Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/06—Antiabortive agents; Labour repressants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• thermoregulation 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.
• 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.
• the method comprises administering to the infant an effective amount of a 2-methoxyestradiol containing composition.
• 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.
• 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.
• FIGURE 2 depicts restoration of physiological levels of 2ME2 in the extremely premature infant to levels found in serum in gestationally age matched controls.
• control 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.
• 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.).
• 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).
• 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.
• 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.
• IVH intraventricular hemorrhage
• NEC necrotizing enterocolitis
• ROP retinopathy of prematurity
• hemangioma hemangioma
• 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.
• 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.
• 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".
• a 2-methoxyestradiol analog exhibits a therapeutic effect when administered to a subject suffering from a complication of premature birth.
• a 2-methoxyestradiol analog exhibits greater stability when administered to a subject than 2-methoxyestradiol itself.
• a 2-methoxyestradiol analog is a compound of Formula I.
• 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.
• analogs of 2-methoxyestradiol include colchicine and combretastatin A-4.
• precursors of 2-methoxyestradiol 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.
• precursors of 2-methoxyestradiol include, for example, estradiol, 2-hydroxyestradiol, etc.
• metabolite 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.
• metabolites of 2- methoxyestradiol include, for example, 2-methoxyestrone, etc.
• 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).
• a subject may be a human subject.
• control level 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.
• Cis configurations are often labeled as (Z) configurations.
• 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.
• 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.
• covalent bond includes coordinate bonds when the bond is with a metal ion.
• therapeutic agent refers to any chemical moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject.
• therapeutic agents also referred to as "drugs”
• drug 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.
• 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.
• 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.
• 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.
• compositions described herein may be administered in a sufficient amount to produce a at a reasonable benefit/risk ratio applicable to such treatment.
• 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.
• 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.
• 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.
• 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.
• 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.
• esters are 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.
• ED 50 is art-recognized, hi certain embodiments, ED 50 means the dose of a drug which produces 50% of its maximum response or effect, or alternatively, the dose which produces a pre-dete ⁇ nined response in 50% of test subjects or preparations.
• LD 5 o is art-recognized, hi certain embodiments, LD 50 means the dose of a drug which is lethal in 50% of test subjects.
• therapeutic index is an art-recognized term which refers to the therapeutic index of a drug, defined as LD 5 o/ED 5O .
• prodrug is art-recognized and is intended to encompass
• a prodrug 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.
• the prodrug is converted by an enzymatic activity of the host animal.
• 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.
• 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.
• a straight chain or branched chain alkyl has about 30 or fewer carbon atoms in its backbone (e.g., C 1 -C 30 for straight chain, C 3 -C 30 for branched chain), and alternatively, about 20 or fewer.
• 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.
• alkyl is also defined to include halosubstituted alkyls.
• aralkyl is art-recognized and refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
• 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.
• 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.
• lower alkenyl and “lower alkynyl” have similar chain lengths.
• 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.
• 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.
• 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, -CF 3 , -CN, or the like.
• 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.
• ortho, rneta and para are art-recognized and refer to 1,2-, 1,3- and
• 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,
• 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, -CF 3 , -CN, or the like.
• substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxy
• 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, -CF 3 , -CN, or the like.
• substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, si
• carrier is art-recognized and refers to an aromatic or non- aromatic ring in which each atom of the ring is carbon.
• nitro is art-recognized and refers to -NO 2 ;
• halogen is art-recognized and refers to -F, -Cl, -Br or -I;
• sulfhydryl is art-recognized and refers to -SH;
• hydroxyl means -OH;
• sulfonyl is art-recognized and refers to -SO 2 " .
• 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.
• 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, R51 and R52 each independently represent a hydrogen, an alkyl, an alkenyl, -(CH 2 ) 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.
• only one of R50 or R51 may be a carbonyl, e.g., R50, R51 and the nitrogen together do not form an imide.
• R50 and R51 each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH 2 ) m -R61.
• 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.
• acylamino is art-recognized and refers to a moiety that may be represented by the general formula:
• R50 is as defined above, and R54 represents a hydrogen, an alkyl, an alkenyl or -(CH 2 ) m -R61, where m and R61 are as defined above.
• R54 represents a hydrogen, an alkyl, an alkenyl or -(CH 2 ) m -R61, where m and R61 are as defined above.
• amido is art recognized as an amino-substituted carbonyl and includes a moiety that may be represented by the general formula:
• alkylthio refers to an alkyl group, as defined above, having a sulfur radical attached thereto.
• the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH 2 ) m -R61, wherein m and
• R61 are defined above.
• Representative alkylthio groups include methylthio, ethyl thio, and the like.
• carbonyl is art recognized and includes such moieties as may be represented by the general formulas:
• X50 is a bond or represents an oxygen or a sulfur
• R55 and R56 represents a hydrogen, an alkyl, an alkenyl, -(CH 2 ) m -R61 or a pharmaceutically acceptable salt
• R56 represents a hydrogen, an alkyl, an alkenyl or -(CH 2 ) m -R61, where m and R61 are defined above.
• X50 is an oxygen and R55 or R56 is not hydrogen
• the formula represents an "ester”.
• X50 is an oxygen
• 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".
• X50 is an oxygen, and R56 is hydrogen
• the formula represents a "formate".
• the oxygen atom of the above formula is replaced by sulfur
• the formula represents a "thiolcarbonyl” group.
• X50 is a sulfur and R55 or R56 is not hydrogen
• the formula represents a "thiolester.”
• X50 is a sulfur and R55 is hydrogen
• the formula represents a "thiolcarboxylic acid.”
• X50 is a sulfur and R56 is hydrogen
• the formula represents a "thiolformate.”
• X50 is a bond, and R55 is not hydrogen
• the above formula represents a "ketone” group.
• X50 is a bond, and R55 is hydrogen
• the above formula represents an "aldehyde” group.
• 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--(CH 2 ) m -R61, where m and R61 are described above.
• sulfonate is art recognized and refers to a moiety that may be represented by the general formula:
• R57 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.
• R57 is as defined above.
• sulfamoyl is art-recognized and refers to a moiety that may be represented by the general formula:
• 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.
• sulfoxido is art-recognized and refers to a moiety that may be represented by the general formula:
• Q50 represents S or O
• R59 represents hydrogen, a lower alkyl or an aryl.
• the phosphoryl group of the phosphorylalkyl may be represented by the general formulas:
• 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.
• 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.
• 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-(CH 2 ) m -R61, m and R61 being defined above.
• triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, ⁇ -toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively.
• triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, j ⁇ -toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.
• Me 5 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.
• compositions described herein may exist in particular geometric or stereoisomeric forms.
• 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.
• a particular enantiomer of a compound described herein 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.
• 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.
• 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.
• substituted is also contemplated to include all permissible substituents of organic compounds.
• 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.
• 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.
• hydrocarbon is contemplated to include all permissible compounds having at least one hydrogen and one carbon atom.
• the permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compounds that may be substituted or unsubstituted.
• protecting group is art-recognized and refers to temporary substituents that protect a potentially reactive functional group from undesired chemical transformations.
• 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).
• 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.
• carboxyl-protecting group 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.
• amino-blocking group 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.
• amino-blocking group 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).
• 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;
• Preferred amino-blocking groups are benzyl (-CH 2 C 6 Hs), acyl [C(O)Rl] or SiRl 3 where Rl is C 1 -C 4 alkyl, halomethyl, or 2-halo-substituted-(C 2 -C 4 alkoxy), aromatic urethane protecting groups as, for example, carbonylbenzyloxy (Cbz); and aliphatic urethane protecting groups such as t-butyloxycarbonyl (Boc) or 9- fluorenylmethoxycarbonyl (FMOC).
• 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.
• electronegative with respect to neighboring atoms 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.
• ⁇ Hammett sigma
• 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.
• 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.
• up regulate e.g., activate or stimulate
• down regulate e.g., inhibit or suppress
• 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.
• treating is art-recognized and refers to curing as well as ameliorating at least one symptom of any condition or disease.
• 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.
• prophylactic or therapeutic treatment of a complication of premature birth refers to administration to the host of one or more of the subject compositions.
• 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).
• a "patient,” “subject” or “host” to be treated by the subject method may mean either a human or non-human animal.
• mammal is known in the art, and exemplary mammals include humans, primates, bovines, porcines, canines, felines, and rodents (e.g., mice and rats).
• 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.
• pharmaceutically-acceptable salts refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds, including, for example, those contained in compositions described herein.
• pharmaceutically acceptable carrier 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.
• a pharmaceutically-acceptable material 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.
• 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;
• 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.
• 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.
• 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.
• 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.
• Premature birth Provided herein are methods and compositions for treating, preventing and/or diagnosing complications of premature birth.
• methods and compositions for treating or preventing a complication of premature birth using 2- methoxyestradiol, or an analog thereof are provided.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• screening for levels of 2-methoxyestradiol may be used to identify subjects that may be candidates for therapeutic treatment with 2-methoxyestradiol, or an analog thereof.
• screening for levels of 2-methoxyestradiol may be used to calculate an effective dose for administering to a subject.
• 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.
• 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.
• IVH intraventricular hemorrhage
• NEC necrotizing enterocolitis
• ROP retinopathy of prematurity
• 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.
• Screening for levels of 2-methoxyestradiol may be used to monitor the course of treatment with 2-methoxyestradiol, or an analog thereof.
• the level of 2-methoxyestradiol 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.
• the level of 2-methoxyestradiol in a subject may be determined directly by measuring the level of 2-methoxyestradiol itself.
• the level of 2- methoxyestradiol may be determined indirectly by measuring the level of a precursor or a metabolite of 2-methoxyestradiol.
• 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.
• the level of a precursor or a metabolite of 2-methoxyestradiol, or an analog thereof may be measured directly for diagnostic or monitoring purposes.
• 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.
• the level of 2-methoxyestradiol, or an analog thereof may be determined in a biologic sample of a subject or a control subject.
• the level of 2-methoxyestradiol may be determined in a urine, blood or plasma sample from a subject.
• the level of 2-methoxyestradiol, or analog thereof, in a biological sample of a subject may be compared directly to a control.
• 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.
• 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 may be determined using any method known in the art.
• 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 gas chromatography- mass spectrometry
• GC-MS is the gold standard for detection and quantitation of low molecular weight steroids, such as 2-methoxyestradiol and/or its analogs.
• 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.
• TLC thin layer chromatography
• HPLC high performance liquid chromatography
• 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.
• 2-methoxyestradiol such as estrogen, estradiol, and the like
• 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.
• hemagglutination-inhibition kit Hi- Estrotec
• Hi- Estrotec hemagglutination-inhibition kit
• Another example of an estrogen assay that may be used in the methods of the invention is Applied Biosystems' HitHunterTM Enzyme Fragment Complementation (EFC) Assay. This assay is based on an engineered ⁇ -galactosidase enzyme that consists of two fragments - the Enzyme Acceptor (EA) and the Enzyme Donor(ED).
• 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 ⁇ -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.
• 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.
• 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.).
• 2-methoxyestradiol or a precursor or metabolite thereof
• a cranial ultrasound on the subject before, during and/or after treatment with the subject 2-methoxyestradiol, or an analog thereof 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.
• 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 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
• 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.
• 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.
• 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®.
• the drugs may be formulated and administered together as a mixture or may be separately formulated and administered.
• 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.
• 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.
• it may be desirable to administer 2-methoxyestradiol and/or an analog thereof to a subject on a monthly, weekly, or daily basis.
• 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.
• 2-methoxyestradiol or the level of 2-methoxyestradiol plus the level of a 2-methoxyestradiol analog
• a 2-methoxyestradiol analog suitable for use in accordance with the methods and compositions described herein comprise a compound of formula I:
• R is H, C 1-6 alkyl, aryl, aralkyl, or carbonyl
• Ri and R 2 are -OR, -SR, or -N(R) 2 ;
• R 3 is H, halide, Ci -6 alkyl, aryl, aralkyl, or carbonyl;
• R 4 , R 5 , and R 6 is are H, halide, Ci -6 alkyl, aryl, aralkyl, or carbonyl;
• m is an integer from 1-5 inclusive.
• n and p are an integer from 1-6 inclusive.
• 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.
• 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 1 is OH.
• 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 2 is OH.
• 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 3 is methyl.
• 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 4 is H.
• 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 5 is H.
• 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 6 is H.
• 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.
• 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 R 2 is OH.
• 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 R 3 is methyl.
• 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, R 1 is OH, and R 2 is OH.
• 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, R 1 is OH, R 2 is OH, and R 3 is methyl.
• 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 4 , R 5 , and R 6 are H.
• 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 R 4 , R 5 , and R 6 are H.
• 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, R 1 is OH, and R 4 , R 5 , and R 6 are H.
• 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, R 1 is OH, R 2 is OH, and R 4 ,
• R 5 , and R 6 are H.
• 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, R 1 is OH, R 2 is OH, R 3 is methyl, and R 4 , R 5 , and R 6 are H.
• compositions and methods described herein are pharmaceutically acceptable addition salts and complexes of formula I.
• the compositions described herein comprise each unique racemic compound, as well as each unique nonracemic compound.
• the meaning of any substituent at any one occurrence is independent of its meaning, or any other substituent's meaning, at any other occurrence.
• prodrugs of 2-methoxyestradiol and the compounds of formula I are also included in the methods and compositions described herein.
• 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.
• 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.
• 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.
• 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-3,17 ⁇ -diol, 2-methoxyestra-l,3,5(10)- triene-3-ol, 17 ⁇ -aminoestra-l,3 3 5,(10)-triene-3,17 ⁇ -diol, 2-methoxy-17-oxime-3- hydroxyestra- 1,3,5(10)-triene- 17-one, 2-methoxy-3 , 17 ⁇ -bis(acetyloxy)estra- 1,3,5,(10)- triene, 2-methoxy-17 ⁇ -propaneestra-l,3,5(10)-triene-3-ol, 2-methoxy-17 ⁇ -methylestra- l,3,5(10)-triene-3-ol, 2-methoxy-17(20)-Z-propyliden
• 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.
• 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.
• compositions described herein may be administered by various means, depending on their intended use, as is well known in the art.
• compositions 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.”
• 2-methoxyestradiol, or an analog thereof is formulated for oral administration as a small tablet or an aqueous solution or suspension.
• liquid compositions of the formulations e.g. aqueous solutions or suspensions, are administered enterally by gavage, e.g. via a stomach- tube.
• 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.
• formulations may be administered parenterally as injections
• compositions 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.
• an excipient 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.
• wetting agents 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.
• 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.
• 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.
• 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.
• 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.
• 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
• pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate
• 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.
• 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.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
• 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.
• 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,
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• aqueous and non-aqueous carriers examples 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.
• polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
• vegetable oils such as olive oil
• 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.
• 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.
• a therapeutic dose is provided in 50, 40, 30, 20, 15, 10, 5 or 3 tablets.
• 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 (ED 50 ), 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.
• 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 ED 50 dose to a patient (human or non-human mammal).
• 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 ED 50 concentration, hi other embodiments less than 100 times, 10 times, or 5 times the ED 50 is provided.
• 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).
• 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).
• 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”).
• 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 ED 50 , or less than 10 or 5 times the ED 50 .
• 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 ED 50 dose to a patient, or less than 100 times the ED 50 , or less than 10 or 5 times the ED 50 .
• 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 ED 50 concentration, or less than 100 times the ED 50 , or less than 10 or 5 times the ED 50 .
• a single dose injection provides about 0.25 mg to 1250 mg of 2- methoxyestradiol, or an analog thereof (or combinations thereof).
• 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).
• Example 1 The role of 2-methoxyestradiol in complications resulting from premature birth
• 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- l ⁇ ). HIF- l ⁇ 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.
• 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.
• 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 ⁇ in response to brief episodes of hypoxia in the placenta and fetus.
• 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).
• the 2ME2 level of a 25 week gestation infant at birth will be -750 pg/mL.
• 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.
• 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 ⁇ , leading to vascular leak and sprout formation. In summary: [00179] Infant born at term exposed to normal third trimester surge of 2ME2:
• ROP hemangioma
• 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.
• 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.
• 2ME2 powerful anti- angiogenic factor 2 methoxyestradiol
• 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?
• amniotic fluid theoretically contains 2ME2 as well as other pro and anti-angiogenic factors, is it a potential source of a balanced angiogenesis "cocktail.”
• 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?
• GC-MS Gas Chromatography- Mass Spectrometry
• Mw 2-methoxyestradiol
• 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.
• 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.
• Example 2A pilot study, mothers of babies delivery by C section at term
• Amniotic fluid 1 spin and freeze
• Example 2B pilot study, mothers and their sick term infants admitted to
• Example 2C pilot study, mothers and their premature infants
• Amniotic fluid 1 spin and freeze 10 cc at delivery
• Goal is to send 1 cc whenever possible
• 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:
• 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.
• 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.
• Conditioned medium from mouse sarcoma 180 cells contains vascular endothelial growth factor. Growth Factors 4, 53-9 (1990).
• VPF/VEGF vascular permeability factor

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