,528,676 and U.S. Patent Application Publication Nos. 2002/0147183 and 2002/0082433).

[00148] In certain embodiments, 2-methoxyestradiol, or analogs thereof, and compositions useful in the methods described herein may be supplied, e.g., in a kit, with printed instructions which direct the user to employ the compositions in the methods and for the purposes described herein. The instructions for use may be printed on a container housing the composition or on a separate sheet which is included with the composition. Among other things, the instructions, may for example, direct the user to employ the composition and may also state that the purpose of such method is to inhibit or otherwise prevent symptoms of, or associated with, a complication of premature birth, such as, for example, IVH, NEC, ROP and hemangioma formation. The instructions may be directed to use in individuals who may be susceptible to (or predisposed to) a complication of premature birth and/or to those already diagnosed as having a complication of premature birth.

[00149] C. Formulations

[00150] The compositions described herein may be administered by various means, depending on their intended use, as is well known in the art. For example, if compositions are to be administered orally, they may be formulated as tablets, capsules, granules, powders or syrups. Liquid formulations of the compositions may also be formulated as breast milk substitutes or "infant formula." In a certain embodiment, 2-methoxyestradiol, or an analog thereof, is formulated for oral administration as a small tablet or an aqueous solution or suspension. [00151] In certain embodiments, liquid compositions of the formulations, e.g. aqueous solutions or suspensions, are administered enterally by gavage, e.g. via a stomach- tube. Methods of gavage administration are routine and well-known to those of skill in the art. Such suspensions and solutions, may be formulated optionally in association with a sweetened vehicle (such as, for example, a syrup or elixir). The suspension or solution may be formulated at a particular concentration and supplied with a measuring/administering apparatus such as, for example, a tube, a spoon or pipette, so that each unit dose, e.g., each milliliter, teaspoon, tablespoon, etc. of liquid may contain, for example, about 0.25 mg to 1250 mg of 2-methoxyestradiol, or an analog thereof (or combinations thereof), hi certain embodiments, the suspension or solution is administered enterally by gavage tube. [00152] Alternatively, formulations may be administered parenterally as injections

(intravenous, intramuscular, transdermal patch, by aerosols or subcutaneous), drop infusion preparations or suppositories. These formulations may be prepared by conventional means, and, if desired, the compositions maybe mixed with any conventional additive, such as an excipient, a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying agent or a coating agent.

[00153] In formulations described herein, wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may be present in the formulated agents.

[00154] Subject compositions may be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of composition that may be combined with a carrier material to produce a single dose vary depending upon the subject being treated, the severity of the complication of premature birth, the medication status, and the particular mode of administration.

[00155] Methods of preparing these formulations include the step of bringing into association compositions described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association agents with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. [00156] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), each containing a predetermined amount of a subject composition thereof as an active ingredient. Compositions described herein may also be administered as a bolus, electuary, or paste. [00157] hi solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the subject composition is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds or absorption descelerators, such as cristaline formulas; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[00158] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. [00159] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the subject composition, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. [00160] Suspensions, in addition to the subject composition, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[00161] Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing a subject composition with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent. Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[00162] Dosage forms for transdermal administration of a subject composition includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[00163] The ointments, pastes, creams and gels may contain, in addition to a subject composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[00164] Powders and sprays may contain, in addition to a subject composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[00165] Compositions and compounds described herein may alternatively be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A non-aqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers may be used because they minimize exposing the agent to shear, which may result in degradation of the compounds contained in the subject compositions.

[00166] Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of a subject composition together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular subject composition, but typically include non-ionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions. [00167] Pharmaceutical compositions that may be suitable for parenteral administration comprise a subject composition in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[00168] Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[00169] In certain embodiments, the subject compounds may be formulated as a tablet, pill capsule or other appropriate ingestible formulation (collectively hereinafter "tablet"), to provide a therapeutic dose in 10 tablets or fewer. In another example, a therapeutic dose is provided in 50, 40, 30, 20, 15, 10, 5 or 3 tablets. [00170] A tablet form of 2-methoxyestradiol, or an analog thereof, may be formulated such that the amount of 2-methoxyestradiol, or an analog thereof (or combinations thereof), provided in 20 tablets, if taken together, would provide a dose of at least the median effective dose (ED₅₀), e.g., the dose at which at least 50% of individuals exhibited the quantal effect of reduction in at least one symptom associated with a complication of premature birth. In a further embodiment, the tablets are formulated such that the total amount of 2-methoxyestradiol, or an analog thereof (or combinations thereof), provided in 10, 5, 2 or 1 tablets would provide at least an ED₅₀ dose to a patient (human or non-human mammal). In other embodiments, the amount of 2-methoxyestradiol, or an analog thereof (or combinations thereof), provided in 20, 10, 5 or 2 tablets taken in a 24 hour time period would provide a dosage regimen providing, on average, a mean plasma level of the 2-methoxyestradiol, or an analog thereof (or combinations thereof), of at least the ED₅₀ concentration, hi other embodiments less than 100 times, 10 times, or 5 times the ED₅₀ is provided. In other embodiments, a single dose of tablets (1-20 tablets) provides about 0.25 mg to 1250 mg of 2-methoxyestradiol, or an analog thereof (or combinations thereof). In an exemplary embodiment, a single dose of tablets (1-20 tablets) provides about 0.5 to 5 mg, about 1 to about 3 mg, or about 2.5 mg of 2-methoxyestradiol, or an analog thereof (or combinations thereof).

[00171] Likewise, 2-methoxyestradiol, or an analog thereof, can be formulated for parenteral administration, as for example, for subcutaneous, intramuscular or intravenous injection, e.g., 2-methoxyestradiol, or an analog thereof, can be provided in a sterile solution or suspension (collectively hereinafter "injectable solution"). The injectable solution is formulated such that the amount of 2-methoxyestradiol, or an analog thereof (or combinations thereof), provided in a 200cc bolus injection would provide a dose of at least the median effective dose, or less than 100 times the ED₅₀, or less than 10 or 5 times the ED₅₀. The injectable solution may be formulated such that the total amount of 2- methoxyestradiol, or an analog thereof (or combinations thereof), provided in 100, 50, 25, 10, 5, 2.5, or 1 cc injections would provide an ED₅₀ dose to a patient, or less than 100 times the ED₅₀, or less than 10 or 5 times the ED₅₀. In other embodiments, the amount of 2- methoxyestradiol, or an analog thereof (or combinations thereof), provided in a total volume of lOOcc, 50, 25, 5 or 2cc to be injected at least twice in a 24 hour time period would provide a dosage regimen providing, on average, a mean plasma level of the 2- methoxyestradiol, or an analog thereof (or combinations thereof), of at least the ED₅₀ concentration, or less than 100 times the ED₅₀, or less than 10 or 5 times the ED₅₀. In other embodiments, a single dose injection provides about 0.25 mg to 1250 mg of 2- methoxyestradiol, or an analog thereof (or combinations thereof). In an exemplary embodiment, a single dose injection provides about 0.5 to 5 mg, about 1 to about 3 mg, or about 2.5 mg of 2-methoxyestradiol, or an analog thereof (or combinations thereof).

EXAMPLES

[00172] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

[00173] Example 1: The role of 2-methoxyestradiol in complications resulting from premature birth

[00174] 2-methoxyestradiol (2ME2) is a natural metabolite of estrogen with little or no estrogen activity. It is a potent angiogenesis inhibitor that suppresses expression of hypoxia inducible factor 1 alpha (HIF- lα). HIF- lα is a pro-angio genie transcription factor that stimulates transcription of multiple pro-angiogenic proteins including vascular endothelial growth factor (VEGF). VEGF is an endothelial mitogen and survival factor that induces permeability in blood vessels. VEGF is 50,000 times more potent than histamine as a vascular leakage agent.

[00175] The median level of 2ME2 in men is < 10 pg/mL and in women of reproductive age is 46 pg/mL in the follicular phase and 70 pg/mL in the luteal phase. During pregnancy, serum 2ME2 in the mother increases dramatically reaching a median of 674 pg/mL at 11-16 weeks. The serum level peaks at 3768 pg/mL by 37-40 weeks gestation; almost a 100 fold increase over pre-pregnancy levels. Rising 2ME2 levels during pregnancy may protect against surges of HIF-I α in response to brief episodes of hypoxia in the placenta and fetus.

[00176] Newborn cord serum contains 2ME2 at a median of 1606 pg/mL, about 40% of the level in the maternal blood stream at term (see FIGURE 1). There are no data regarding the levels of 2ME2 in cord blood or serum of premature babies, amniotic fluid of preterm or term infants, or their mothers' colostrum or breast milk. Furthermore, there are no published data about when this level falls back to "normal" in the neonate, whether preterm or term. However, by extrapolating from the data above with the fetus's 2ME2 level 40% that of the mother's, we predict that the 2ME2 level of a 25 week gestation infant at birth will be -750 pg/mL. Furthermore, if the half life of 2ME2 is similar in premature infants as the 10 hour half life in adults, then 2ME2 would fall to less than 25 pg/mL within the first 2.5 days postnatally.

[00177] We expect that the 2ME2 levels at birth are directly proportional to gestational age; the more preterm the infant, the lower the 2ME2 level at birth, rising with duration in utero and eventually reaching that of a term newborn. Since preterm babies are unlikely to make significant quantities of their own 2ME2, they would be limited to that provided by maternal and placental sources, and levels would likely drop off rapidly based on the approximately 10 hour half- life of 2ME2. Because 2ME2 is necessary to protect against HIF- lα, hypoxic episodes in a baby with low 2ME2 levels would lead to rapid burst of HIF-lα with subsequent down stream events such as increases in circulating VEGF. [00178] The biological effects of low levels of 2ME2 and consequent high levels of

VEGF could contribute to many of the complications of prematurity. It could be causal for intraventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP) and hemangioma for two reasons. First, the loss of a direct anti- angiogenic effect of the 2ME2 and second, the resulting high levels of VEGF, due to lack of inhibition of HIF-I α, leading to vascular leak and sprout formation. In summary: [00179] Infant born at term exposed to normal third trimester surge of 2ME2:

[00180] φ 2ME2 ->Φ HIF-I (->Ψ VEGF (and normal blood vessel formation)

[00181] Preterm infant delivered prior to third trimester surge of 2ME2:

[00182] Φ 2ME2-> φ HIF-I ( -> φVEGF-> φvascular leak (IVH, NEC) and

[00183] φvascular sprout formation (ROP, hemangioma)

[00184] The amniotic fluid, cord blood, or maternal serum may have protein biomarkers that could predict if a baby is at increased risk of developing IVH, NEC and ROP prior to clinical signs of the disease. Such a panel of markers will help determine which patients are at higher risk of developing these complications. We hypothesize that the balance of pro and anti-angiogenic factors is relatively tipped towards excess angiogenesis in infants born prematurely compared to in-utero, gestationally age matched controls. This excess angiogenesis may be due to a relative deficiency of the powerful anti- angiogenic factor 2 methoxyestradiol (2ME2) in preterm infants, and this may lead to increased risk of IVH, retinal neovascularization and ROP, NEC, and potentially life- threatening hemangiomas. [00185] We hypothesize that the steadily rising blood levels of 2-methoxyestradiol in the blood of a pregnant woman which are paralleled in the blood of a fetus, protects the fetus against surges of hypoxia inducible factor- 1 alpha (HIF-I alpha), in response to brief episodes of hypoxia in the placenta.

[00186] We will collect blood, amniotic fluid and breast milk from preterm infants and their mothers as well as healthy term controls in order to compare an array of pro and anti-angiogenic factors and collect outcome data regarding IVH, NEC, ROP and hemangioma, as described in the Examples below. A unifying molecular basis for the increased incidence of the pathologic states associated with prematurity may be provided and may lead to preventive therapy.

[00187] The identification at birth of which of the babies born prematurely will develop IVH, NEC, or ROP prior to the development of clinical symptoms would allow for a tailored treatment for these babies. Replacement therapy with a correct angiogenic cocktail administered orally or parenterally may be sufficient to prevent these complications (FIGURE 2). Currently, 2ME2 is being tested in clinical trials (phase 2 and phase 3) for advanced prostate cancer, with no side effects reported. Of note, it appears to inhibit the growth of abnormal (e.g. oncologic) blood vessels but does not affect normal vessel growth or activity. This offers the theoretical opportunity to eventually supplement 2ME2 if we learn that prematurely born infants in fact have a relative deficiency of this anti-angiogenesis factor.

[00188] Li particular, the following questions may be addressed during this study:

[00189] 1. Is there an association between preterm labor and low 2ME2 levels? By comparing 2ME2 levels of pregnant women in preterm labor with controls matched by gestational age who are not in preterm labor, we can determine if there is an association between 2ME2 levels and pre-term labor (PTL), raising the causal question of whether low 2ME2 triggers premature delivery.

[00190] 2. Betamethasone given to mothers prior to pre-term delivery in an effort to mature pulmonary function also decreases IVH rate, for unclear reasons. Does betamethasone stimulate 2ME2 production by the placenta, a potential mechanism for the decreased IVH rate?

[00191] 3. Can premature infants who will eventually develop IVH, NEC, ROP or hemangiomas be identified at birth, prior to the signs of disease, using a panel of protein biomarkers of maternal serum, amniotic fluid, or cord blood? [00192] 4. Do premature infants who develop IVH, NEC, ROP or hemangiomas have decreased 2ME2 levels at birth compared to age-matched premature babies without these complications?

[00193] 5. What is the half-life of the infants' 2ME2? Does a shorter half-life create a risk of inappropriately short protection against hypoxic outbursts?

[00194] 6. Does the rate of fall of 2ME2 of the mother of a premature infant predict the rate of fall of the infant?

[00195] 7. Since amniotic fluid theoretically contains 2ME2 as well as other pro and anti-angiogenic factors, is it a potential source of a balanced angiogenesis "cocktail."

[00196] 8. Breast milk may contain 2ME2 that could offer protection to premature infants. What are 2ME2 levels in breast milk and are breast-fed premature infants protected against IVH, NEC, ROP and hemangiomas relative to non-breast fed premature infants?

[00197] Prophetic Example 2: Determination of the levels of 2-methoxyestradiol at birth (cord blood, amniotic fluid, maternal plasma), as well as the 2ME2 half-life in infant's plasma and maternal breast milk

[00198] Gas Chromatography- Mass Spectrometry (GC-MS) may be used to measure the levels of 2ME2 or analogs thereof in cord blood, amniotic fluid, plasma, breast milk, and the like. GC-MS is the gold standard for detection and quantitation of low molecular weight steroids, such as 2-methoxyestradiol (Mw=^:302 g/mol).

[00199] We will start with a pilot study to refine our laboratory procedure including determining the rough range and kinetics of 2ME2 levels in pregnant women and newborn infants in order to fine tune how many samples we will need from each baby. Therefore, we will measure 2ME2 in -270 samples, derived from 5 mothers of term infants (37-42 weeks gestation) delivered by C section (Example 2a), 5 mothers and their term infants requiring hospitalization in the NICU (Example 2b) and 10 mothers and their extremely premature (< 28 weeks gestation) infants (Example 2c). These samples, as well as all others through the study will be 2 - 10 niL in volume.

[00200] Because blood samples cannot be collected from normal term infants (as they do not require any for conventional analyses), we will only collect cord blood, maternal serum, amniotic fluid, and maternal colostrum, from normal term babies born after

C-section. The 2ME2 half-life will be determined in infants born at term (37-40 weeks of gestation) but that require hospitalization at the NICU and that require multiple blood analyses over time. [00201] The data from the term infants will serve as controls in order to determine range of 2ME2 levels and to assess the rate of fall of 2ME2 levels in the newborn and in breast milk. The data from the premature infants will be analyzed to determine whether 2ME2 in premature infants follows the same kinetics as in term infants. These data will help us refine our laboratory assays and determine how many samples need to be drawn from the rest of the preterm infants based on when 2ME2 levels become undetectable in premature and term infants.

[00202] Example 2A, pilot study, mothers of babies delivery by C section at term

[00203] Patient inclusion/exclusion criteria; 37 0/7 - 42 completed weeks gestation at birth, delivered by C section, healthy post delivery, preferably with expectation to nurse [00204] Number of patients: 5 mothers

[00205] Samples to be collected: Total samples = 50

[00206] Table I

Samples/patient Sample Volume Time point

Maternal serum 4 (spin, freeze) 2 cc #1: before delivery

#2: post partum day 1

#3: post partum day 2

#4: post partum day 3

Amniotic fluid 1 (spin and freeze) lOcc at delivery

Cord blood 1 (spin and freeze) 2-5cc

Colostrum (if possible) 1 (freeze) 2-5 cc within first 24 hours

Breast milk (if possible) 3 (freeze) 2-5 cc #1: post partum day 1

#2: post partum day 2

#3: post partum day 3

[00207] Example 2B, pilot study, mothers and their sick term infants admitted to

NICU

[00208] Patient inclusion/exclusion criteria; 37 0/7 - 42 completed weeks gestation at birth, admitted to NICU for any reason with expected length of stay > 4 days and intention to breast feed.

[00209] Number of patients: 5 mothers and 5 babies

[00210] Samples to be collected: Total samples = 60.

[00211] Table II

Samples/patient Sample Volume Time point

Maternal serum 3 2 cc #1: post partum day 1

#2: post partum day 2

#3: post partum day 3

Infant's blood^Λ 4 (spin and freeze) 1 cc #1:DOL # 1

#2: DOL # 2 #3: DOL # 4

#4: DOL # 10*

Colostrum 1 (freeze) 2-5 cc within first 24 hours

Breast milk 3 (freeze) 2-5 cc #1:DOL # 1

#2: DOL # 2

#3: DOL # 4

#4: DOL # 10*

DOL = day of life

^Λ ALWAYS to be coordinated with clinically indicated blood draw.

* 10 or time of discharge whichever comes first.

[00212] Example 2C, pilot study, mothers and their premature infants

[00213] Patient inclusion/exclusion criteria: < 28 weeks gestation (e.g. through 28

6/7 weeks), preferentially delivered by C section with intention to breast feed.

[00214] Number of patients: 10 mothers and 10 babies

[00215] Samples to be collected: Total samples = 160

[00216] Table III

Samples/patient Sample Volume Time point

Maternal serum 5 (spin and freeze) 2 cc #1: pre steroids, pre delivery

#2: post steroids, pre delivery

#3: post partum day 1

#4: post partum day 2

#5: post partum day 3

Amniotic fluid 1 (spin and freeze) 10 cc at delivery

Cord blood 1 (spin and freeze) 2-5 cc at delivery

Infant's blood^Λ 4 (spin and freeze) 0.3 - 1 cc #1:DOL #1

< 1 kg: 0.5 cc #2:DOL #2

> l kg: 1.0 cc #3:DOL #4

#4:DOL #10*

Colostrum 1 (freeze) 2-5 cc within first 24 hours

Breast milk 4 (freeze) 2-5 cc #1:DOL #1

#2: DOL #2

#3: DOL #4

#4: DOL #10*

DOL = day of life

^Λ ALWAYS to be coordinated with clinically indicated blood draw.

* 10 or time of discharge whichever comes first.

Details of blood volume:

Goal is to send 1 cc whenever possible

Requirement: 3cc/kg every 8 weeks

For 0.5 kg baby, max is 1.5 cc = 4 x 0.37 cc

For 1 kg baby, max is 3 cc = 4 x 0.75 cc samples

For 1.5 kg, max is 4.5 cc = 4 x 1.1 cc samples

For 2 kg, max is 6 cc, so 4 x 1.5 cc samples [00217] Samples will be collected in Li-heparine tubes spun (with the exception of breast milk) and the plasma phase will be frozen at -70 degrees C, preferably within 30 minutes after blood withdrawal.

[00218] Prophetic Example 3: Correlation of a panel of pro and anti-angiogenic factors measured in cord blood with outcome data regarding IVH, NEC, ROP and hemangioma formation.

[00219] Using multiplex antibody platforms and ELISAs₃ we will determine the relative abundance of different growth factors in order to assess the net "angiogenic balance" by gestational age, and correlate this with IVH, NEC, ROP and infantile hemangioma formation. For this aim, we will collect amniotic fluid and cord blood samples, or use archived samples if available. 2-5 mL samples from babies at < 32 weeks gestation (e.g. through 32 6/7 weeks), with cord blood available for collection as discarded human material, will be used.

[00220] Based on a 5-10% prevalence rate of IVH (any grade), NEC and ROP (any stage), and a goal of collecting cord blood from 15-20 babies who go onto develop each of the above complications, we estimate that we will need to collect approximately 300 samples of cord blood.

[00221] We will collect cord blood as discarded human material from mother/infant diads enrolled in this part of the study. Based on our hypothesis, these four complications arise from predominantly pro-angiogenic environment. The cord blood will be analyzed for the following factors, and correlated with outcome data regarding IVH, NEC, ROP and hemangioma formation:

[00222] 1. 2ME2 plasma levels, to establish if babies that develop these four complications have lower levels of this angio genesis inhibitor at birth. [00223] 2. Plasma levels of multiple angiogenic and anti-angiogenic markers.

We would like to determine if babies that develop IVH, NEC, ROP, or hemangioma have at the time of birth decreased levels of other known anti-angiogenic factors (such as thrombospondin) or increased levels of angiogenic growth factors (such as VEGF, bFGF, PlGF), relative to gestationally-matched control infants. We will use multiplex monoclonal antibody microarray systems that allow the simultaneous measurement of dozens to hundreds of biomarkers with a small sample size.

[00224] 3. Biomarker discovery. If we find that the angiogenic/antiangio genie profiling does not reveal a set of biomarkers that can accurately predict the patients' outcome, we will then use 2-dimensional gel electrophoresis and the DIGE System, to identify novel proteins that could serve as biomarkers for IVH₅ NEC, ROP, or hemangioma. [00225] Prophetic Example 4: Discovery and quantification of protein biomarkers in cord blood:

[00226] Detection of panels of biomarkers may provide higher sensitivities and specificities for diagnosis and prognosis than single markers. We will determine whether alterations in protein abundance in cord blood plasma can be predictive indicators of IVH, NEC, ROP and infantile hemangioma formation in premature infants prior to the development of clinical symptoms. We will use the DIGE System (GE) to discover novel protein biomarkers.

EQUIVALENTS

[00227] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The appended claims are not intended to claim all such embodiments and variations, and the full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. [00228] All publications and patents mentioned herein, including those listed below, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. [00229] Ward, R.M. & Beachy, J.C. Neonatal complications following preterm birth.

Bjog 110 Suppl 20, 8-16 (2003).

[00230] D'Amato, RJ., Lin, CM., Flynn, E., Folkman, J. & Hamel, E. 2-

Methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site. Proc Natl Acad Sd USA 91, 3964-8 (1994). [00231] Fotsis, T. et al. The endogenous oestrogen metabolite 2-methoxyoestradiol inhibits angiogenesis and suppresses tumour growth. Nature 368, 237-9 (1994). [00232] Mabjeesh, NJ. et al. 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulatiiig HIF. Cancer Cell 3, 363-75 (2003). [00233] Kieran, M. W., Folkman, J. & Heymach, J. Angiogenesis inhibitors and hypoxia. Nat Med 9, 1104; author reply 1104-5 (2003). [00234] Liu, Y., Cox, S.R., Morita, T. & Kourembanas, S. Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells. Identification of a 5' enhancer. Circ Res 77, 638-43 (1995).

[00235] Forsythe, J. A. et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. MoI Cell Biol 16, 4604-13 (1996).

[00236] Rosenthal, R.A., Megyesi, J.F., Henzel, WJ., Ferrara, N. & Folkman, J.

Conditioned medium from mouse sarcoma 180 cells contains vascular endothelial growth factor. Growth Factors 4, 53-9 (1990).

[00237] Shulman, K., Rosen, S., Tognazzi, K., Manseau, EJ. & Brown, L.F.

Expression of vascular permeability factor (VPF/VEGF) is altered in many glomerular diseases. JAm Soc Nephrol 7, 661-6 (1996).

[00238] Xiao, Z., Prieto, D., Conrads, T.P., Veenstra, T.D. & Issaq, HJ. Proteomic patterns: their potential for disease diagnosis. MoI Cell Endocrinol 230, 95-106 (2005).

[00239] Nelson, R.E., Grebe, S.K., DJ, O.K. & Singh, RJ. Liquid chromatography- tandem mass spectrometry assay for simultaneous measurement of estradiol and estrone in human plasma. Clin Chem 50, 373-84 (2004).

Claims

Claimed are:

1. A method for treating or preventing a complication of premature birth comprising administering to a subject in need thereof an effective amount of 2- methoxyestradiol (2-ME), an analog thereof, or a pharmaceutically acceptable salt or prodrug thereof.

2. The method of claim 1, wherein the method further comprises first identifying a subject susceptible to a complication of premature birth.

3. The method of claim 1, wherein the method further comprises first identifying a subject suffering from a complication of premature birth.

4. The method of claim 1, wherein the method further comprises determining the level of 2-methoxyestradiol and/or an analog thereof in a biological sample from said subject.

5. The method of claim 4, wherein the level of 2-methoxyestradiol and/or an analog thereof is determined by determining the level of a precursor or metabolite of 2- methoxyestradiol and/or an analog thereof.

6. The method of claim 4, wherein the method further comprises comparing said level of 2-methoxyestradiol and/or an analog thereof to the level of 2- methoxyestradiol in a control.

7. The method of claim 1, wherein the method further comprises determining the level of a precursor of 2-methoxyestradiol in a biological sample from said subject.

8. The method of claim 7, wherein the method further comprises comparing said level of the 2-methoxyestradiol precursor to the level of the 2-methoxyestradiol precursor in a control.

9. The method of claim 1, wherein the method further comprises determining the level of a metabolite of 2-methoxyestradiol and/or an analog thereof in a biological sample from said subject.

10. The method of claim 9, wherein the method further comprises comparing said level of a metabolite of 2-methoxyestradiol and/or an analog thereof to the level of the metabolite of 2-methoxyestradiol in a control.

11. The method of claim 4, wherein said biological sample is urine, blood or plasma.

12. The method of claim 4, wherein said control is a predetermined standard from at least one subject not suffering from a complication of premature birth.

13. The method of claim 6, wherein if the level of 2-methoxyestradiol in said subject sample is lower than the level of 2-methoxyestradiol in said control, the method further comprises administering to said subject an amount of 2-methoxyestradiol, or an analog thereof, effective to increase the level of 2-methoxyestradiol and/or an analog thereof in said subject to a level that is about the same as the level of 2-methoxyestradiol in said control.

14. The method of claim 6, wherein the method comprises determining the level of 2-methoxyestradiol and/or an analog thereof in said subject as compared to a control on a regular basis.

15. The method of claim 14, wherein said regular basis is once about every 1, 2, 3, 4, 5, 6, 7, 10, 14, 28, or 35 days.

16. The method of claim 14, which further comprises adjusting the level of 2- methoxyestradiol, or analog thereof, administered to said subject on a regular basis so that the level of 2-methoxyestradiol and/or an analog thereof is maintained in said subject at a level that is about the same as the level of 2-methoxyestradiol in a control.

17. The method of claim 1 , wherein said subject is a human.

18. The method of claim 1, wherein about 0.1, 0.3, 1, 2.5, or 5 mg of said 2- methoxyestradiol, or analog thereof, is administered to said subject.

19. The method of claim 1, wherein said 2-methoxyestradiol, or analog thereof, is administered orally, nasally, parenterally or transdermally.

20. The method of claim 1, wherein 2-methoxyestradiol, or analog thereof, is administered enterally by gavage tube.

21. The method of claim 1, which further comprises monitoring said subject for one or more symptoms of a complication of premature birth.

22. The method of claim 1, wherein said complication of premature birth is selected from the group consisting of: intraventricular hemorrhage, necrotizing enterocolitis, retinopathy of prematurity and hemangioma.

23. The method of claim 1, which further comprises administering to said subject at least one second therapeutic agent that is not 2-methoxyestradiol, an analog thereof, or a pharmaceutically acceptable salt or prodrug thereof.

24. The method of claim 23, wherein said second therapeutic agent is administered to said subject concurrently with said 2-methoxyestradiol, or an analog thereof.

25. The method of claim 1, wherein the 2-methoxyestradiol analog is a compound of Formula I:

I wherein, independently for each occurrence: R is H, alkyl, aryl, aralkyl, or carbonyl; Ri and R₂ are -OR, -SR, or -N(R)₂; R₃ is H, halide, alkyl, aryl, aralkyl, or carbonyl;

R₄, R₅, and R₆ is are H, halide, hydroxy, alkoxy, amino, thiol, alkyl, aryl, aralkyl, or carbonyl; m is an integer from 1-5 inclusive; and n and p are an integer from 1-6 inclusive.

26. The method of claim 1, wherein the 2-methoxyestradiol analog is colchicine or combretastatin A-4.

27. A method for treating or preventing a complication of premature birth, comprising: a) determining the level of 2-methoxyestradiol in a biological sample from a preterm subject and comparing said level to the level of 2-methoxyestradiol in a control, wherein said control is a predetermined standard from at least one non-preterm subject or a preterm subject having the same gestational age as said preterm subject that does not have a complication of premature birth; b) administering to said preterm subject an amount of 2-methoxyestradiol, or an analog thereof, sufficient to raise the level of 2-methoxyestradiol and/or an analog thereof in a biological sample of said subject to a level that is about the same as the level of 2-methoxyestradiol in said control; and c) repeating steps a) and b) on a regular basis thereby maintaining the level of 2-methoxyestradiol in said subject at a level that is about the same as the level of 2- methoxyestradiol in the control.

28. The method of claim 27, wherein said biological sample is urine, blood or plasma.

29. The method of claim 27, wherein said method further comprises the first identifying a subject susceptible to, or suffering from, a complication of premature birth.

30. A method for diagnosing, or predicting the development of, a complication of premature birth in a subject comprising determining the level of 2- methoxyestradiol in a biological sample from said subject and comparing said level to the level of 2-methoxyestradiol in a control, wherein said control is a predetermined standard from at least one subject not having a complication of premature birth at about the gestational age as said subject or at least one subject that is a term infant, and wherein a lower level of 2-methoxyestradiol in said subject sample as compared to the control is indicative of a subject susceptible to, or suffering from, a complication of premature birth.

31. The method of claim 30, wherein the severity of the complication of premature birth is determined based on the degree of difference in the level of 2- methoxyestradiol in said subject as compared to the control and wherein a greater deviation from the control is indicative of a more severe complication of preterm birth.

32. The method of claim 31, wherein said biological sample is urine, blood or plasma.

33. The method of claim 32, wherein said subject is a human.

34. A method for diagnosing, or predicting the development of, a complication of premature birth in a subject comprising determining the level of a 2- methoxyestradiol metabolite in a biological sample from said subject and comparing said level to the level of the 2-methoxyestradiol metabolite in a control, wherein said control is a predetermined standard at least one subject not having a complication of premature birth at about the gestational age as said subject or at least one subject that is a term infant, and wherein a lower level of the 2-methoxyestradiol metabolite in said subject sample as compared to the control is indicative of a subject susceptible to, or suffering from, a complication of premature birth.

35. A method for diagnosing, or predicting the development of, a complication of premature birth in a subject comprising determining the level of a 2- methoxyestradiol precursor in a biological sample from said subject and comparing said level to the level of the 2-methoxyestradiol precursor in a control, wherein said control is a predetermined standard from at least one subject not having a complication of premature birth at about the gestational age as said subject or at least one subject that is a term infant, and wherein a change in the level of the 2-methoxyestradiol precursor in said subject sample as compared to the control is indicative of a subject susceptible to, or suffering from, a complication of premature birth.

36. The method of claim 35, wherein a higher level of the 2-methoxyestradiol precursor in said subject sample as compared to the control is indicative of a subject susceptible to, or suffering from, a complication of premature birth.