HK1146645B - Chemically modified small molecules - Google Patents

Description

chemically modified small molecules

Technical Field

The present invention provides chemically modified small molecules and related methods, which have certain advantages over small molecules that have not been chemically modified. The chemically-modified small molecules described herein are related to and/or have application in the fields of drug discovery, pharmacotherapy, physiology, organic chemistry, polymer chemistry, and others.

Background

In recent years, there has been development in using proteins as active agents due to: techniques for identifying, isolating, purifying proteins and/or producing proteins by recombinant means have been improved; with the advent of proteomics, people have more knowledge about the role of proteins in living organisms; as well as formulations of chemically modified proteins, drug delivery vehicles and methods for enhancing their pharmacokinetic or pharmacodynamic properties.

In improving the chemical modification of proteins, polymers, such as poly (ethylene glycol) or PEG, are covalently attached to proteins to increase circulation half-life, reduce immunogenicity, and/or reduce proteolytic degradation. The process of covalently attaching PEG to proteins or other active agents is commonly referred to as pegylation. Proteins for injection that are modified by covalent attachment of PEG are generally modified by attachment to PEG polymers of relatively high molecular weight, typically between about 5,000 and about 40,000 daltons.

Modification of larger molecules to enhance drug efficacy is perhaps one of the most common applications of pegylation. Pegylation is also used to a limited extent to improve the bioavailability of poorly water-soluble small molecule drugs and to ease formulation. For example, the water solubility of a water soluble polymer, such as PEG, covalently conjugated to artemisinic acid is increased. See U.S. Pat. No. 6,461,603. Similarly, PEG is covalently bound to a triazolyl compound (e.g., trimemol), which has improved water solubility and chemical stability. See international patent publication WO 02/043772. Covalent attachment of PEG to diindolylmaleimides is used to enhance the poor bioavailability of such compounds due to low water solubility. See international patent publication WO 03/037384. PEG chains conjugated to small molecule drugs in order to improve water solubility are generally between about 500 daltons and about 5000 daltons in size, depending on the molecular weight of the small molecule drug.

The active agent can be administered by one of several routes, including injection, oral, inhalation, nasal, and transdermal. Oral administration is one of the most preferred routes of administration due to its ease. Oral administration is the most common method of administration of small molecule drugs (i.e., non-protein based drugs) and is not only convenient, but also often preferred by patients over other methods of administration. Unfortunately, many small molecule drugs have certain attributes (e.g., low oral bioavailability) that make oral administration impractical. The properties of small molecule drugs required for dissolution and selective diffusion through various biological membranes often directly conflict with those required for optimal target adsorption and administration. The major biological membranes that restrict the entry of small molecule drugs into certain organs or tissues are membranes associated with certain physiological barriers such as the blood-brain barrier, the blood-placenta barrier and the blood-testis barrier.

The blood-brain barrier protects the brain, preventing the invasion of most toxins. The specialized cell, called the stellate cell, has many small branches that form a barrier between the capillary endothelium and the brain nerve cells. The very tight junctions between lipids on the stellate cell wall and adjacent endothelial cells limit the passage of water soluble molecules. Although the blood-brain barrier allows the passage of essential nutrients, the barrier effectively cuts off the passage of some foreign substances, and can reduce the rate of other substances entering the brain tissue.

The placental barrier protects the developing, sensitive fetus from the invasion of toxins that may be present in the maternal circulation. This barrier is composed of multiple cell layers within the placenta between the maternal and fetal circulation vessels. Lipids on the cell membrane limit the diffusion of water-soluble toxins. Other substances, such as nutrients, gases and waste products produced by the developing fetus, may pass through the placental barrier. Like the blood-brain barrier, the placental barrier, while not completely impermeable, effectively reduces the diffusion of many toxins from the mother to the fetus.

For many oral drugs, penetration across certain biological membranes (such as the blood-brain barrier or the blood-placental barrier) should be prevented to the utmost, as this may lead to serious side effects such as neurotoxicity, insomnia, headache, confusion, nightmare or fetal malformations. These side effects, when severe, interrupt drug development and lead to brain or placenta absorption which we want to prevent. Thus, there is a need for new methods of administration that are not only effective in administering drugs, particularly small molecule drugs, to a patient, but also reduce the adverse and often toxic side effects of small molecule drugs. In particular, there is a need for improved methods of administration that can find an optimal balance between good oral bioavailability, bioactivity, and pharmacokinetic profile. The present invention fulfills this and other needs.

Summary of The Invention

The present invention is based on the development and discovery of chemically modified small molecule drugs that are unique (e.g., low biological membrane penetration), and methods for the preparation and administration of these drugs.

In one aspect, the invention provides compositions comprising monodisperse or bimodal conjugates, each conjugate comprising a moiety derived from a small molecule drug covalently attached to a water-soluble oligomer by a stable linkage. The oligomers are preferably taken from monodisperse (i.e. monomolecular) or bimodal, even trimodal or tetramodal compositions. Conjugates formulated from monodisperse oligomer compositions are referred to as monodisperse conjugates, conjugates formulated from bimodal oligomer compositions are referred to as bimodal conjugates, and so on.

Advantageously, the water-soluble oligomer, when combined with a small molecule drug, is effective in reducing the ability to cross certain biological membranes (e.g., membranes associated with the blood-brain barrier or the blood-placenta barrier). In one or more embodiments, conjugates are provided that exhibit a lower biological membrane penetration rate than small molecule drugs that are not conjugated to water-soluble oligomers.

Conjugates can generally be described as having the structure O-X-D, where O corresponds to a water-soluble oligomer, X corresponds to a stable linkage, and D corresponds to a moiety derived from a small molecule drug.

In one or more embodiments, the small molecule drug is orally bioavailable. In addition, the conjugates also have oral bioavailability. Where both the small molecule drug and the corresponding small molecule drug-oligomer conjugate are bioavailable, preferably the conjugate has an oral bioavailability that is at least 10% of the oral bioavailability of the unconjugated form of the small molecule drug. Typical ratios of oral bioavailability maintained by conjugates compared to unconjugated forms of small molecule drugs include: at least about 20%; at least about 30%; at least about 40%; at least about 50%; at least about 60%; at least about 70%; at least about 80%; at least about 90%.

In one or more embodiments, the conjugated drug exhibits reduced first pass metabolism as compared to the unconjugated form of the small molecule drug. Thus, the present invention provides (among other things) a method of reducing metabolism of an active agent, the method comprising the steps of: providing monodisperse or bimodal conjugates, each conjugate comprising a moiety derived from a small molecule drug covalently attached to a water-soluble oligomer by a stable linkage, wherein the conjugate exhibits a lower metabolic rate than the small molecule drug not attached to the water-soluble oligomer; and administering the conjugate to a patient.

The water-soluble oligomer used in the preparation of the conjugates may vary and the invention is not particularly limited in this regard. Typical oligomers include oligomers containing monomers selected from the group consisting of: alkylene oxides, alkenols, vinylpyrrolidone, hydroxyalkyl methacrylamide, hydroxyalkyl methacrylate, sugars, alpha hydroxy acids, phosphazenes, oxazolines, amino acids, monosaccharides, and N-acryloylmorpholine. In one or more preferred embodiments, the water-soluble oligomer includes ethylene oxide monomers.

The oligomeric component of the conjugates described herein comprises a sequence of bound individual monomers. A typical oligomer may comprise a plurality of monomers of repeating sequence, the number of monomers corresponding to one or more of the following ranges: 1 to 25; 1 to 20; 1 to 15; 1 to 12; 1-10; and 2-9. The oligomer may have a plurality of monomers corresponding to any of the following values: 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; and 12.

The oligomeric components of the conjugates described herein can have a variety of geometries, structures, and features. Non-limiting examples include linear and branched oligomer structures.

In one or more embodiments, each of the conjugates described herein has a single water-soluble oligomer covalently bound to a single moiety derived from a small molecule drug. That is, the ratio of oligomer to moiety derived from the small molecule drug is 1: 1. However, in one or more embodiments, the conjugate may have 1, 2, or 3 oligomers covalently bound to a moiety derived from a small molecule drug.

Although covalent bonding (through one or more atoms) is preferred, the bonding that connects the water-soluble oligomer and the moiety derived from the small molecule drug can be any suitable bonding that binds the molecule. Suitable covalent linkages between the water-soluble oligomer and the small molecule drug include, but are not limited to, the following: an ether; an amide; a urethane; an amine; thioethers and carbon-carbon bonds.

The compositions described herein may comprise only a single conjugate, or two, three, four or more different conjugates. For example, a composition can comprise a single species of conjugate, while other conjugate species (e.g., conjugate species that differ in molecular weight, molecular structure, etc.) are substantially absent. In addition, the compositions described herein may also comprise, for example, two different conjugates mixed together, characterized by: (a) the same moiety obtained from the small molecule drug is present in all conjugates in the composition, and (b) the oligomer size of one conjugate is different from the oligomer size of the other conjugates. For those compositions comprising a mixture of conjugates of different species, each species will be present in the composition in known, defined amounts. Although the conjugate species within any particular composition may vary in the size of the oligomers described above, the difference in the conjugate species may also be based on the oligomer type, the moiety derived from the small molecule drug, the stereoisomer of the conjugate, and the like.

In another aspect, the invention provides a method of administering such a composition as described herein. The method of this aspect comprises the step of administering a composition comprising monodisperse or bimodal conjugates, each conjugate comprising a moiety derived from a small molecule drug covalently attached to a water-soluble oligomer by a stable linkage, wherein the conjugate exhibits a lower biological membrane penetration rate than the small molecule drug not attached to the water-soluble oligomer. Conveniently, the step of administering may be selected from any one of several methods of administration, including for example, the group consisting of: oral, transdermal, buccal, transmucosal, vaginal, rectal, parenteral, and pulmonary.

In yet another aspect, the present invention provides a method of optimizing selective biofilm penetration of small molecule drugs. The method in this aspect includes the step of covalently attaching oligomers derived from the monodisperse or bimodal oligomer composition to the small molecule drug via a stable linkage covalent bond, thereby forming a conjugate that exhibits a biofilm penetration rate that is lower than the biofilm penetration rate of the small molecule drug prior to conjugation.

In yet another aspect, the present invention provides a method for optimizing the reduction of the penetration of a small molecule drug through a biological membrane, the method comprising the steps of: (a) preparing a series of monodisperse or bimodal conjugates, each conjugate in the series comprising a moiety derived from a small molecule drug covalently attached to a water-soluble oligomer by a stable linkage, wherein each conjugate in the series differs only in oligomer size, based on the number of monomers in the oligomer; (b) characterizing each conjugate prepared in step (a) to the maximum extent that the conjugate does not penetrate a biological membrane; and (c) based on the results of (b), finding conjugates of the series of conjugates prepared in step (a) that have the best reduction in biofilm penetration rate.

The invention also provides a method of preparing a conjugate comprising the step of covalently attaching a water-soluble oligomer from a monodisperse or bimodal oligomer composition to a small molecule drug. The process produces a conjugate comprising a stable linkage linking the oligomer and a moiety derived from the small molecule drug. An exemplary method of providing conjugates includes the step of reacting, in one or more synthetic steps, a water-soluble oligomer derived from a monodisperse or polymolecular oligomer composition, wherein the oligomer has a reactive group a, with a small molecule drug comprising a reactive group B suitable for reaction with a, under conditions effective to form a hydrolytically stable linkage resulting from the reaction of a and B, thereby forming a small molecule drug-water-soluble oligomer conjugate.

Insofar as the method of preparing the conjugates produces a mixture of isomers (or other conjugate species), the additional step of separating the isomers (or other conjugate species) to obtain a single conjugate isomer (or conjugate species) can be performed. Alternatively, for any two or more compositions each having a single conjugate isomer (or conjugate species), the step of combining the two or more separate compositions can be performed to provide a composition having a known and defined number of each conjugate isomer (or conjugate species).

The invention also provides a process for preparing monodisperse water-soluble oligomers such as oligo (ethylene oxide). The process comprises the step of reacting a halo-terminated oligo (ethylene oxide) having (m) monomers with a hydroxy-terminated oligo (ethylene oxide) having (n) monomers under conditions effective to displace the halo group to form an oligomer having (m) + (n) monomeric subunits (OEG)_m+n) Wherein (m) and (n) each lie in the range from 1 to 10. Although not necessary, the range of (m) is preferably 2 to 6 (more preferably 1 to 3) and the range of (n) is preferably 2 to 6.

The process for preparing monodisperse water-soluble oligomers is generally carried out in the presence of strong bases, such as sodium, potassium, sodium hydride, potassium hydride, sodium methoxide, potassium methoxide, sodium tert-butoxide or potassium tert-butoxide, which are suitable for converting the hydroxyl groups of the hydroxyl-terminated oligo (ethylene oxide) into the corresponding alkoxide.

With respect to the halogen (or halo group) associated with the halo-terminated oligo (ethylene oxide) (or other halo-terminated oligomer), the halogen is typically selected from chlorine, bromine, and iodine. Furthermore, the capping of the halo-terminated oligo (ethylene oxide) is typically, for example, methyl or ethyl to provide the corresponding methyl or ethyl ether end. The preferred halo-terminated oligo (ethylene oxide) is H₃CO-(CH₂CH₂O)_m-Br, wherein (m) is as defined above.

With respect to hydroxy-terminated oligo (ethylene oxide), such hydroxy-terminated oligo (ethylene oxide) corresponds to the structure HO- (CH)₂CH₂O)_n-H, wherein (n) is as defined above.

The process for preparing monodisperse water-soluble oligomers may also comprise the step of subjecting OEG to_m+nIs converted to a halo group to form an OEG_m+n-X, wherein X is halo. After the above steps, OEG can be performed_m+nReaction of X with a hydroxy-terminated oligo (ethylene oxide) having (n) monomers under conditions effective to displace the halo group thereby forming an oligomer having (m) +2(n) monomer subunits (OEG)_m+n) The ranges of oligo (ethylene oxide), (m) and (n) of (a) are as described above. Alternatively, the above steps may be repeated until a monodisperse oligo (ethylene oxide) having the desired discrete monomer amount is obtained.

The present invention also provides a method of preparing conjugates using the monodisperse oligo (ethylene oxide) compositions prepared by the above method. Although the monodisperse oligomer of ethylene oxide described above is preferred for use in preparing conjugates of the invention, it may also be used in conjunction with any of a number of active agents or surfaces. Preferred bioactive agents that bind to monodisperse oligo (ethylene oxide) s prepared as described above include small molecule therapeutics, diagnostics, dyes, imaging agents, targeting agents, surfactants, emollients, cosmeceuticals, nutritional supplements, and the like.

These and other objects, aspects, specific embodiments and features of the present invention will become more apparent when read in conjunction with the following detailed description.

Brief description of the drawings

FIG. 1 is a graph of the use of 13-cis retinoic acid (abbreviated as "13-cis-RA") and its typical small PEG conjugates (PEG conjugates) to Sprague Dawley rats₃-13-cis viaminate (retinamide), "PEG₃-13-cis RA”；PEG₅-13-cis viaminate, "PEG₅-13-cis RA；PEG₇-13-cis viaminate, "PEG₇-13-cis RA; and PEG₁₁-13-cis viaminate, "PEG₁₁-13-cis RA ") plasma concentration over time. See example 7 for a detailed description.

FIG. 2 is a graph of plasma concentration over time using 6-naloxonol (naloxonol) and its typical small PEG conjugates (3-matrix, 5-matrix, 7-matrix) to Sprague Dawley rats. See example 7 for a detailed description.

FIG. 3 depicts the effect of PEG chain length on the intestinal delivery (as an indicator of oral bioavailability) of various PEG-13-cis-RA conjugates and 13-cis-RA in Sprague-Dawley rats.

FIG. 4 depicts the effect of covalent binding of PEG-matrices of different sizes on blood brain barrier transport of 13-cis-RA and various PEG-13-cis-RA conjugates.

FIG. 5 depicts covalent binding of PEG-matrices of different sizes to naloxone and PEG_nThe effect of intestinal transport of Nal (as an indicator of oral bioavailability).

FIG. 6 shows covalent binding of PEG-matrices of different sizes to naloxone and PEG_n-the effect of blood brain barrier transport of Nal.

FIG. 7 depicts naloxone and PEG after oral gavage_nPharmacokinetics of Nal in rats.

FIGS. 8 and 9 depict covalent binding of PEG-matrices of different sizes to naloxone metabolite and PEG_n-effect of Nal metabolite levels.

FIG. 10 is a mass spectrum of methoxy-PEG-350 obtained from a commercial source (Sigma-Aldrich). It can be seen by analysis that although the reagent is sold as methoxy-PEG having a molecular weight of 350, the reagent is actually a mixture of 9 different PEG oligomers with a number of monomeric subunits in the range of about 7 to about 15.

Detailed description of the invention

It must be noted that, unless the context clearly shows that it is only singular, the singular forms in this specification include plural referents.

In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below:

"Water-soluble" in "water-soluble oligomer" means that the oligomer is at least 35% (by weight) soluble in water, preferably more than 95% soluble in water, at room temperature. Typically, the amount of light transmitted by an unfiltered "water-soluble" oligomeric water-soluble formulation is at least 75%, and more preferably 95% or more of the amount of light transmitted by the same solution after filtration. On a weight basis, a "water-soluble" oligomer is preferably at least 35% (by weight) water-soluble, more preferably at least 50% (by weight) water-soluble, even more preferably at least 70% (by weight) water-soluble, and even more preferably at least 85% (by weight) water-soluble. Most preferably, however, the water-soluble oligomer is at least 95% (by weight) soluble or completely soluble in water.

The terms "monomer", "monomeric subunit" and "monomeric unit" are used interchangeably herein and refer to one of the basic structural units of a polymer or oligomer. In the case of a homo-oligomer, the monomer units are defined as the structural repeat units of the oligomer. In the case of co-oligomers, it is more useful to define the monomer units as monomer residues that form oligomers by oligomerization, since the structural repeat unit may include more than one monomer unit type. Preferred oligomers of the present invention are homooligomers.

An "oligomer" is a molecule having from about 1 to about 30 monomers. The structure of the oligomer may vary. Specific oligomers for use in the present invention include those having various geometries (e.g., linear, branched, or branched), as described in more detail below.

As used herein, "PEG" or "polyethylene glycol" is intended to include all water-soluble poly (ethylene oxide). Unless otherwise indicated, a "PEG oligomer" or an oligoethylene glycol is one in which all of the monomer subunits are ethylene oxide subunits. Typically, almost all or all of the monomer subunits are ethylene oxide subunits, although the oligomers may contain different end-capping moieties or functional groups, e.g., for conjugation. Typically, the PEG oligomers used in the present invention will comprise one of the following two structuresThe method comprises the following steps: "- (CH)₂CH₂O)_n- "or" - (CH)₂CH₂O)_n-1CH₂CH_2-Depending on whether the terminal oxygen is replaced, for example, during synthesis switching. As mentioned above, for the PEG oligomers of the invention, the variable (n) ranges from 1 to 30, and the structure of the terminal group and the overall PEG can vary. When the PEG further comprises functional group a for attachment to, for example, a small molecule drug, the functional group does not result in the formation of (i) an oxygen-oxygen bond (-O-, peroxide linkage), or (ii) a nitrogen-oxygen bond (N-O, O-N), when covalently bound to the PEG oligomer.

The "capping group" is typically a non-reactive carbon-containing group that binds to the terminal oxygen of the PEG oligomer. For the purposes of the present invention, preferred blocking groups have a relatively low molecular weight, for example methyl or ethyl. The end capping group may also comprise a detectable label. Such labels include, but are not limited to, fluorescent agents, chemiluminescent agents, enzyme labeling components, colorimetric labels (e.g., dyes), metal ions, and radioactive components.

"branched" when referring to the geometry or overall structure of an oligomer means that the oligomer has two or more polymeric "arms" extending from the branching point.

"branched" when referring to the geometry or overall structure of the oligomer means that the oligomer has two or more functional groups extending from the branching point, typically through one or more atoms.

"branching point" refers to a branching point comprising one or more atoms at which an oligomer branches or bifurcates from a linear structure into one or more additional arms.

The term "reactive" or "reactive" means that the functional group reacts readily or at a practicable rate under the conventional conditions of organic synthesis. This is relative to groups that do not react or require strong catalysts or impractical reaction conditions to react (i.e., "non-reactive" or "inert" groups).

"unreactive" when referring to a functional group present on a molecule in a reaction mixture means that the functional group remains substantially unchanged under conditions effective to produce a desired reaction in the reaction mixture.

A "protecting group" is a moiety that prevents or blocks a particular functional group in a molecule that is chemically reactive from reacting under certain reaction conditions. Protecting groups may vary depending on the chemical reactive group being protected and the reaction conditions to be employed, as well as whether other reactive or protecting groups are present in the molecule. For example, functional groups that may be protected include carboxylic acid groups, amino groups, hydroxyl groups, thiol groups, carbonyl groups, and the like. For carboxylic acids, representative protecting groups include esters (such as p-methoxybenzyl ester), amides and hydrazides; carbamate (e.g., t-butyloxycarbonyl) and amide for amino; for hydroxy, ethers and esters; thioethers and thioesters for thiol groups; acetals and ketals for carbonyl groups; and so on. These protecting groups are familiar to the person skilled in the art and are described, for example, in "protecting groups in organic Synthesis" by T.W. Greene and G.M.Wuts (third edition, Wiley, New York, 1999) and in the references cited therein.

The functional group in the "protected form" refers to a functional group having a protecting group. As used herein, the term "functional group" or any synonym thereof, encompasses the protected state of a functional group.

A "physiologically cleavable" or "hydrolyzable" or "degradable" bond is a relatively unstable bond that will react with (i.e., hydrolyze) water under physiological conditions. The tendency of a bond to decompose in water depends not only on the type of bond linkage connecting the two central atoms, but also on the substituents bound to these central atoms. Suitable hydrolytically unstable or weak linkages include, but are not limited to, carboxylate esters, phosphate esters, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, oligonucleotides, thioesters, thiol esters, and carbonates.

"enzymatically degradable linkage" refers to linkage that can be degraded by one or more enzymes.

By "hydrolytically stable" linkage or bond is meant that a chemical bond, typically a covalent bond, is very stable in water, that is, does not undergo hydrolysis to any appreciable extent under physiological conditions over an extended period of time. Examples of hydrolytically stable bonds include, but are not limited to: carbon-carbon bonds (e.g., in an aliphatic chain), ethers, amides, urethanes, amines, and the like. Generally, hydrolytically stable linkages are linkages that exhibit a hydrolysis rate of less than about 1-2% per day under physiological conditions. The hydrolysis rates of representative chemical bonds can be found in most standard chemical textbooks.

"substantially" or "essentially" means nearly entirely or completely, for example, 95% or more, more preferably 97% or more, still more preferably 98% or more, still more preferably 99% or more, still more preferably 99.9% or more, and most preferably 99.99% or more of a given amount.

"monodisperse" refers to an oligomer composition wherein substantially all of the oligomers in the composition have a defined single (i.e., the same) molecular weight and a defined number of monomers, as determined by chromatography or mass spectrometry. The monodisperse oligomer composition is pure in the sense that it has essentially a single, well-defined monomer amount (which is an integer), rather than a large scale distribution. The monodisperse oligomer composition of the present invention has a MW/Mn value of 1.0005 or less, more preferably a MW/Mn value of 1.0000. Further, a composition comprising monodisperse conjugates means that substantially all oligomers of all conjugates in the composition have a single, well-defined monomer number (which is an integer), rather than a large scale distribution, and have a MW/Mn value of 1.0005 or less, more preferably 1.0000 if the oligomers are not bound to a moiety derived from a small molecule drug. However, compositions comprising monodisperse conjugates can include one or more unconjugated substances, such as solvents, reagents, excipients, and the like.

"bimodal," when referring to an oligomer composition, refers to an oligomer composition having the following characteristics, wherein substantially all of the oligomer has one of two distinct and different monomer amounts (which are integers), rather than a large-scale distribution, and wherein the molecular weight distribution, when plotted as a fraction against molecular weight, exhibits two separately identifiable peaks. For the bimodal oligomer compositions described herein, although the two peaks may be of different sizes, it is preferred that each peak be symmetrically distributed on either side of the respective mean. The polydispersity index Mw/Mn of each peak in the bimodal distribution is desirably 1.01 or less, more preferably 1.001 or less, still more preferably 1.0005 or less, and the most preferred value of MW/Mn is 1.0000. Further, a composition comprising bimodal conjugates means that substantially all oligomers of all conjugates in the composition have one of two distinct, different monomer numbers (which are integers), rather than a large scale distribution, and if the oligomers are not combined with moieties derived from small molecule drugs, have a MW/Mn value of 1.01 or less, more preferably 1.001 or less, even more preferably 1.0005 or less, and most preferably 1.0000. However, compositions comprising bimodal conjugates may include one or more unconjugated substances, such as solvents, agents, excipients, and the like.

As used herein, a "small molecule drug" refers to an organic, inorganic, or organometallic compound having a molecular weight generally below about 1000. The small molecule drugs of the present invention comprise oligopeptides and other biomolecules having a molecular weight of less than about 1000.

"moiety derived from a small molecule drug" and "small molecule drug moiety" are used interchangeably herein to refer to that portion or residue of the source small molecule drug (or its active or chemically modified state) up to the linkage-associated covalent bond that results from its covalent attachment to the oligomer of the invention.

A "biofilm" is any membrane, typically composed of specialized cells or tissues, that is a barrier to the passage of at least some xenobiotics or other undesirable substances. As used herein, "biofilm" includes membranes associated with physiological protective barriers, including, for example: the blood brain barrier; a blood and cerebrospinal fluid barrier; a blood-placenta barrier; a blood milk barrier; a blood testis barrier; and mucosal barriers including vaginal mucosa, urethral mucosa, anal mucosa, buccal mucosa, sublingual mucosa, rectal mucosa, and the like. Unless the context clearly indicates otherwise, the term "biofilm" does not include membranes associated with the intermediate gastrointestinal tract (e.g., the stomach and small intestine).

Herein, "biofilm penetration" provides a measure of the ability of a compound to penetrate a biological barrier (e.g., the blood brain barrier, BBB). Various methods can be used to assess the transport ability of molecules to penetrate any particular biofilm. Methods for assessing the rate of biological membrane penetration associated with any particular biological barrier (e.g., blood cerebrospinal fluid barrier, blood placental barrier, blood milk barrier, intestinal barrier, etc.) are known and described herein and/or in the relevant literature and/or can be determined by one of ordinary skill in the art.

A "blood brain barrier penetrating" compound according to the present invention is a compound that penetrates the BBB at a rate greater than atenolol using the methods described herein.

In the present invention, "reduced metabolic rate" means that the metabolic rate of a conjugate of a water-soluble oligomer and a small molecule drug is significantly reduced as compared with the metabolism rate of a small molecule drug not bound to a water-soluble oligomer (i.e., the small molecule drug itself) or a new metabolic rate of a standard reference substance. In the special case of "reduced first pass metabolic rate", the same "lower metabolic rate" is required except in the case of oral administration of the small molecule drug (or standard reference) and the corresponding conjugate. Oral drugs are absorbed into the portal circulation through the gastrointestinal tract and must first pass through the liver before reaching the systemic circulation. Since the liver is the primary site of drug metabolism or biotransformation, a substantial portion of the drug can be metabolized at this site before reaching the systemic circulation. The extent of first pass metabolism, and therefore any reduction thereof, can be measured in a number of different ways. For example, animal blood samples can be taken at regular intervals and plasma or serum analyzed by liquid chromatography/mass spectrometry for metabolite levels. Other methods of measuring "reduced metabolic rate" associated with first pass metabolism and other metabolic processes are known and described herein and/or in the relevant literature and/or can be determined by one of ordinary skill in the art. Preferably, the conjugates of the invention may provide a reduced metabolic rate reduction which satisfies at least one of the following values: at least about 5%, at least about 10%, at least about 15%; at least about 20%; at least about 25%; at least about 30%; at least about 40%; at least about 50%; at least about 60%; at least about 70%; at least about 80%; and at least about 90%.

A compound having "oral bioavailability" (e.g., a small molecule drug or conjugate thereof) is a compound that has about 1% bioavailability, preferably greater than 10%, when administered orally, wherein the bioavailability of the compound refers to the portion of the administered drug that reaches the systemic circulation in an unmetabolized form.

"alkyl" refers to a hydrocarbon chain, generally between about 1 and 20 atoms in length. Such hydrocarbon chains are preferably, but not necessarily, saturated and may be branched or straight, but are preferably straight. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like. Herein, when referring to three or more carbon atoms, "alkyl" includes cycloalkyl.

"lower alkyl" means an alkyl group containing from 1 to 6 carbon atoms and may be straight or branched, for example methyl, ethyl, n-butyl, i-butyl, t-butyl.

"non-interfering substituents" refer to those groups that are generally not reactive with other functional groups in a molecule when present in the molecule.

"alkoxy" refers to the group-O-R, wherein R is alkyl or substituted alkyl, preferably C₁-C₂₀Alkyl (e.g., methoxy, ethoxy, propoxy, benzyl, etc.), preferably C₁-C₇。

"electrophile" refers to an ionic or neutral collection of ions, atoms, or atoms that has an electrophilic center (i.e., a center that is electron seeking and is capable of reacting with a nucleophile).

"nucleophile" refers to an ionic or neutral collection of ions, atoms, or atoms having a nucleophilic center (i.e., a center that seeks an electrophilic center and is capable of reacting with an electrophile).

As used herein, "drug" includes any agent, compound, combination of substances, or mixture that is capable of providing a pharmacological, often beneficial, effect in vivo or in vitro. Including foods, food supplements, nutrients, nutraceuticals, pharmaceuticals, vaccines, antibodies, vitamins, and other beneficial agents. In this context, these terms further include any physiologically or pharmacologically active substance capable of producing a local or systemic effect in a patient.

By "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is meant an excipient that the composition of the present invention may include and that does not cause significant adverse toxicological effects to the patient.

"pharmacologically effective amount," "physiologically effective amount," and "therapeutically effective amount" are used interchangeably herein and refer to the amount of conjugate of a water-soluble oligomer and a small molecule drug present in a composition required to provide a desired level of active agent and/or conjugate in the bloodstream or target tissue. The precise amount will depend on a variety of factors, e.g., the particular active agent, the ingredients and physical characteristics of the composition, the intended patient population, patient considerations, and the like, and can be readily determined by one skilled in the art based on the information provided herein and in the relevant literature.

A "bifunctional" oligomer is an oligomer having two functional groups, typically at its ends. When the functional groups are the same, the oligomer is referred to as a homobifunctional oligomer. When the functional groups are different, the oligomer is called a heterobifunctional oligomer.

The basic or acidic reactants described herein include neutral, charged reactants and any corresponding salt forms thereof.

The term "patient" refers to a living organism, including humans and animals, suffering from or susceptible to a condition that can be prevented or treated by administration of a conjugate described herein, typically but not necessarily in the form of a conjugate of a water-soluble oligomer and a small molecule drug.

"optional" or "optionally" means that the subsequently described circumstance may or may not occur, and that the description thus includes instances where the circumstance occurs and instances where it does not.

The present invention relates to chemically modified small molecule pharmaceutical compositions obtained by, inter alia, covalent bonding of water-soluble oligomers obtained from monodisperse or bimodal water-soluble oligomer compositions. Since the water-soluble oligomer is obtained from a monodisperse or bimodal water-soluble oligomer composition, the composition of the small molecule drug and the oligomer of the present invention is very pure and definite from a structural point of view.

One advantage of the conjugates described herein is that they exhibit reduced biological membrane penetration rates as compared to the corresponding active agents in the unconjugated form. Without wishing to be bound by theory, it is believed that molecular size is an important factor in determining whether and to what extent any particular molecule is able to pass through or cross any particular biological membrane. For example, most, if not all, protective barriers rely at least in part on the highly dense cells that make up the membrane, which has tight junctions through which only smaller molecules can pass. Thus, for a particular small molecule drug, the combination of a water-soluble polymer with the small molecule drug provides a necessarily larger conjugate, and it is expected that the conjugate will either be prevented from passing through a biological membrane, or will have a reduced biological penetration rate as compared to the unconjugated small molecule drug.

As will be described in more detail below and in the experimental section, however, increasing the molecular size by conjugation of water-soluble oligomers with small molecule drugs, and thus decreasing the rate of biological membrane penetration, generally does not provide a completely satisfactory conjugate. Ideally, the conjugate is provided as a composition comprising a monodisperse or bimodal conjugate. To reiterate, without wishing to be bound by theory, it is believed that even very small differences in the number of monomers between conjugates result in large differences in properties such as pharmacological activity, metabolism, oral bioavailability, biofilm penetration, solubility, and others.

In addition, as shown by the mass spectra provided in FIG. 10, commercially available oligomer compositions, such as PEG-350, are relatively impure in nature because of the wide variety of oligomer sizes present in the composition. Thus, the use of such relatively impure oligomer compositions (without further purification) in conjugate synthesis results in a wide variety of conjugate molecular weight sizes (due to the wide range of molecular weights of the compositions used to form the conjugates). As a result, the resulting conjugate composition comprises a plurality of conjugates, wherein each conjugate may have different properties. From a medical regulatory and medical point of view, it is desirable to avoid compositions comprising parts with significantly different properties.

Thus, the conjugates provided by the present invention are not only relatively large (as compared to the corresponding unconjugated small molecule drug) to reduce biological membrane penetration (as compared to the corresponding unconjugated small molecule drug), but are also substantially pure to ensure that the compositions have consistent desired activity and other attributes. Thus, the present invention provides compositions comprising monodisperse or bimodal conjugates, each conjugate comprising a moiety derived from a small molecule drug covalently attached to a water-soluble oligomer by a stable linkage, wherein the conjugate exhibits a lower biological membrane penetration rate than the small molecule drug not attached to the water-soluble oligomer.

As previously described, the use of discrete oligomers derived from a defined oligomer composition to form conjugates can advantageously alter certain properties associated with the corresponding small molecule drug. For example, the conjugates of the invention exhibit reduced penetration of a biological membrane (e.g., a biological membrane associated with the blood-brain barrier and the blood-placental barrier) when administered by any of several suitable routes of administration, such as parenteral, oral, transdermal, buccal, pulmonary, or nasal administration. Preferably, the conjugate exhibits reduced, minimal or virtually no penetration of a biological membrane, such as that associated with the blood-brain barrier and the blood-placental barrier, but, when administered orally, penetrates the Gastrointestinal (GI) wall and enters the systemic circulation. If pulmonary administration is employed, it is preferred that the conjugate be administered without penetration into the systemic circulation or with a low rate of penetration of the pulmonary tissue-blood barrier, thereby maintaining the drug level in the lungs and producing local pharmacological activity in the lungs. In addition, the conjugates of the present invention maintain a degree of bioactivity and bioavailability in the conjugated state.

With respect to the blood brain barrier ("BBB"), this barrier limits the blood-to-brain transport of drugs. This barrier consists of a continuous layer of distinct endothelial cells connected by tight junctions. Brain capillaries, which contain more than 95% of the total surface area of the BBB, are the major route for most solutes and drugs to enter the central nervous system.

Although some compounds are present in brain tissue at sufficient concentrations to produce pharmacological effects in the brain are desirable results, many other compounds that are not pharmacologically active in brain tissue are ultimately able to reach central nervous system tissues. By reducing the rate of penetration of these non-centrally acting compounds into the central nervous system, the risk of side effects on the central nervous system is reduced and possibly even the therapeutic effect is improved.

For compounds in which the extent of blood-brain barrier penetration is not readily known, this ability can be determined using a suitable animal model, such as the in situ murine brain perfusion ("RBP") model described herein. Briefly, the RBP method involves catheterization of the carotid artery, perfusion of a solution of the compound under controlled conditions, followed by a flushing phase to remove compounds remaining in the vascular space. (for example, analysis can be performed by research-mandated tissues such as Absorption Systems, Exton, PA, etc.) more specifically, in the RBP model, a catheter is placed in the left carotid artery and the side branch vessels are tied. In a single channel perfusion assay, physiological buffer containing compound (5 micromolar) was perfused at a flow rate of 10 ml/min. After 30 seconds, the perfusion was stopped and the contents of the cerebral vessels were flushed away by rinsing with buffer without compound for 30 seconds. Brain tissue was then excised for analysis and the concentration of compound was determined by liquid chromatography using tandem mass spectrometry detection (LC/MS). In addition, blood brain barrier penetration can also be estimated by calculating the compound's molecular polar surface area ("PSA"), i.e., the sum of the surface distributions of the polar atoms (usually oxygen, nitrogen, and attached hydrogen) within the molecule. PSA is known to be associated with compound transport properties, such as blood brain barrier transport. Methods for determining PSA of compounds can be found, for example, in the following documents: ertl, p, et, j.med.chem.2000, 43, 3714-; and Kelder, j., et., pharm. res.1999, 16, 1514-.

One barrier similar to the blood brain barrier is the blood brain spinal fluid barrier. This barrier forms a barrier that reduces the amount of toxins or undesirable materials that reach the cerebrospinal fluid, which is mostly located in the ventricular system and in the arachnoid space. To determine whether and to what extent a compound (e.g., a small molecule drug or conjugate) administered to a patient is able to cross the blood and cerebrospinal fluid barrier, a known amount of the compound can be injected into a mouse. Several days after compound injection, a sample of the cerebrospinal fluid of the mouse can be analyzed to determine the presence and amount of the compound.

The blood-placental barrier protects the developing fetus from most toxins distributed within the maternal circulation. This barrier consists of several layers of cells within the placenta between the maternal and fetal circulation vessels. Like the blood-brain barrier, the placental barrier is not completely impermeable, but can effectively slow the diffusion of most toxins. To determine whether and to what extent a compound (e.g., a small molecule drug or conjugate) administered to a pregnant mammal is able to cross the blood-placental barrier, a known amount of the compound can be injected into pregnant mice. Several days after compound injection, samples of mouse fetal tissue can be analyzed to determine the presence and amount of the compound.

The blood-milk barrier isolates and restricts the penetration of certain substances in the systemic circulation by means of biological membranes, similar to the blood-brain barrier. With respect to the blood milk barrier, biofilms prevent certain substances from passing through into the mammary gland. To determine whether and to what extent a compound (e.g., a small molecule drug or conjugate) administered to a pregnant mammal is able to cross the blood-milk barrier, a known amount of the compound can be injected into a mammalian mouse. Several days after injection of the compound, a sample of milk taken from the mammary gland can be analyzed to determine the presence and amount of the compound.

The blood testis barrier comprises strut cells (sertoli cells) that are distributed around the male reproductive tract and are joined together by tight junctions. To determine whether and to what extent a compound (e.g., a small molecule drug or conjugate) administered to a male mammal is able to cross the blood-testicular barrier, a known amount of the compound can be injected into male mice. Several days after compound injection, the mouse testes can be excised and analyzed for the presence and amount of compound.

The mucosal barrier represents another biological membrane that typically prevents or reduces the entry of undesirable substances into the systemic circulation. Administration of a compound to a particular mucosal area, followed by analysis of the blood sample to determine the presence and amount of the compound, allows determination of whether and to what extent the compound has penetrated the particular mucosal area.

For any biofilm, the water-soluble oligomer-small molecule drug conjugate exhibits a lower biofilm penetration rate than a small molecule drug not bound to the water-soluble oligomer. Exemplary reductions in biofilm penetration include a reduction in: at least about 5%; at least about 10%; at least about 25%; at least about 30%; at least about 40%; at least about 50%; at least about 60%; at least about 70%; at least about 80%; or at least about 90%, these reductions are compared to the biofilm penetration rate of small molecule drugs that are not bound to the water-soluble oligomer. The reduction in the biological membrane penetration rate of the conjugate is preferably at least about 20%. In some cases, it is preferred that the small molecule drug itself can penetrate one or more of the biological membranes described herein.

Conjugates exhibiting reduced biological membrane penetration typically comprise a structure

O-X-D

Wherein: o corresponds to a water-soluble oligomer, X corresponds to a stable linkage, and D corresponds to a moiety derived from a small molecule drug.

The moiety derived from the small molecule drug is different from the original small molecule drug of origin in the sense that the small molecule drug moiety is attached (typically by a covalent bond) to an atom not associated with the small molecule drug of origin. However, the moiety derived from the small molecule drug is fundamentally the same as the small molecule drug and has a similar pharmacological mechanism of action, except that it is different from the point of attachment to another atom. Therefore, the discussion of small molecule drugs is equivalent to describing the moieties derived from small molecule drugs.

The active agent in the conjugate is a small molecule drug, i.e., a pharmacologically active compound having a molecular weight of less than about 1000 daltons. For purposes of the present invention, small molecule drugs include oligopeptides, oligonucleotides, and other biomolecules having a molecular weight of less than about 1000 daltons. "Small molecule drugs" also include fragments of peptides, proteins, or antibodies, including native sequences and variants falling within the above molecular weight ranges.

Exemplary molecular weights for small molecule drugs include the following: less than about 950; less than about 900; less than about 850; less than about 800; less than about 750; less than about 700; less than about 650; less than about 600; less than about 550; less than about 500; less than about 450; less than about 400; less than about 350; and less than about 300.

The small molecule drugs used in the present invention, if the molecules are chiral, can be obtained from racemic mixtures, optically active forms such as single optical enantiomers, or enantiomers in any combination or ratio. In addition, small molecule drugs may have one or more geometric isomers. With respect to geometric isomers, the composition may comprise only one geometric isomer or a mixture of two or more geometric isomers. The small molecule drugs for use in the current invention may be in a conventional active state or may be modified to some extent. For example, a small molecule drug may have a target agent, adjunct or delivery vehicle bound to it, either before or after covalent attachment to the oligomer. Small molecule drugs may also have lipophilic moieties bound to them, such as phospholipids (e.g., distearoylphosphatidylethanolamine or "DSPE", dipalmitoylphosphatidylethanolamine or "DPPE", etc.) or small fatty acids. However, in some cases, it is preferred that the small molecule drug moiety does not include binding to a lipophilic moiety.

The small molecule used to bind to the conjugates of the invention can be any of the following small molecules. Suitable agents may be selected from, for example, respiratory drugs, anticonvulsants, muscle relaxants, anti-inflammatory agents, appetite suppressants, migraine agents, muscle contractants, anti-infectives (antibiotics, antivirals, antifungals, and vaccines), antirheumatics, antimalarials, antiemetics, bronchodilators, antithrombotic agents, antihypertensives, cardiovascular agents, antiarrhythmics, antioxidants, antiasthmatics, diuretics, lipid regulators, antiandrogens, antiparasitics, anticoagulants, neoplastic agents, antineoplastics, hypoglycemic agents, nutritional agents and additives, growth supplements, anti-enteritis agents, vaccines, antibodies, diagnostic agents, and contrast agents.

More specifically, the active agent may belong to one of several structures, including but not limited to small molecules, oligopeptides, polypeptides or protein mimetics, fragments or analogues, steroids, nucleosides, oligonucleotides, electrolytes, and the like. Preferably, the active agent associated with the oligomer of the present invention has a free hydroxyl, carboxyl, thio, amino or the like (i.e., the "handle") suitable for covalent attachment to the oligomer. The drug may also be modified by introducing a suitable "handle", preferably by converting one of its existing functional groups into a functional group suitable for forming a stable covalent bond between the oligomer and the drug. Both methods are exemplified in the experimental section.

Suitable active agents for covalent attachment to the oligomers of the invention include small molecule mimetics and active fragments (including variants) of: asparaginase, amdoxovir (dapd), antadine, carpronine, calcitonin, cyanobacterial toxin, dinierein-toxin linker, Erythropoietin (EPO), EPO agonists for EPO (e.g., peptides of about 10-40 amino acids in length comprising a specific core sequence as described in WO 96/40749), deoxyribonuclease alpha, a protein that stimulates erythropoiesis (NESP), coagulation factors (e.g., factor V, factor VII, factor vila, factor VIII, factor IX, factor X, factor XII, factor XIII), von Willebrand factor; ceredase, imiglucerase, alpha-glucosidase, collagen, cyclosporine, alpha-defensin, beta-defensin, exedin-4, Granulocyte Colony Stimulating Factor (GCSF), Thrombopoietin (TPO), alpha-1 protease inhibitor, elcatin, Granulocyte Macrophage Colony Stimulating Factor (GMCSF), fibrinogen, filgrastim, the growth hormone human growth hormone (hGH), Growth Hormone Releasing Hormone (GHRH), GRO-beta antibodies, bone morphogenetic proteins (e.g., bone morphogenetic protein-2, bone morphogenetic protein-6, OP-1); acidic fibroblast growth factor, basic fibroblast growth factor, CD-40 ligand, heparin, human serum albumin, Low Molecular Weight Heparin (LMWH), interferon (e.g., interferon alpha, interferon beta, interferon gamma, interferon omega, interferon tau, consensus interferon); interleukins and interleukin receptors, such as the interleukin-1 receptor, interleukin-2 fusion protein, interleukin-1 receptor antagonist, interleukin-3, interleukin-4 receptor, interleukin-6, interleukin-8, interleukin-12, interleukin-13 receptor, interleukin-17 receptor; lactoferrin and lactoferrin fragments, luteinizing hormone-releasing hormone (LHRH), insulin, proinsulin, insulin analogs (such as the monoacylated insulin described in U.S. Pat. No. 5,922,675), amylin, C-peptide, somatostatin analogs (including octreotide), vasopressin, Follicle Stimulating Hormone (FSH), influenza vaccine, insulin-like factor (I GF), insulin opsonin, macrophage colony stimulating factor (M-CSF), plasminogen activators (such as alteplase, urokinase, reteplase, streptococcase, pamiprinase, lanoteplase, and teneteplase; Nerve Growth Factor (NGF), osteoprotegerin, platelet-derived growth factor, tissue growth factor, transforming growth factor-1, vascular endothelial cell growth factor, leukemia inhibitory factor, insulin, and combinations thereof, Keratinocyte Growth Factor (KGF), Glial Growth Factor (GGF), T cell receptor, CD molecules/antigen, Tumor Necrosis Factor (TNF), monocyte chemoattractant protein-i, endothelial growth factor, parathyroid hormone (PTH), glucagon-like peptide, growth hormone, thymosin alpha 1IIb/IIIa inhibitor, thymosin beta 10, thymosin beta 9, thymosin beta 4, alpha-1 antitrypsin, Phosphodiesterase (PDE) compounds, VLA-4 (very late antigen-4), VLA-4 inhibitors, bisphosphonates, respiratory polynuclear virus antibodies, cystic fibrosis transmembrane regulator (CFrR) gene, deoxyribonuclease (Dnase), bactericidal/permeability-enhancing protein (BPI), and anti-CMV antibodies. Typical monoclonal antibodies include etanercept (a two-molecule fusion protein consisting of the extracellular ligand-binding portion of the human 75kD TNF receptor linked to the Fc portion of IgG 1), abciximab, afuromab, sulley, cenipine, infliximab, teimomab, mitumomab, moromono-CD 3, iodine 131 descicumumab conjugates, erfuzumab, rituximab, and trastuzumab (herceptin).

Other agents suitable for covalent binding to the oligomers of the invention include, but are not limited to, amifostine, amiodarone, aminomethylbenzoic acid, p-Aminonurate, aminoglutethimide, aminolevulinic acid, aminosalicylic acid, amsacrine, anagrelide, anastrozole, asparaginase, anthracycline, retinal, bicalutamide, bleomycin, buserelin, cabergoline, capecitabine, carboplatin, carmustine, chlomambucin, cilastatin sodium, cisplatin, cladribine, disodium clodronate, cyclophosphamide, desacetyl cyclopropanoderogesterone, cytarabine, camptothecin, 13-cis-retinoic acid, all-trans retinoic acid; dacarbazine, actinomycin D, daunorubicin, deferoxamine, dexamethasone, diclofenac, stilbestrol, docetaxel, doxorubicin, epirubicin, estramustine, etoposide, exemestane, fexofenadine, fludarabine, fludrocortisone, fluorouracil, fluorohydroxymethyltestosterone, flutamide, gemcitabine, epinephrine, L-dopa, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, itraconazole, goserelin, letrozole, leucovorin, levamisole, lisinopril, levothyroxine sodium, lomustine, azacitine, luteolin, megestrol, melphalan, mercaptopurine, metahydroxydesmethylephedrine ditartrate, methotrexate, metoclopramide, mezzolazine, mitomycin, mitotane, mitoxantrone, naloxone, nimoramide, doxycycline, doxyc, Octreotide, oxaliplatin, pamidronic acid, pentostatin, pilampicin, porphine, prednisone, procarbazine, prucloperazine, ondansetron, letrothiolane, sirolimus, streptozotocin, tacrolimus, tamoxifen, temozolomide, teniposide, testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa, topotecan, retinoic acid, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, dolasetron, granisetron; formoterol, fluticasone, linden, midazolam, alprazolam, amphotericin B, podophylotoxins, nucleoside antiviral agents, aroylhydrazone, sumatriptan; macrolides such as erythromycin, oleandomycin, roxithromycin, clarithromycin, daunomycin, azithromycin, flurubicin, dirithromycin, josamycin, spiramycin, meridamycin, bleomycin, milocarmycin, rokitamycin, andrazithromycin, and swinolide A; fluoroquinolones, such as ciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrovafloxacin, moxifloxacin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin, iloxacin, pazufloxacin, clinafloxacin and sitafloxacin; aminoglycoside antibiotics, such as gentamicin, netilmicin, paramecium, tobramycin, amikacin, kanamycin, neomycin and streptomycin, vancomycin, teicoplanin, lanoplanin (rampolanin), mediterranin, cleistan, daptomycin, gramicin, polymyxin E methanesulfonic acid; polymyxins such as polymyxin B, capreomycin, bacitracin, penems; penicillins, including penicillinase-sensitive agents, such as penicillin G, penicillin V; penicillinase inhibitors, such as methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin; gram (stain) negative microbial agents such as ampicillin, amoxicillin and natacillin, penicillin and glatiramer; anti-pseudomonas penicillins, such as carbenicillin, ticarcillin, azlocillin, mezlocillin and piperacillin; cephalosporins, such as cefpodoxime proxetil, cefprozil, cephalobutene, ceftizoxime, ceftriaxone, cephalothin, cefapirin, cephalexin, cephradine, cefoxitin, cefamandole, cefazolin, cephradine, cefaclor, cefadroxil, cefalexin, cefuroxime, ceforanide, cefotaxime, ceftriaxone, cefotaxime, cefepime, cefixime, cefonicid, cefoperazone, cefotetan, cefmetazole, ceftazidime, chlorocefprozil and moxalactam, the monobactal ring (e.g. aztreonam); and carbapenems such as imipenem, meropenem, pentamidine-isothiourea, salbutamol sulfate, lidocaine, metaproterenol sulfate, prednisolone dipropionate, triamcinolone acetonide, budesonide acetonide, fluticasone, ipratropium bromide, flunisolide, cromolyn sodium, and ergotamine tartrate; taxanes, such as paclitaxel; SN-38, and tefosetyl.

The above exemplary drugs, where applicable, include analogs, agonists, antagonists, inhibitors, isomers, polymorphs, and pharmaceutically acceptable salts formed from these substances. Thus, for example, if the above-mentioned typical drug is relatively large, it does not belong to the class of small molecule drugs, but is still included because the large molecule analogs, which have similar activities but are small, can be used.

Small molecule drugs particularly suitable for the present invention are those that can significantly penetrate biological membranes. Small molecule drugs that are capable of penetrating the skin barrier are also contemplated for use in the present invention. In some cases, small molecule drugs, when administered orally or even parenterally, have an unfavorably large number of crossing biological membranes. For example, small molecule drugs that have unfavorable blood-brain barrier penetration are those that exhibit greater brain absorption than atenolol. In this regard, small molecule drugs having a brain absorption rate ("BUR") (measured as described herein) greater than about 15pmol/gm brain/sec are non-limiting examples of those small molecule drugs that have unfavorable blood-brain barrier penetration.

Thus, small molecule drugs for non-central nervous system indications but which penetrate the blood brain barrier are preferred subjects in terms of the blood brain barrier, since the conjugation of these drugs provides molecules with fewer central nervous system side effects. For example, structurally related nucleotides and nucleosides (e.g., 8-azaguanine, 6-mercaptopurine, azathioprine, thioinosinate, 6-methylthioinosinate, 6-thiouric acid, 6-thioguanine, vidarabine, cladribine, ancitabine, azacytidine, erythro-9- (2-hydroxy-3-nonyl) adenine, fludarabine, gemcitabine, and the like) are preferred.

With respect to fludarabine, this small molecule drug exhibits an oral bioavailability of about 70% and is useful for the treatment of chronic lymphatic leukemia as well as hairy cell leukemia, non-hodgkin's lymphoma and mycosis fungoides. Fludarabine also exhibits central nervous system related side effects with severe neurological effects including blindness, coma and even death. Animal studies in mice and rabbits have shown that drugs may also cause deformities. Thus, the fludarabine conjugates are expected to be effective in preventing the penetration of the blood brain barrier and/or the blood placental barrier by the drug, or at least to reduce the rate of penetration of these barriers, thereby ameliorating the undesirable side effects of fludarabine.

Another class of small molecule drugs, while typically used as peripheral neuroactive agents, have common central nervous system related side effects and are antihistamines. Structurally, antihistamines are self-associated as aminoalkyl ethers. The small molecule medicine includes diphenhydramine, diphenhydramine bromide, doxylamine, carbinoxamine, clemastine, dimenhydrinate, tripheniramine, pyrilamine, mesalamine, azolamine, pheniramine, chlorpheniramine, dexchlorpheniramine, brompheniramine, dexbrompheniramine, piretamine, triprolidine, promethazine, alismazine, methdilazine, cyclizine, clocrizine, diphenylaline, phenindamine, dimetindidine, minoxidil, buclizine, azaconazole, cyproheptadine, azatadine, terfenadine, fexofenadine, astemizole, cetirizine, azelastine, azatadine, loratadine, and desloratadine.

Yet another class of small molecule drugs are opioid antagonist small molecule drugs that are expected to reduce blood-brain barrier penetration. Opioid antagonists include naloxone, N-methylnaloxone, 6-amino-14-hydroxy-17-allylnordesomorphine, naltrexone, nalmexadone, N-methylnaltrexone, nalbuphine, butorphanol, cyclazocine, tebuconazole, nalprofen, naltrendole, nornaloxone, oxorphan, 6-amino-6-deoxy-naloxone, tebuconazole, levorphanol methylnaltrexone, buprenorphine, seco-clofane, levorphanol and nalprofen, and opioid antagonists described in U.S. patent nos. 5,159,081, 5,250,542, 5,270,328 and 5,434,171 and Knapp et al (l.f. tseng ed, p.15, hartwood acad Academic papers, 1995). But in general includes any member of the oxymorphone chemical class (including the opioid antagonists described above, as well as oxymorphone, codeine, oxomidine, morphine, ethylmorphine hydrochloride, diacetylmorphine, hydromorphone, dihydrocodeine, dihydromorphine, and dihydromorphine).

Another class of chemical small molecule drugs are platinum coordination complex-based drugs. These include, for example, cis-platinum, hydrogen platinum, carboplatin and oxaliplatin.

Another class of small molecule drugs that are particularly suitable for conjugation are steroidal drugs. Preferred steroids have a hydroxyl group (or an acyl group that can be reduced to a hydroxyl group) within the molecular structure. Examples of steroids without restricting the invention include aldosterone, deoxycorticosterone, fludrocortisone, cortisone, hydrocortisone, prednisolone, prednisone, medrysone, methylprednisolone, acanisolone, thiocyanometasone, beclomethasone, betamethasone, dexamethasone, diflorasone, diflunisal, methylprednisolone, paramethasone, amcinonide, desonide, fluocinolone, flunisolide, flurandrenolone acetonide, triamcinolone, clobetasol, halcinolone acetonide, mometasone, clocortolone and desoximetasone.

Fluoroquinolones and related small molecule drugs of this class are useful in forming conjugates. Typical fluoroquinolones include ciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrefloxacin, moxifloxacin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin, iloxacin, pazufloxacin, clinafloxacin and sitafloxacin.

Yet another class of drugs commonly used for peripheral neurological indications, some of which are known to cause deformities, are retinoid-like small molecule drugs. Structurally related classes of retinoids include, but are not limited to, vitamin a, retinal, 3-dehydroretinol, alpha-carotene, beta-carotene, gamma-carotene, delta-carotene, cryptoxanthin, tretinoin, isotretinoin, etretinate, and eretin. Because of the potential teratogenicity of such small molecule drugs (or any class of drugs that cause malformations), we would like to reduce the potential harm to the fetus by completely eliminating or reducing the penetration of agents suspected of being teratogens across the blood-placental barrier.

Small molecule drugs for use herein as part of a conjugate include phenothiazines, dibenzodiazepinesGalactogues (e.g. metoclopramide) and thiazine compounds. Examples of phenothiazines include prochlorperazine, chlorpromazine, trichloromepromazine, and fluphenazine. DibenzodiazepinesExamples of (a) are clozapine, olanzapine and quetiapine. Other small molecule drugs include amlodipine, nifedipine, nimodipine, 5-hydroxytryptophan, tretinoid and isotretinoin. Another preferred drug is nevirapine, which readily penetrates the placental barrier.

Other small molecule drugs suitable for use in the present invention may be found in several documents, such as: "Merck index", 13 th edition, Merck & co, Inc. (2001); "AHFS handbook of medicines, 2 nd edition", American society of health and System pharmacists and Lippincott, Williams and Wilkins; "physician desk reference", Thomson Healthcare inc, 2003; and "Remington: pharmaceutical and practice ", 19 th edition, 1995.

The delivery and pharmacological properties of the small molecule drugs can be significantly changed after the small molecule drugs are modified by covalent bonding with water-soluble oligomers taken from monodisperse or bimodal oligomer components. With water-soluble oligomers taken from monodisperse or bimodal oligomer components, the properties of the drug can be tailored because the resulting conjugates form well-defined compositions, not in the form of a distribution of a range of small molecule drug-oligomer conjugate species with a distribution of monomeric subunits (and thus molecular weights). As noted above, it has been observed that increasing or decreasing the number of monomers can have a significant effect on the properties of the resulting conjugate. Discrete oligomer matrices of different sizes (from 1 to 30 monomer subunits) can be screened in a reasonable time, enabling the tailoring of conjugates with optimal properties.

When combined with small molecule drugs, the oligomers can provide different source moleculesThe properties of the sub-drugs. Small oligomers (as compared to 5000 to 6 million polymer chains typically associated with proteins) may also be used to increase the likelihood that the drug maintains at least some, and preferably a significant, degree of biological activity. This feature is illustrated in Table VI (example 10), which provides the biological activity (EC) of a typical conjugate of the invention₅₀) And (4) data. The illustrative PEG oligomer-naloxone/naloxonol conjugates have biological activities in the range of about 5% to about 35% of the unmodified parent drug, further demonstrating the advantageous properties of the compounds of the present invention.

Oligomers generally comprise two or more monomer types that combine in series to form a monomer chain. The oligomer may be composed of one monomer type (i.e., a homooligomer) or two or three monomer types (i.e., a co-oligomer). Preferably, each oligomer is a co-oligomer of two monomers, more preferably a homo oligomer. The monomers employed form oligomers which are soluble in water, the oligomers being > 95% and preferably > 99% soluble in water at physiological pH values of about 7.2 to 7.6 at room temperature.

Accordingly, each oligomer comprises up to three different monomer types: alkylene oxides, such as ethylene oxide or propylene oxide; alkenols, such as vinyl alcohol, 1-propenol or 2-propenol; (ii) vinylpyrrolidone; hydroxyalkyl methacrylamide or hydroxyalkyl methacrylate, wherein the alkyl group is preferably methyl; alpha-hydroxy acids such as lactic acid or glycolic acid; phosphazenes, oxazolines, amino acids, carbohydrates (such as monosaccharides, sugars, or mannitol); and N-acryloyl morpholine. Preferred monomer types include alkylene oxides, alkenyl alcohols, hydroxyalkyl methacrylamides or methacrylates, N-acryloyl morpholines, and alpha-hydroxy acids. Preferably, each oligomer is individually a co-oligomer of two monomer types selected from the above group, or, more preferably, is a homo oligomer of one monomer type selected from the above group.

The two monomer types in the co-oligomer may be the same monomer type, for example, two alkylene oxides, such as ethylene oxide and propylene oxide. Homogeneous oligomers in which the oligomer is ethylene oxide are preferred. Typically, but not necessarily, the ends of the oligomers that are not covalently bound to the small molecule are capped to render them unreactive. The terminus may also include a reactive group. When the terminal reactive group is selected, the reactive group is either unreactive under the conditions of final oligomer formation or during covalent attachment of the oligomer to the small molecule drug, or is protected as necessary. One common terminal functional group is a hydroxyl group or-OH, especially for oligoethylene oxides.

The water-soluble oligomer ("O" in the formula of conjugate O-X-D) may have any of a number of different geometries. For example, "O" (in the formula O-X-D) may be linear, branched or forked. Most typically, the water-soluble oligomer is linear or branched, e.g., has a branch point. While the discussion herein is more directed to the use of poly (ethylene oxide) as the illustrated oligomer, the discussion and structure herein can be readily extended to include any of the above water-soluble oligomers.

The molecular weight of the water-soluble oligomer, excluding the linker moiety, is generally lighter. Exemplary molecular weights of the water-soluble polymer include: less than about 1500; less than about 1400; less than about 1300; less than about 1200; less than about 1100; less than about 1000; less than about 900; less than about 800; less than about 700; less than about 600; less than about 500; less than about 400; less than about 300; less than about 200; and less than about 100 daltons.

Exemplary molecular weight (excluding linkers) ranges for the water-soluble oligomers include: between about 100 and about 1400 daltons; between about 100 and about 1200 daltons; between about 100 and about 800 daltons; between about 100 and about 500 daltons; between about 100 and about 400 daltons; between about 200 and about 500 daltons; between about 200 and about 400 daltons; between about 75 and about 1000 daltons; and between about 75 to about 750 daltons;

the amount of monomer in the water-soluble oligomer is preferably in the range of one or more of: between about 1 and about 30 (including 30); between about 1 and about 25; between about 1 and about 20; between about 1 and about 15; between about 1 and about 12; between about 1 and about 10. In some cases, the number of consecutive monomers in the oligomer (and corresponding conjugate) is 1, 2, 3, 4, 5,6, 7, or 8. In other embodiments, the oligomer (and corresponding conjugate) comprises 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive monomers. In yet other embodiments, the oligomer (and corresponding conjugate) has 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive monomers.

These values correspond to a molecular weight of 75, 119, 163, 207, 251, 295, 339, 383, 427 and 471 daltons for the methoxy-terminated oligo (ethylene oxide) when the water-soluble oligomer has 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 monomers, respectively. When the oligomer has 11, 12, 13, 14 or 15 monomers, these values correspond to methoxy-terminated oligo (ethylene oxide) having molecular weights of 515, 559, 603, 647 and 691 daltons, respectively.

In the case of bimodal oligomers, the oligomers will be bimodal centered on any two of the above amounts of monomers. The polydispersity index Mw/Mn of each peak in the bimodal distribution is preferably 1.01 or less, more preferably 1.001 or less, still more preferably 1.0005 or less. The MW/Mn value is most preferably 1.0000. For example, a bimodal oligomer may have an exemplary combination of any one of the following monomeric subunits: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, etc.; 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, etc.; 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, etc.; 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, and the like; 5-6, 5-7, 5-8, 5-9, 5-10, and the like; 6-7, 6-8, 6-9, 6-10, and the like; 7-8, 7-9, 7-10, and the like; and 8-9, 8-10, and so on.

In addition, the oligomers of the present invention can be trimodal or even tetramodal and have monomer units in the ranges described above. Oligomer compositions having a well-defined oligomer mixture (i.e., bimodal, trimodal, tetramodal, etc.) can be prepared by mixing purified monodisperse oligomers to obtain the desired oligomer characteristics (two oligomer mixtures differing only in monomer amount are bimodal; three oligomer mixtures differing only in monomer amount are trimodal; four oligomer mixtures differing only in monomer amount are tetramodal), or can be obtained from column chromatography of polydispersed oligomers by recovering a "central cut" to obtain oligomer mixtures in the desired and well-defined molecular weight range. As shown in FIG. 10, commercially available PEG is generally a polydisperse mixture, even for low molecular weight species. The illustrated methoxy-PEG sample, although labeled methoxy-PEG-350, was analyzed by mass spectrometry and found to contain 9 different PEG oligomer components, each differing in the number of monomeric subunits. For the purposes of the present invention, that is, for the preparation of conjugates having the properties described herein, polydisperse polymers are not particularly preferred, since slight variations in the number of monomers have a dramatic effect on the resulting conjugate. These effects may be suppressed or even not found in conjugate mixtures prepared using polydisperse oligomers. Furthermore, commercial batches of polydisperse polymers (or oligomers) often vary widely in composition and for this reason are not particularly preferred for use in the present invention, and batch-to-batch compositional consistency is a desirable characteristic of the oligomers described herein.

As mentioned above, the water-soluble oligomer is preferably obtained from a monomolecular or monodisperse composition. That is, the oligomers in the composition have the same discrete molecular weight values, rather than the molecular weights being distributed. Some monodisperse oligomers can be purchased commercially, for example from Sigma-Aldrich, or prepared directly using starting raw materials available from commercial sources, such as Sigma-Aldrich. For example, the oligoethylene glycols of the present invention may be prepared as described in Chen Y., Baker, G.L., J.org.Chem., 6870-6873(1999) or WO02/098949A 1. Such oligomers may also be prepared as described in example 9 herein.

As noted above, one aspect of the present invention is an improved process for preparing monodisperse oligomers, such as oligo (ethylene oxide). These oligomers are useful in a variety of applications including, but not limited to, the preparation of small molecule drug-water soluble oligomer conjugates having the aforementioned advantageous properties.

To provide the desired monodisperse oligomers, a new process is used. It was found that the halogen-terminated oligomer reagents were more reactive than the aforementioned reagents and were capable of producing more monofunctional products.

Accordingly, the present invention also includes a method of preparing a monodisperse oligomer compound. The process comprises reacting a halo-terminated oligomer having (m) monomers (such as oligo (ethylene oxide) with a hydroxy-terminated oligo (ethylene oxide) having (n) monomers, generally speaking, the halo group on the halo-terminated oligomeric ethylene glycol is chlorine, bromine or iodine, however, the halo group is preferably bromine the reaction is conducted under conditions effective to replace the halo group derived from the halo-terminated oligomer, thereby forming an oligo (ethylene oxide) having (m) + (n) monomeric subunits (OEGm + n), wherein (m) and (n) range from 1 to 10, that is, (m) and (n) are 1, 2, 3, 4, 5,6, 7, 8, 9 or 10, (m) and (n) are each 1 to 6, preferably, (m) is 1, 2 or 3, and (n) is 1 to 6, in selected embodiments, (m) is 1, 2 or 3, and (n) is between 2 and 6. The reaction is generally carried out in the presence of a strong base suitable for converting the hydroxyl groups of the hydroxy-terminated oligoethylene oxide into the corresponding alkoxide. Suitable strong bases include sodium, potassium, sodium hydride, sodium methoxide, potassium methoxide, sodium t-butoxide, and potassium t-butoxide. In a preferred embodiment, the halo-terminated oligoethylene glycol has a capping group such as methoxy or ethoxy.

Representative hydroxy-terminated oligo (ethylene glycol) s correspond to HO- (CH)₂CH₂O)_n-H structure, wherein (n) is as described above. The process preferably comprises converting the terminal hydroxyl group OEG_m+nIs halo-X to form OEG_m+n-X. The above steps are then repeated until a unimolecular oligomer with the desired number of subunits is obtained.

An exemplary reaction scheme is as follows:

X＝Cl，Br，I

2.CH₃O-(CH₂CH₂O)_m+n-H→CH₃(O-CH₂CH₂)_m+n-X

4.CH₃O-(CH₂CH₂O)_m+2n-H→CH₃(O-CH₂CH₂)_m+2n-X

as indicated above, the process involves combining two unimolecular oligomers by a substitution reaction in which the halide on one oligomer, preferably an oligoethylene oxide, more preferably a halogen-derived oligoethylene oxide methyl ether, is reacted with an oligoethylene glycol-alkoxide to form the corresponding oligomer (see reaction 1 above).

Alkoxides are typically formed from the conversion of a terminal hydroxyl group to the corresponding alkoxide by the corresponding oligomeric ethylene oxide in the presence of a strong base. The reaction generally takes place in an organic solution such as tetrahydrofuran "THF" at a temperature of about 0 ℃ to about 80 ℃. The reaction time is typically between about 10 minutes and about 48 hours. The product of the above exemplary reaction, the capped oligoethylene oxide, contains the sum of the amount of monomer containing the halogen-derived oligomer and the amount of monomer in the oligoethylene glycol-alkoxide [ (m) + (n) ]. In the case of the purified conjugate, the yield is typically about 25% to 75%, most typically about 30% to 60%.

In the above example, the hydroxyl end of the product of reaction 1 is then activated, if necessary, to bind small molecules. Furthermore, the hydroxyl end of the above exemplified products [ in the above example with (m) + (n) subunits ] is then converted to a halide, preferably bromide, if desired. The conversion of ethanol to alkyl halide can be carried out directly or via an agent such as a sulfonate or haloformate. Suitable conditions and reagents for carrying out such a transformation are found, for example, in Larock, R. "Comprehensive Organic Transformations", VCH, 1994, pages 353 to 363.

The preferred method is the method described in example 11. The stepwise addition of oligoethylene oxide halide to oligoethylene oxide is repeated as described above to form an oligoethylene oxide having (m) +2(n) monomers. In this way, discrete oligomeric ethylene oxide subunits are gradually added to existing monomeric (single molecule) oligomeric ethylene oxide products in a controlled manner, ensuring the production of well-defined oligomers with the correct number of subunits.

Commonly available are monomolecular oligoethylene glycols (Sigma-Aldrich) having about 1-3 monomeric subunits. The use of halogenated oligoethylene glycol reagents represents an improvement over prior methods, such as the use of methanesulfonic acid, because the methods described herein can increase yields, shorten reaction times, and reduce reaction conditions due to the more reactive halides, especially brominated oligoethylene glycol reagents. The oligomers so prepared are typically purified prior to further use, for example, by one or more of the following methods: chromatography (such as HPLC), ion exchange chromatography, column chromatography, precipitation or recrystallization. Purity can then be determined using a variety of analytical techniques, such as NMR, GPC, and FTIR. The product thus formed is suitable for further use.

The linker or bond linkage of the present invention may be a single atom, such as oxygen or sulfur, two atoms or more. The linker is typically, but not necessarily, linear in nature. The "X" bond (X in the formula O-X-D) is hydrolytically stable, preferably also enzymatically stable. Preferably, the "X" bond is a bond with a chain length of less than about 12 atoms, more preferably less than about 10 atomsPreferably, less than about 8 atoms and even more preferably, less than about 5 atoms and even more preferably, wherein length refers to the number of atoms in the single chain, not counting substituents. For example, like Roligomer-NH- (C ═ O) -NH-R'_MedicineSuch urea bonds are considered to have a chain length of 3 atoms-NH-C(O)-NH-). In selected embodiments herein, no further spacer groups are included between the bonds. Small-bond linkages are preferred, and are suitable for use in the present invention, because such small-bond linkages do not dominate or outweigh the effect of adding one or a small number of monomeric subunits on the differences in the delivery properties of the conjugates of the present invention.

In some cases, the "X" linker is hydrolytically stable and comprises an ether, amide, carbamate, amine, thioether, urea, or carbon-carbon bond. The functional groups described below and illustrated in the working examples are typically used to form a bond linkage. As described further below, the key linkage may also preferably comprise (or be adjacent to or flanked by) spacers. The spacer is most effective in cases where the biological activity of the conjugate is significantly reduced by the location of the oligomer on the parent drug.

More specifically, in selected embodiments herein, the linker of the invention may be any one of the following:

-O-，-NH-，-S-，-C(O)-，C(O)-NH，NH-C(O)-NH，O-C(O)-NH，-C(S)-，-CH₂-，-CH₂-CH₂-，

-CH₂-CH₂-CH₂-，-CH₂-CH₂-CH₂-CH₂-，-O-CH₂-，-CH₂-O-，-O-CH₂-CH₂-，-CH₂-O-CH₂-，

-CH₂-CH₂-O-，-O-CH₂-CH₂-CH₂-，-CH₂-O-CH₂-CH₂-，-CH₂-CH₂-O-CH₂-，

-CH₂-CH₂-CH₂-O-，-O-CH₂-CH₂-CH₂-CH₂-，-CH₂-O-CH₂-CH₂-CH₂-，

-CH₂-CH₂-O-CH₂-CH₂-，-CH₂-CH₂-CH₂-O-CH₂-，-CH₂-CH₂-CH₂-CH₂-O-，

-C(O)-NH-CH₂-，-C(O)-NH-CH₂-CH₂-，-CH₂-C(O)-NH-CH₂-，-CH₂-CH₂-C(O)-NH-，

-C(O)-NH-CH₂-CH₂-CH₂-，-CH₂-C(O)-NH-CH₂-CH₂-，-CH₂-CH₂-C(O)-NH-CH₂-，

-CH₂-CH₂-CH₂-C(O)-NH-，-C(O)-NH-CH₂-CH₂-CH₂-CH₂-，

-CH₂-C(O)-NH-CH₂-CH₂-CH₂-，-CH₂-CH₂-C(O)-NH-CH₂-CH₂-，

-CH₂-CH₂-CH₂-C(O)-NH-CH₂-，-CH₂-CH₂-CH₂-C(O)-NH-CH₂-CH₂-，

-CH₂-CH₂-CH₂-CH₂-C(O)-NH-，-NH-C(O)-CH₂-，-CH₂-NH-C(O)-CH₂-，

-CH₂-CH₂-NH-C(O)-CH₂-，-NH-C(O)-CH₂-CH₂-，-CH₂-NH-C(O)-CH₂-CH₂，

-CH₂-CH₂-NH-C(O)-CH₂-CH₂，-C(O)-NH-CH₂-，-C(O)-NH-CH₂-CH₂-，-O-C(O)-NH-CH₂-，

-O-C(O)-NH-CH₂-CH₂-，-NH-CH₂-，-NH-CH₂-CH₂-，-CH₂-NH-CH₂-，-CH₂-CH₂-NH-CH₂-，

-C(O)-CH₂-，-C(O)-CH₂-CH₂-，-CH₂-C(O)-CH₂-，-CH₂-CH₂-C(O)-CH₂-，

-CH₂-CH₂-C(O)-CH₂-CH₂-，-CH₂-CH₂-C(O)-，

-CH₂-CH₂-CH₂-C(O)-NH-CH₂-CH₂-NH-，

-CH₂-CH₂-CH₂-C(O)-NH-CH₂-CH₂-NH-C(O)-，

-CH₂-CH₂-CH₂-C(O)-NH-CH₂-CH₂-NH-C(O)-CH₂-, divalent cycloalkyl, -N (R6) -, R6 is H or an organic radical selected from the group consisting of: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl.

However, for the purposes of the present invention, a sequence of atoms is considered not to be a bond linkage when it is directly adjacent to an oligomer segment, but rather is simply another monomer, and thus a bond linkage will represent a purely oligomeric chain extension.

The "X" bond linkage between the oligomer and the small molecule is typically formed by the reaction of a functional group at the end of the oligomer with a corresponding functional group in the small molecule drug. Exemplary reactions are briefly described below. For example, the amino group "O" on an oligomer can react with a carboxylate or a reactive carboxylate derivative on a small molecule, or vice versa, to form an amide linkage. Alternatively, the amine on the oligomer reacts with an activated carbonate (e.g., succinimide or benzenetriaminocarbonate) on the drug, or vice versa, to form a urethane linkage. The amine on the oligomer reacts with the isocyanate on the drug (R-N ═ C ═ O) to form a urea linkage (R-NH- (C ═ O) -NH-R'). Further, the ethanol (alkoxide) groups on the oligomer react with the alkyl halide or halide groups in the drug to form ether linkages. In yet another conjugation method, small molecules with aldehyde functionality are conjugated to oligomer amino groups by reductive amination, thereby forming secondary amine bonds between the oligomer and the small molecule.

Particularly preferred oligomers are those having aldehyde functionality. In this regard, the oligomer has the following structure: CH (CH)₃O-(CH₂-CH₂-O)_n-(CH2)_p-c (o) H, wherein (n) is any one of 1, 2, 3, 4, 5,6, 7, 8, 9 and 10 and (p) is any one of 1, 2, 3, 4, 5,6 and 7. Preferred (n) values include 3, 5 and 7, and preferred (p) values include 2, 3 and 4. In addition, the carbon atom α on the-C (O) H moiety may optionally be replaced by an alkyl group. Preferably, the oligomer reagent is provided as a monodisperse composition.

Typically, the oligomer ends without functional groups are capped, rendering them unreactive. If the oligomer has a functional group at the end other than that used to form the conjugate, the selected functional group is either unreactive under the conditions used to form the "X" bond or protected during the formation of the "X" bond.

As described above, the oligomers include functional groups for forming small molecule conjugates having the attributes described herein. Depending on the reactive group within or entering the small molecule, the functional group typically comprises an electrophilic or nucleophilic group to covalently bind to the small molecule. Examples of nucleophilic groups that may be present in an oligomer or small molecule include hydroxyl, amine, hydrazine (-NHNH)₂) Hydrazide (-C) (O) NHNH)₂) And mercaptans. Preferred nucleophiles include amines, hydrazines, hydrazides and thiols, especially amines. Most small molecule drugs covalently bound to oligomers will have free hydroxyl, amino, thio, aldehyde, ketone, or carboxyl groups.

Examples of electrophilic functional groups that may be present in an oligomer or small molecule include carboxylic acids, carboxylates, imides, orthoesters, carbonates, isocyanates, isothiocyanates, aldehydes, ketones, sulfur, olefins, acrylates, methacrylates, acrylamides, sulfones, maleimides, disulfides, iodine, epoxies, sulfonates, silanes, alkoxysilanes, and halosilanes. More specific examples of such groups include succinimidyl ester or carbonate, imidazolyl ester or carbonate, benzotriazole ester or carbonate, vinyl sulfone, chloroethyl sulfone, vinyl pyridine, pyridine disulfide, iodoacetamide, glyoxal, diketone, methanesulfonate, methyl tosylate, and triacrylate (2, 2, 2-trifluoroethane sulfonate).

Also included are sulfur analogs of several of these groups, such as thiones, thionehydrates, ketals, and the like, as well as hydrates or protected derivatives of any of the foregoing (e.g., aldehyde hydrates, hemiacetals, acetals, ketone hydrates, hemiketals, ketals, thioacetals). Another effective conjugation reagent is 2-thiazolethione.

As noted above, an "activated derivative" of a carboxylic acid refers to a derivative of a carboxylic acid that readily reacts with nucleophiles, generally much more readily than an underivatized carboxylic acid. Reactive carboxylic acids include, for example, acid halides (e.g., acid chlorides), anhydrides, carbonates, and esters. These esters include the general form (- (CO) O-N [ (CO) -]₂) An imide ester of (a); for example, N-hydroxysuccinimide (NHS) ester or N-hydroxyphthalimide ester. Also preferred are imidazolyl esters and benzotriazole esters. Particularly active propionic or butyric esters, as described in commonly owned U.S. patent No. 5,672,662. These include- (CH)₂)_2-3A C (═ O) O-Q form, wherein Q is selected from the group consisting of N-succinimide, N-thiosuccinimide, N-phthalimide, N-glutarimide, N-tetrahydrophthalimide, N-norborneol-2, 3-dicarboximide, benzotriazole, 7-azobenzotriazole and imidazole.

Other preferred electrophilic groups include succinimide carbonate, maleimide, benzotriazole carbonate, glycidyl ether, imidazole carbonate, p-nitrophenyl carbonate, acrylate, triacrylate, aldehyde, and orthopyridyl disulfide.

These electrophilic groups react with nucleophiles (such as hydroxyl, thio, or amino groups) to form various binding types. The preferred reaction of the present invention is one that is capable of supporting the formation of hydrolytically stable linkages. For example, carboxylic acids and their reactive derivatives, including orthoesters, succinimidyl esters, imidazolyl esters, and benzotriazole esters, react with nucleophiles of the above type to form esters, thioesters, and amide compounds, respectively, wherein the amide compounds have the highest decomposition stability. As mentioned above, conjugates having hydrolytically stable linkages between the oligomer and the drug are most preferred. Carbonates, including succinimide, imidazolyl and benzotriazole carbonates, react with the amino group to form a carbamate salt. Isocyanate (R-N ═ C ═ O) reacts with hydroxyl OR amino groups to form carbamate (RNH-C (O) -OR ') OR urea (RNH-C (O) -NHR') bonds, respectively. Aldehydes, ketones, glyoxals, diketones and hydrates or ethanol adducts thereof (i.e., aldehyde water, hemiacetals, acetals, ketone hydrates, hemiketals and ketals) are preferably reacted with amines, and then, if necessary, the resulting imines are reduced to provide amine linkages (reductive amination).

Nucleophilic groups such as thiols can be added to some electrophilic functional groups, including electrophilic double bonds, to form electrophilic double bonds, such as thioether bonds. These groups include maleimides, vinyl sulfones, vinyl pyrimidines, acrylates, methacrylates, and acrylamides. Other groups comprise leaving groups that can be substituted with nucleophiles; these groups include chloroethyl sulfone, pyridine disulfide (including a cleavable S-S bond), iodoacetamide, methanesulfonic acid, methyl tosylate, thiosulfonate, and triacrylate. Epoxides react with nucleophiles by ring opening to form, for example, ether or amine linkages. Reactions occurring on oligomers and small molecules involving complementary reactive groups as described above are used to prepare conjugates of the invention.

For example, example 1 details the preparation of oligomeric conjugates of retinoid acids. Small molecules, i.e., retinoid acids containing reactive carboxyl groups, are conjugated with amino-active oligoethylene glycols to provide conjugates having amino groups that covalently link the small molecule to the oligomer linkage. Example 1 also describes the covalent attachment of PEG 3-mer (an oligoethylene glycol having 3 ethylene glycol subunit), PEG 7-mer and PEG 11-mer to a retinoid acid.

Example 4 describes the preparation of oligomer-naloxone conjugates. In this representative synthesis, after protecting the aromatic hydroxyl group, the keto group in naloxone is reduced to the corresponding hydroxyl group and then conjugated to an oligoethylene glycol to form an ether (-O-) which is linked to a small molecule conjugate. Interestingly, in this example, the reduction of the hydroxyl group in naloxone results in the formation of two isomers that differ in hydroxyl orientation. The corresponding oligomeric conjugates were prepared and isolated and shown to have several different properties, which will be discussed in detail below. This represents another feature of the present invention, namely, the preparation/isolation of the individual monomers of the oligomer-small molecule conjugates and their use.

As previously described, the conjugates of the present invention exhibit low biological barrier penetration rates. In addition, the conjugate retains at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or more of the activity of the unmodified parent small molecule drug. For certain small molecule drugs having multiple reactive sites suitable for modification, it may be necessary to perform molecular modeling, or biological activity assays in vivo or in vitro, to assess the biological activity of the resulting conjugate and to determine the site most suitable for covalent binding to the oligomer. See Table VI for exemplary biological activity data for various naloxone and derivative naloxone, 6-NH 2-naloxone, and 6-OH-naloxonol oligomeric conjugates. In this study, variables included the site of chemical modification on the parent drug, the type of covalent bond, the stereochemistry, and the size of the oligomer to which the drug moiety was covalently bound. As the data show, the biological activity of the conjugates was about 5% to about 35% of the parent drug.

It has been found that stable covalent association of water-soluble small oligomers with orally bioavailable small molecule drugs can significantly alter the properties of these small molecules, making them clinically more effective. More specifically, covalent attachment to monodisperse oligomers such as oligomeric ethylene oxides can effectively reduce, or in some cases, eliminate, drug delivery across the blood-brain barrier, thereby significantly reducing central nervous system related side effects. A typical selection method for the best size oligomer is as follows.

First, oligomers obtained from monodisperse or bimodal water-soluble oligomers are conjugated with small molecule drugs. It is preferred that the drug have oral bioavailability and exhibit biofilm penetration rate by itself. Next, the ability of the conjugate to penetrate a biological membrane was determined using an appropriate model and compared to the unmodified parent drug. If the results were good, that is, if the penetration rate was significantly reduced, the biological activity of the conjugate was further evaluated. Advantageous conjugates according to the invention should be biologically active because the linkage is hydrolytically stable and does not result in the release of the unmodified drug upon administration. Thus, the drug in the conjugated state should have biological activity, preferably at a higher level of biological activity relative to the parent drug, i.e., greater than about 30% and more preferably greater than about 50% of the biological activity of the parent drug.

The above procedure is then repeated using oligomers of the same monomer type but with different numbers of subunits.

Because the gastrointestinal tract ("GIT") limits the transport of food and drugs from the digestive lumen to the blood and lymph, GIT represents another barrier that conjugates must be tested for. However, when the conjugate is to be delivered orally in the systemic circulation, the GIT barrier represents a barrier that must not prevent the conjugate. The GIT barrier is composed of a continuous layer of intestinal cells connected by tight junctions in the intestinal epithelial cells.

Oral bioavailability was then assessed for each conjugate that had a reduced ability to penetrate biological membranes compared to the unconjugated small molecule drug. Based on these results, that is, based on the sequential addition of increasing numbers of discrete monomers to a particular small molecule at a particular location or site within the small molecule, it is possible to determine the oligomer size that most effectively provides a conjugate having an optimal balance between reduced biological membrane penetration, oral bioavailability, and biological activity. This screening is made feasible due to the small oligomers, which allows us to effectively tailor the properties of the resulting conjugates. By making small incremental changes in oligomer size, and using experimental design methods, we can effectively find conjugates with a favorable balance between biofilm penetration, bioactivity, and oral bioavailability. In some cases, conjugation to oligomers as described herein can be effective in improving the oral bioavailability of the drug.

For example, by first preparing a series of oligomers of varying weights and having different functional groups, and then administering the conjugate to a patient and periodically sampling the blood and/or urine to obtain the necessary clearance profile, one of ordinary skill in the art can find the most appropriate molecular size and linkage to enhance oral bioavailability using routine experimentation. Once a series of clearance profiles for each of the conjugates tested is obtained, the appropriate conjugate can be determined.

Animal models (rodents and dogs) may also be used to study oral drug delivery. In addition, abiotic in vivo methods include rodent eversion of visceral excised tissue and Caco-2 cell monolayer tissue-culture models. These models are useful in predicting oral drug bioavailability.

The invention also includes pharmaceutical formulations of the conjugates described herein in combination with a pharmaceutical excipient. In general, the conjugates themselves will be in a solid state (e.g., a precipitate) and may be held together by suitable pharmaceutical excipients in either a solid or liquid state.

Typical excipients include, but are not limited to, carbohydrates, inorganic salts, antibacterial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.

Carbohydrates (e.g., sugars), derivatized sugars (e.g., alditols), alduronic acids, esterified sugars, and/or sugar polymers may be present as excipients. Specific carbohydrate excipients include, for example: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch and the like; and alditols such as mannitol, xylitol, maltitol, lactitol, sorbitol (glucitol), glucopyranosyl sorbitol, inositol, and the like.

Excipients may also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, monosodium phosphate, disodium phosphate, and combinations thereof.

The preparation may also include an antimicrobial agent to prevent or retard the growth of microorganisms. Non-limiting examples of antimicrobial agents suitable for the present invention include algicidal amines, algicidal chlorides, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof.

Antioxidants may also be present for the preparation. Antioxidants are used to prevent oxidation and thus deterioration of the conjugate or other components of the formulation. Antioxidants suitable for use in the present invention include, for example, ascorbyl palmitate, butanol-modified methoxyphenol, butanol-modified hydroxytoluene, hypophosphorous acid, thioglycerol, propyl gallate, sodium bisulfite, sodium aldehydite, sodium metabisulfite, and combinations thereof.

The surfactant may be present as an excipient. Exemplary surfactants include: polysorbates, such as "Tween 20" and "Tween 80", and pluronics, such as F68 and F88 (both available from BASF, Mount Olive, and New Jersey); sorbitan esters; lipids such as phospholipids, lecithin and other phosphatidylcholines, phosphatidylethanolamines (although not preferred as liposomes), fatty acids and aliphatic ethers; steroids, such as cholesterol; and chelating agents such as EDTA, zinc and other suitable such cations.

The acid and base groups which may be present as excipients are prepared. Non-limiting examples of acids that may be used include acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, sulfuric acid, fumaric acid, and combinations of these acids. Examples of suitable bases include, but are not limited to, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium dihydrogen phosphate, sodium citrate, sodium formate, sodium sulfate, potassium butenedioate, and combinations thereof.

The amount of conjugate in this composition may vary depending on several factors, but will be the optimal therapeutically effective dose when the composition is stored in a unit dose container. A therapeutically effective dose can be determined by repeating experiments with increasing amounts of conjugate to determine which amount produces a clinically desirable endpoint.

The amount of any individual excipient in the composition will vary based on the activity of the excipient and the particular needs of the composition. Typically, the optimum amount of any individual excipient is determined by routine experimentation, i.e., by preparing a composition containing varying amounts of excipient (from low to high), examining stability and other parameters, and then determining in which range the optimum performance can be obtained without serious adverse effects.

Generally, however, the excipient comprises from about 1% to about 99% by weight of the composition, with 5% -98% being preferred, 15-95% being more preferred, and concentrations below 30% being most preferred.

The aforementioned pharmaceutical excipients and others are described in the following documents: "Remington: pharmacy & practice ", 19 th edition, Williams & Williams, 1995," physician's desk reference ", 52 nd edition, health economics, Montvale, NJ (1998), and kibbe a.h., handbook of pharmaceutical excipients, 3 rd edition, american society for pharmaceuticals, washington city, 2000.

The pharmaceutical composition may be in a variety of forms, and the invention is not limited in this respect. Typical formulations preferably take a form suitable for oral administration, such as tablets, caplets, capsules, gel pills, tablets, dispersions, suspensions, solutions, elixirs, syrups, lozenges, transdermal patches, sprays, suppositories, and powders.

The orally active conjugates are preferably in an oral dosage form, including tablets, caplets, capsules, gel pellets, suspensions, solutions, elixirs and syrups, and may also comprise granules, beads, powders or pellets which may optionally be encapsulated. These dosage forms are prepared using conventional methods known to those skilled in the art of pharmaceutical formulation and described in the relevant text.

For example, tablets and caplets can be produced using standard tablet processing methods and equipment. Preferably, tablets or caplets comprising the conjugates described herein are prepared using direct compression and granulation processes. In addition to the conjugate, the tablets and caplets will generally comprise pharmaceutically acceptable inert carrier materials such as binders, lubricants, disintegrants, fillers, stabilizers, surfactants, colorants, and the like. The binder serves to enhance the cohesive properties of the tablet, thereby ensuring that the tablet remains intact. Suitable binder materials include, but are not limited to, starches (including corn starch and pregelatinized starch), gelatin, sugars (including sucrose, glucose, dextrose, and lactose), polyethylene glycols, waxes, and natural and synthetic gums, such as acacia alginate, polyvinylpyrrolidone, cellulosic polymers (including hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, ethyl cellulose, hydroxyethyl cellulose, and the like), and Veegum. Lubricants are used to facilitate tablet manufacture, improve powder flowability and prevent particles from capping the tip (i.e., particle breakage) when the pressure is released. Effective lubricants are magnesium stearate, calcium magnesium and stearic acid. Disintegrants are used to facilitate disintegration of tablets and are typically starches, clays, celluloses, algins, colloids, or cross-linked polymers. Fillers include silicon dioxide, titanium dioxide, alumina, talc, kaolin, powdered cellulose, microcrystalline cellulose and the like, and soluble substances such as mannitol, urea, sucrose, lactose, glucose, sodium chloride and sorbitol. Stabilizers well known in the art are used to inhibit or slow decomposition reactions, including oxidation reactions.

Capsules are also desirable oral dosage forms, and the conjugate-containing compositions may be encapsulated as a liquid or gel (for gelcaps) or a solid (including microparticles, such as granules, beads, powders or pellets). Suitable capsules include hard and soft capsules, typically made from gelatin, starch or fibrous materials. The two halves of the hard gel capsule are preferably sealed, for example with a gel tape or the like.

Including parenteral formulations in substantially dry form (typically as lyophilisates or precipitates, which may be in powder or bulk form), as well as formulations prepared for injection, typically as liquids, requiring reconstitution of the dry state of the parenteral formulation. Suitable diluents include bacteriostatic water for injection, water with 5% glucose, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.

In some instances, compositions for parenteral administration may be in the form of a non-aqueous solution, suspension, or emulsion, each of which is generally sterile. Examples of nonaqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil and corn oil), and injectable organic esters (such as ethyl oleate).

The parenteral formulations described herein may also contain adjuvants such as preservatives, wetting agents and dispersing agents. These formulations are provided in a sterile manner by filtration, irradiation or heating of the bacteria retaining filter using a bactericide.

The conjugate may also be administered through the skin using a conventional patch or other transdermal delivery mechanism, wherein the conjugate is contained within a layered structure that serves as a delivery device and is adhered to the skin. Within this structure, the conjugate is contained in one layer, the "storage layer," below the upper back layer. The layered structure may comprise a single storage layer, or a plurality of storage layers.

The invention also provides a method of administering a conjugate to a patient suffering from a disorder responsive to treatment with a conjugate described herein. The method comprises administering (typically orally) the conjugate (preferably provided as part of a pharmaceutical formulation) in an amount necessary to achieve a therapeutic effect. Other modes of administration may also be employed, such as pulmonary, nasal, buccal, rectal, sublingual, transdermal and parenteral administration. Herein, "parenteral administration" includes subcutaneous injection, intravenous injection, arterial injection, intraperitoneal injection, intracardiac injection, intrathoracic injection, and intramuscular injection.

In the case of parenteral administration, it may be necessary to use slightly larger oligomers than the aforementioned oligomers, i.e., in the molecular weight range of between about 500 and 3 kilodaltons (e.g., molecular weights of about 500, 1000, 2000, 2500, 3000, 5000, 7500, 10000, 15000, 20000, 25000, 30000 daltons or even greater).

The method of administration may be used to treat any condition that may be treated or prevented by the use of the particular conjugate. One of ordinary skill in the art will know which conditions a particular conjugate can effectively treat. The actual dosage employed may vary depending upon the age, weight and general condition of the subject, as well as the severity of the condition, the judgment of the medical practitioner, and the conjugate used. The amounts required to achieve a therapeutic effect are known to those skilled in the art and/or described in the relevant references and literature. Generally, the amount required to achieve a therapeutic effect will be between 0.001 mg and 100 mg, with dosages between 0.01 mg/day and 75 mg/day being preferred, and between 0.10 mg/day and 50 mg/day being more preferred.

Depending upon the judgment of the clinician, the needs of the patient, etc., unit doses of the particular conjugate (again preferably provided as part of a pharmaceutical formulation) may be administered in a variety of dosing regimens. The specific dosing regimen is known to those of ordinary skill in the art or can be determined experimentally using routine methods. Typical dosing regimens include, but are not limited to, five times per day, four times per day, three times per day, two times per day, once per day, three times per week, two times per week, once per month, and any combination of the above frequencies. Once the clinical endpoint is reached, administration of the synthetic agent should be discontinued.

One benefit of administering the conjugates of the invention is that first-pass metabolism can be reduced relative to the parent drug. For an example, please see the supporting results in example 8. This result is advantageous for many oral drugs that are metabolized substantially by the gut. In this way, the gap of the conjugate can be adjusted by selecting the molecular size of the oligomer, the linkage, and the location of the covalent bond to provide the desired gap properties. Based on the teachings herein, one of ordinary skill in the art will be able to determine the desired molecular size of the oligomer. Preferred conjugates with reduced first-pass metabolism compared to the corresponding unconjugated small molecule drug include: at least about 10%, at least about 20%, at least about 30%; at least about 40%; at least about 50%; at least about 60%, at least about 70%, at least about 80%, and at least about 90%.

Accordingly, the present invention provides a method of reducing metabolism of an active agent. The method comprises the following steps: providing monodisperse or bimodal conjugates, each conjugate comprising a moiety derived from a small molecule drug covalently attached to a water-soluble oligomer by a stable linkage, wherein the conjugate exhibits a metabolic rate that is lower than the metabolic rate of the small molecule drug not attached to the water-soluble oligomer; and administering the conjugate drug to the patient. Typically, administration is by one of the administration types selected from the group consisting of: oral, transdermal, buccal, transmucosal, vaginal, rectal, parenteral, and pulmonary.

While conjugates can be effective in reducing many types of metabolism (including stage I and stage II metabolism), conjugates are particularly effective when the small molecule drugs within the conjugate are metabolized by liver enzymes (e.g., one or more P450 cytochrome isomers) and/or by one or more intestinal enzymes.

Experiment of

While the invention has been described in connection with certain preferred embodiments, the foregoing description and the following examples are intended to illustrate, but not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention relates.

Unless otherwise indicated, all chemical reagents in the examples attached are commercially available. Example 9 describes an example of a unimolecular PEG-matrix agent. All of the oligoethylene glycol methyl ethers used in the examples below were monodisperse and chromatographically pure, as determined by reverse phase chromatography.

All of¹H NMR (nuclear magnetic resonance) data were generated by a 300MHz NMR spectrometer manufactured by Bruker. Some of the compounds and the sources of these compounds are listed below.

2-bromoethyl methyl ether, 92%, Aldrich;

1-bromo-2- (2-methoxyethoxy) ethane, 90%, Aldrich;

CH3(OCH2CH2)3Br, prepared from CH3(OCH2CH2)3 OH;

tris (ethylene glycol) monomethyl ether, 95%, Aldrich;

di (ethylene glycol), 99%, Aldrich;

tri (ethylene glycol), 99%, Aldrich;

tetra (ethylene glycol), 99%, Aldrich;

penta (ethylene glycol), 98%, Aldrich;

hexa (ethylene glycol), 97%, Aldrich;

sodium hydride, 95% dry powder, Aldrich;

methanesulfonyl chloride, 99%, ACE;

tetrabutylammonium bromide, Sigma.

Example 1

CH ₃ (OCH ₂ CH ₂ ) ₃ -NH-13-cis-viaminate (PEG) ₃ -13-cis-RA) synthesis

Preparation of PEG₃-13-cis-RA. The synthesis is summarized as follows.

0.1085 g CH₃(OCH₂CH₂)₃-NH₂(0.6656 mmol), 0.044 g of 1-hydroxybenzyltriazole ("HOBT", 0.3328 mmol) and 0.200 g of 13-cis-retinoid ("13-cis-RA", 0.6656 mmol) are dissolved in 10 ml of benzene. To the resulting solution was added 0.192 g of 1, 3-dicyclohexylcarbodiimide ("DCC", 0.9318 mmol) and the reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered and the solvent was removed by rotary evaporation. After further drying of the crude semifinished product under vacuum, it was dissolved in 20 ml of dichloromethane to give an organic phase which was washed twice with 15 ml of deionized water. In Na₂SO₄Dried, filtered, and the solvent removed using rotary evaporation. To the resulting product was added 2 drops of dichloromethane containing 50ppm of butylated hydroxytoluene and the product was dried under vacuum. Yield 0.335 g¹H NMR (DMSO): delta.1.02 (singlet, 2 CH)₃) 1.67 (singlet, CH)₃) 3.5 (broad multiplex, PEG), 6.20(m，3H)。

Example 2

CH₃-(OCH₂CH₂)₇Synthesis of-NH-13-cis-viaminate (PEG 7-13-cis-RA)

0.2257 g CH₃(OCH₂CH₂)7-NH₂(0.6656 mmol), 0.044 g of 1-hydroxybenzyltriazole (0.3328 mmol) and 0.200 g of 13-cis-retinoid (0.6656 mmol) are dissolved in 10 ml of benzene. To the resulting solution was added 0.192 g of 1, 3-dicyclohexylcarbodiimide (0.9318 mmol), and the resultant reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered, the solvent removed by rotary evaporation, the product dried under vacuum, dissolved in 20 ml of dichloromethane and the organic phase washed twice with 15 ml of deionized water. Organic phase in Na₂SO₄Dried, filtered and the solvent removed by rotary evaporation. To the resulting product was added 2 drops of dichloromethane containing 50ppm of butylated hydroxytoluene and the product was dried under vacuum. Yield 0.426 g¹H NMR (DMSO): delta.1.01 (singlet, 2 CH)₃) 1.68 (singlet, CH)₃) 3.5 (broad multiplex, PEG), 6.20(m, 3H).

CH₃-(OCH₂CH₂)₅-NH-13-cis-viaminates can be prepared analogously using this method, but CH₃(OCH₂CH₂)₇-NH₂By CH₃(OCH₂CH₂)₅-NH₂(″mPEG₅-NH₂") instead.

Example 3

CH ₃ -(OCH ₂ CH ₂ ) ₁₁ Synthesis of-NH-13-cis-viaminate (PEG) ₁₁ -13-cis-RA)

0.349 g CH₃(OCH₂CH₂)₁₁-NH₂(0.6789 mmol), 0.044 g of 1-hydroxybenzyltriazole (0.3328 mmol) and 0.204 g of 13-cis-retinoid (0.6789 mmol) are dissolved in 10 ml of benzene. To the resulting solution was added 0.192 g of 1, 3-dicyclohexylcarbodiimide (0.9318 mmol), and the resultant reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered, the solvent was distilled off by rotary evaporation, the product was dried under vacuum, dissolved in 20 ml of dichloromethane and the solution was washed twice with 15 ml of deionized water to give an organic phase. Organic phase in Na₂SO₄Dried, filtered and the solvent distilled off by rotary evaporation. To the resulting product was added 2 drops of dichloromethane containing 50ppm of butylated hydroxytoluene and the product was dried under vacuum. Yield 0.541 g¹H NMR (DMSO): delta.1.01 (singlet, 2 CH)₃) 1.68 (singlet, CH)₃) 3.5 (broad multiplex, PEG), 6.20(m, 3H).

Example 4

PEG ₃ Synthesis of (E) -3-naloxonol

The structure of naloxonol (a typical small molecule drug) is as follows:

naalol

This molecule (with protected hydroxyl group) was prepared as part of the larger synthetic scheme described in example 5.

Example 5

α，β-6-CH ₃ -(OCH ₂ CH ₂ ) ₁ -naloxonol (α, β -PEG) ₁ -Nal) Synthesis

Preparation of alpha, beta-PEG₁-naloxonol. The synthesis is summarized as follows:

synthesis of A.3-MEM-naloxone

Diisopropylethylamine (390 mg, 3.0 mmol) was added to naloxone HCl.2Hl₂O (200 mg, 0.50 mmol) in CH₂Cl₂(10 ml) to the solution, the mixture was stirred while adding. Methoxyhexyl chloride ("MEMC 1", 250 mg, 2.0 mmol) was then added dropwise to the above solution. Solution N at room temperature₂The atmosphere was stirred overnight.

Analysis of the crude product by HPLC showed the yield of 3-MEM-O-naloxone (1) to be 97%. The solvent was removed by rotary evaporation to give a viscous oil.

Synthesis of a mixture of alpha and beta epimers of B.3-MEM-naloxonol (2)

3 ml of 0.2N NaOH was added to 5 ml of 3-MEM-naloxone (1) (obtained from 5.A above and used without further purification) in ethanol. Adding NaBH dropwise into the solution₄(76 mg, 2.0 mmol) in water (1 ml). The resulting solution was stirred at room temperature for 5 hours. The ethanol was removed by rotary evaporation and then 0.1N HCl solution was added to eliminate excess NaBH₄The pH was adjusted to 1. With CHCl₃The solution was washed to remove excess methoxyhexyl chloride and its derivatives (3X 50 ml) and K was added₂CO₃The pH of the solution was raised to 8.0. With CHCl₃(3X 50 ml) the product was isolated in Na₂SO₄And drying.The solvent was removed by evaporation to yield a colorless, viscous solid (192 mg, 0.46 mmol, based on naloxone HCl · 2H₂Isolated yield of O92%).

HPLC showed the product to be a mixture of alpha and beta epimers of 3-MEM-naloxonol (2).

5.C.6-CH₃-OCH₂CH₂Synthesis of a mixture of alpha and beta epimers of (3a) O-3-MEM-naloxonol

NaH (60% in mineral oil, 55 mg, 1.38 mmol) was added to a solution of 6-hydroxy-3-MEM-naloxonol (2) (192 mg, 0.46 mmol) in dimethylformamide ("DMF", 6 mL). Mixture at room temperature N₂Stirring was carried out under atmosphere for 15 minutes, followed by addition of 2-bromoethyl ether (320 mg, 2.30 mmol) in DMF (1 ml). Then, the solution was at room temperature N₂Stirred under atmosphere for 3 hours.

HPLC analysis shows the formation of alpha-and beta-6-CH₃-OCH₂CH₂-O-3-MEM-naloxonol (3) mixture in about 88% yield. DMF was removed by rotary evaporation to yield a viscous white solid. The product was used directly in the subsequent conversion without further purification.

5.D.6-CH₃-OCH₂CH₂Synthesis of a mixture of alpha and beta epimers of naloxonol (4)

The crude alpha-and beta-6-CH are added₃-OCH₂CH₂-O-3-MEM-Narol (3) dissolved in 5 ml CH₂Cl₂A cloudy solution was formed, and then 5 ml of trifluoroacetic acid ("TFA") was added to the solution. The resulting solution was stirred at room temperature for 4 hours. The reaction was complete as determined by HPLC assay. Removal of CH with rotary evaporator₂Cl₂Then 10 ml of water was added. Adding a sufficient amount of K to the solution₂CO₃Excess TFA was eliminated and the pH adjusted to 8. Then using CHCl₃(3X 50 ml) the solution was extracted, the extracts were mixed and further extracted with 0.1N HCl solution (3X 50 ml) to give an aqueous phase. Adding K₂CO₃The pH of the aqueous phase was adjusted to 8 and then adjusted with CHCl₃(3X 50 ml) was further extracted. Mixing the obtained organic layers, and adding Na₂SO₄And (5) drying. The solvent was removed to yield a colorless, viscous solid.

The solid product was eluted in two passes to CHCl₃/CH₃Purification of OH (30: 1) on a silica gel column (2 cm. times. 30 cm) gave the product as a viscous solid. By using¹H NMR confirmed that the purified product was 6-CH containing ca.30% alpha epimer and ca.70% beta epimer₃-OCH₂CH₂-alpha-and beta epimers of naloxonol (4) [100 mg, 0.26 mmol ], isolated yield 56% based on 6-hydroxy-3-MEM-naloxonol (2)]。

¹H NMR(δ，ppm，CDCl₃): 6.50-6.73(2H, multiploid, aromatic proton of naloxonol), 5.78(1H, multiploid, olefinic proton of naloxone), 5.17(2H, multiploid, olefinic proton of naloxonol), 4.73(1H, doublet, C of alpha naloxonol)₅Proton), 4.57(1H, C of dual-state, beta-naloxonol)₅Proton), 3.91(1H, multiple, C of naloxonol)₆Proton), 3.51-3.75(4H, multiploid, PEG), 3.39(3H, singlet, methoxy proton of PEG, α epimer), 3.36(3H, singlet, methoxy proton of PEG, β epimer), 3.23(1H, multiploid, C of β naloxonol)₆Proton), 1.46-3.22(14H, multiplet, naloxonol proton).

Example 6

6-CH ₃ -(OCH ₂ CH ₂ ) ₃ Synthesis of-Nalo alcohol (α, β -PEG 3-Nalo alcohol)

6.A.6-CH₃-(OCH₂CH₂)₃Mixture of alpha and beta epimers of (E) -O-3-MEM-naloxonolSynthesis of (2)

NaH (60% in mineral oil, 38 mg, 0.94 mmol) was added to 3-MEM-naloxonol [98 mg, 0.24 mmol ], obtained as in example 5, shown as (2) in the schematic diagram of example 5]In dimethylformamide ("DMF" 8 ml). Solution N at room temperature₂Stirred for 15 minutes under atmosphere and then CH was added₃-(OCH₂CH₂)₃Br (320 mg, 1.41 mmol) in DMF (1 ml). The resulting solution is then in N₂The lower oil bath was heated for 2 hours.

HPLC analysis shows the desired products-alpha-and beta-6-CH₃-(OCH₂CH₂)₃The yield of the mixture of-O-3-MEM-naloxonol was about 95%. DMF was removed by rotary evaporation to yield a viscous white solid. The crude product was used without further purification.

6.B.6-CH₃-(OCH₂CH₂)₃-O-naloxonol (α, β -PEG)₃Nal) Synthesis of alpha and beta epimers

Alpha-and beta-6-CH obtained by crude preparation of the above-mentioned 6.A₃-(OCH₂CH₂)₃-O-3-MEM-Naro alcohol mixture dissolved in 3 ml of CH₂Cl₂To form a cloudy solution, 4 ml of trifluoroacetic acid ("TFA") was added. The resulting solution was stirred at room temperature for 4 hours. HPLC analysis showed the reaction was complete. Removal of solvent CH by rotary evaporation₂Cl₂. To the resulting solution was added 5 ml of water followed by addition of K₂CO₃Excess TFA was eliminated and the pH was adjusted to 8. Then, using CHCl₃(3X 50 ml) the solution was extracted. CHCl₃After the extracts were mixed, the mixture was extracted with 0.1N HCl solution (3X 50 mL). Adding K again to the remaining aqueous phase₂CO₃The pH was adjusted to 8 and then adjusted with CHCl₃(3X 50 ml) was extracted. The organic extracts were mixed and then added to Na₂SO₄And drying. Then, the solvent was removed to obtain a colorless viscous solid.

Two passes of the above solidsThe eluent is CHCl₃/CH₃OH (30: 1) was purified on a silica gel column (2 cm. times. 30 cm). The purified product is 6-CH₃-(OCH₂CH₂)₃-a mixture of α and β epimers of O-naloxonol wherein the amounts of α and β epimers are approximately equal and the mixture has NMR properties. (46 mg, 0.097 mmol, isolated yield 41% based on 6-hydroxy-3-MEM-O-naloxone).¹H NMR(δ，ppm，CDCl₃): 6.49-6.72(2H, multiploid, aromatic proton of naloxonol), 5.79(1H, multiploid, olefinic proton of naloxonol), 5.17(2H, multiploid, olefinic proton of naloxonol), 4.71(1H, doublet, C of alpha naloxonol)₅Proton), 4.52(1H, C of dual-state, beta-naloxonol)₅Proton), 3.89(1H, multiple, C of alpha naloxonol)₆Proton), 3.56-3.80(12H, multiploid, PEG), 3.39(3H, singlet, methoxy proton of PEG, alpha epimer), 3.38(3H, singlet, methoxy proton of PEG, beta epimer), 3.22(1H, multiploid, C of beta naloxonol)₆Proton), 1.14-3.12(14H, multiplet, naloxonol proton).

6.C.α-6-CH₃-(OCH₂CH₂)₃-O-naloxonol and β -6-CH₃-(OCH₂CH₂)₃Isolation of-O-naloxonol

About 80 mg of alpha and beta PEG₃-natural mixture of naloxonol epimers dissolved in minimal amount of CHCl₃Then injecting CHCl₃Prepared in silica gel column (2 cm. times.30 cm). With CHCl₃/CH₃The OH mixture (60: 1) was carefully eluted through the silica gel column. The first elution was pure alpha-PEG₃-naloxonol (26 mg, 33% isolated yield) followed by pure β -PEG₃Naloxonol (30 mg, 38% isolated yield). Both compounds were colorless, viscous solids. alpha-PEG₃-Nal，¹H NMR(δ，ppm，CDCl₃): 6.49-6.73(2H, two doublets, aromatic protons of naloxonol), 5.79(1H, multiplets, olefinic protons of naloxonol), 5.17(2H, triplet, olefinic protons of naloxonol), 471(1H, doublet, C of naloxonol)₅Proton), 3.81(1H, multiple, C of naloxonol)₆Proton), 3.57-3.80(12H, multiplet, PEG), 3.40(3H, singlet, methoxy proton of PEG), 1.13-3.12(14H, multiplet, naloxone proton). beta-PEG₃-Nal，¹H NMR(δ，ppm，CDC₃): 6.54-6.72(2H, two doublets, aromatic protons of naloxonol), 5.77(1H, multiplet, olefinic protons of naloxonol), 5.15(2H, triplet, olefinic protons of naloxonol), 4.51(1H, doublet, C of naloxonol)₅Proton), 3.58-3.78(12H, multiploid, PEG), 3.39(3H, singlet, methoxy proton of PEG), 3.20(1H, multiploid, C of naloxonol)₆Proton), 1.30-3.12(13H, multiplet, naloxonol proton).

α，β-6-CH₃-(OCH₂CH₂)₅-O-naloxonol ("α, β -PEG)₅Nal') and alpha, beta-6-CH₃-(OCH₂CH₂)₇-O-naloxonol ("α, β -PEG)₇Nal ") and the separation of the respective isomers.

Example 7

Oral bioavailability of cis-retinoid and naloxonol PEG-matrix

Female SpragueRats (150-200 g) were obtained from the Harlan laboratory. The study was restarted by inserting a catheter into the external jugular vein of the rat and allowing the rat to acclimate for at least 72 hours. Rats were fasted for one night (day 1), but were fed water ad libitum.

On the morning of the day of dosing (day 0), each rat was weighed and the catheter was flushed with heparin (1000U/ml). An aqueous formulation containing either pegylated or free drug was administered orally (gavage) to rats using a feeding tube. The dose is determined in mg/kg body weight. The total dose does not exceed 10 ml/kg. At specified time intervals (1, 2 and 4 hours), blood samples (approximately 1.0 ml) were drawn through the catheter, placed in a 1.5 ml centrifuge tube containing 14 microliters of heparin, mixed and centrifuged to separate the plasma. Plasma samples were frozen (< -70 ℃) prior to assay. Plasma samples were purified by precipitation, analytes extracted and assayed by high performance Liquid Chromatography (LC) using a Mass Spectrometer (MSD). The standard sample was prepared in the same way and a standard curve was generated from which the concentration of the unknown sample could be deduced (see results in table two). Where appropriate, analysis was performed using internal standards.

Table one summarizes selected attributes (e.g., molecular weight and solubility) of the test compounds. Table one also shows the IC_s0Values represent the in vitro enzyme binding activity of some test compounds.

Watch 1

Selected attributes of test compounds

Medicine	Molecular weight	Soluble (mu M)	IC50(nM)*
				13-cis-retinoid (parent drug)	300.45	0.47	-
PEG3-13-cis-RA	445.64	3.13	-
				PEG5-13-cis-RA	549.45	Soluble in water	-
PEG7-13-cis-RA	621.45	58.3	-
				PEG11-13-Cis-RA	797.45	Soluble in water	-
Naloxone "Nal" (parent drug)	327.37	Is as soluble as the HCl salt	6.8
				PEG3The alpha isomer of Nal	475.6	Soluble in water	7.3
PEG3Beta isomer of Nal	475.6	Soluble in water	31.7
				PEG5The alpha isomer of Nal	563.0	Soluble in water	31.5
PEG5Beta isomer of Nal	563.0	Soluble in water	43.3
				PEG7The alpha isomer of Nal	652.0	Soluble in water	40.6
PEG7Beta isomer of Nal	652.0	Soluble in water	93.9
				Alpha isomer of PEG9-Nal	740.0	Soluble in water	64.4
Beta isomer of PEG9-Nal	740.0	Soluble in water	205.0
				Hydroxyzine "Hyd" (parent drug)	374.91	Is as soluble as the HCl salt	48.8
PEG1-Hyd	433.0	Soluble in water	70.3
				PEG3-Hyd	521.0	Soluble in water	105.0
PEG5-Hyd	609.0	Soluble in water	76.7
				Cetirizine "Cet" (parent drug)	388.89	Is as soluble as the HCl salt	77.1
PEG1-Cet	446.0	Soluble in water	61.0
				PEG3-Cet	534.0	Soluble in water	86.4
PEG5-Cet	622.0	Soluble in water	128.0

Mu-opiate binding activity of naloxone series compounds

Histamine H-1 binding Activity of Hydroxylazine and cetirizine series of Compounds

Table II calculates and shows the oral bioavailability of the retinoid acid series compounds. All data were normalized to 6 mg/kg dose. The plasma concentration/time profiles of these compounds are shown in figure 1.

Watch two

Oral bioavailability of retinoid acid series compounds

Table three calculates and gives the oral bioavailability of each isomer in the naloxone series of compounds. The dose of naloxone for oral administration was 5 or 10 mg/kg and the dose of pegylated compound was normalized to 1 mg/kg. The plasma concentration/time curves of these compounds are shown in figure 2.

Watch III

Oral bioavailability of naloxone series compounds

The above results show that pegylation of small lipophilic compounds (free base forms) such as retinoids and naloxone can improve solubility and oral bioavailability. On the other hand, conjugation to oligomeric PEG also increases the molecular weight of the parent compound (greater than about 500 daltons), particularly the increase in length of the PEG-matrix, limiting oral penetration of highly water soluble compounds, which can be obtained from, for example, PEG₇-13-cis-RA and PEG₁₁Two examples of-13-cis-RA.

Example 8

Transport of cis-retinoids and naloxone PEG-matrix across the Blood Brain Barrier (BBB)

In these experiments, the in situ brain perfusion method employed intact rat brain, (i) to determine the ability of the drug to penetrate the BBB under normal physiological conditions, and (ii) to study delivery mechanisms, such as passive diffusion and carrier-mediated delivery.

Perfusion was performed using a single time point method. Briefly, a perfusion fluid (perfusate) containing the test compound was injected into the rat body through the left lateral carotid artery at a constant rate using an infusion pump (20 ml/min). The perfusion flow rate is set to deliver a complete flow of fluid to the brain at normal physiological pressures (80-120mm Hg). The perfusion time lasted 30 seconds. After completion of the perfusion, the cerebral vasculature was immediately reinfused with the drug-free perfusate for 30 seconds to clear residual drug. The infusion pump was turned off and the brain was immediately removed from the skull. The left brain sample from each rat was weighed and then homogenized using a Polytron homogenizer. Four (4) ml of 20% methanol was added to each rat brain for homogenization. After homogenization, the total amount of the homogenized mixture was measured and recorded.

The measured amount of homogeneous mixture was diluted with organic solution and then centrifuged. The supernatant was purged and evaporated in a nitrogen stream and recombined and analyzed by LC/MS. The concentration of drug in the brain homogenate tested was quantified based on a calibration curve generated from the injection of drug into a blank (i.e., no drug) brain homogenate. The concentration of drug in the three brain homogenates was analyzed and the resulting values were used to calculate the brain uptake (unit: pmol/g rat brain/sec perfusion).

Each perfusion solution contained atenolol (target concentration, 50. mu.M), antipyrine (target concentration, 5. mu.M) and test compound (13-cis-retinoid, PEG) at a target concentration of 20. mu.M_n-13-cis-retinoid, naloxone or PEG_n-Nal)。

Table four BBB absorption was calculated, normalized and recorded for each test compound. All data were normalized to a 5 μ M dose solution infusion at a perfusion rate of 20 ml/min for 30 seconds.

Watch four

Blood Brain Barrier (BBB) absorption of test compounds

Medicine	Normalized brain absorption rate, units: p moles/gm brain/sec (mean. + -. SD)	N (rat)
			Atenolol (Low standard)	0.7±0.9	4
Antipyrine (high standard)	17.4±5.7	4
			13-cis-retinoid acids	102.54±37.31	4
PEG3-13-cis-RA	79.65±20.91	4
			PEG5-13-cis-RA	58.49±13.44	3
PEG7-13-cis-RA	24.15±1.49	3
			PEG11-13-cis-RA	17.77±1.68	3
Naloxone	15.64±3.54	3
			PEG3-Nal	4.67±3.57	3
PEG5-Nal	0.96±0.36	3
			PEG7Nal (alpha isomer)	0.94±0.32	3
PEG7Nal (. beta. -isomer)	0.70±0.19	3
			Hydroxyzine	355.89±59.02	3
PEG5-Hyd	131.60±15.84	3
			PEG7-Hyd	12.01±2.97	3
Cetirizine	1.37±0.37	3
			PEG5-Cet	4.32±0.26	3
PEG7-Cet	1.13±0.05	3

The above results show that pegylation of lipophilic compounds such as 13-cis-retinoid can significantly reduce the brain absorption rate ("BUR") of the lipophilic compounds, e.g., PEG, compared to the parent compound "13-cis-retinoid₇The brain absorption of-13-cis-RA was reduced by a quarter, PEG₁₁The brain absorption coefficient of-13-cis-RA decreased by one fifth. For naloxone, PEG₅Nal and PEG₇The BUR of Nal is reduced by one sixteenth. As regards hydroxyzine, as PEG₇BUR is reduced to approximately one twentieth of the original when Hyd is administered. As PEG₇The relatively minimal transport across the blood-brain barrier of cetirizine does not change much upon Cet administration.

Thus, in general, we have surprisingly found that by combining water-soluble small polymers with such small molecule drugs, modifying their ability to penetrate biological membranes, such as membranes associated with the gastrointestinal barrier, the blood-brain barrier, the placental barrier, and the like, the transport properties of the drug can be optimized. More importantly, when drugs are administered orally, the ability of these drugs to penetrate biological barriers such as the blood-brain barrier can be significantly reduced by the incorporation of one or more small water-soluble polymers. Ideally, the delivery of such modified drugs through the gastrointestinal tract is not greatly affected, while delivery across biological barriers such as the blood-brain barrier is significantly impeded, and the oral bioavailability of the modified drug remains above clinically effective levels.

The generated data in examples 7 and 8 were plotted in order to compare the effect of PEG size on the relative oral bioavailability of 13-cis-retinoid and naloxone and BBB delivery, respectively. See fig. 3-7. FIG. 3 examines the effect on oral bioavailability of 13-cis-retinoid when combined with PEG 3-body, PEG 5-body, PEG 7-body and PEG 11-body, respectively. Figure 4 examines the effect of covalent binding to these different PEG-matrices on blood brain barrier transport of 13-cis-retinoid acids. FIG. 5 examines the effect of covalent binding to PEG3-, PEG 5-and PEG 7-bodies on the oral bioavailability of naloxone. FIG. 6 shows the effect of covalent binding to these PEG-matrices on blood brain barrier transport of naloxone. FIG. 7 shows, PEG_nThe Nal compound has higher oral bioavailability than naloxone. As can be seen from these figures, as PEG oligomers are increased, BBB absorption is significantly reduced, while oral bioavailability is improved relative to the parent molecule.

The difference in oral bioavailability between the alpha-and beta-isomers of naloxone may be due to their differences in physicochemical properties. One isomer is slightly more lipophilic than the other, resulting in small differences in oral bioavailability.

Example 9

In vitro metabolism of PEG-naloxonol

To investigate the effect of pegylation on the phase II metabolism (glycosylation) of naloxone, an in vitro method was developed. This method requires the preparation of a NADPH Regenerating System (NRS) solution. Sodium bicarbonate (22.5 mg) was dissolved in 1 ml of deionized water to prepare an NRS solution. To the solution was added B-nicotinamide adenine dinucleotide phosphate sodium salt or NADP (1.6 mg), glucose-6-phosphate (7.85 mg), glucose-6-phosphate dehydrogenase (3. mu.L), uridine 5-diphosphonate glucuronic acid trisodium salt or UDPGA (2.17 mg), adenosine 3 '-phosphate 5' -phosphate lithium sulfate or PAPS (0.52 mg), and 1M magnesium chloride solvent (10. mu.L). After the solid was completely dissolved, the solution was stored in an ice bath.

Adding appropriate amount of naloxone HCl and 6-mPEG₃-O-naloxone, alpha-6-mPEG₅-O-naloxone and alpha-mPEG₇O-naloxone was dissolved in 1 ml of deionized water to prepare a 30mM stock solution of the test substance.

Male Sprague Dawley rat microsomes (0.5 mL, 20 mg/mL; M00001 from In-vitro Technologies, Baltimore, Md.) were removed from the freezer and thawed In an ice bath. Forty microliters of liver microsomes were diluted to 100 microliters by adding 60 microliters of deionized water to the test flask. Tris buffer, pH 7.4 (640. mu.l) and stock solution of the test substance (10. mu.l) were added to the test flask to give a capacity of 750. mu.l.

Each vial and NRS solution was placed separately in a 37 ℃ water bath for 5 minutes. NRS solution (250. mu.L) was added to each vial. When NRS is added to the first vial, a reaction timer is started. Each sample (200. mu.l) was collected, and then perchloric acid (20. mu.l) was added to the collected sample, respectively, to terminate the reaction. Samples were collected at the following time points: 0-2, 20, 40 and 60 minutes. All vials with the reaction terminated were stored in an ice bath.

Acetonitrile (100. mu.l) was added to each vial, followed by centrifugation at 3000Xg for 5 minutes. The supernatant (230. mu.l) was removed and 10. mu.l of the test solution was assayed by LC/MS. At each time point, the concentration of the test substance in each sample was measured and recorded.

Table five lists the percent active residues after liver microsome culture.

Watch five

Percentage of active residue after liver microsome culture

Time (minutes)	Naloxone	α-PEG3-Nal	β-PEG3-Nal	α-PEG5-Nal	α-PEG7-Nal
						0	100.0	100.0	100.0	100.0	100.0
20	47.1	64.8	83.9	84.1	87.4
						40	27.6	51.7	75.2	75.6	81.6
60	15.6	45.7	69.6	69.2	76.9

[0329] From the results in table five, it is possible to conclude that: pegylation with oligomers can reduce the rate of glycosylation of small molecules such as naloxone. In addition, as the PEG oligomer chain increases, the rate of glycosylation decreases. In addition, PEG₃Comparison of the α -isomer with the β -isomer of naloxonol shows that the β -isomer is a poor substrate for cytochrome P450 isozymes in isolated rat liver microparticles. This observation reconfirmed the in vivo data shown in fig. 7.

Turning to fig. 8 and 9, the data show that conjugation to small PEG can effectively reduce the rate of drug metabolism (as shown by glucuronide formation in the case of naloxone). It is possible that the level of β -isomer in blood is higher than α -isomer because the first-pass effect is largely prevented, that is, the extent of first-pass metabolism is significantly prevented due to covalent binding to oligomeric PEG molecules (fig. 7). The PEG molecule may have steric hindrance and/or hydrophilic or hydrophobic effects, and PEG binding to the β -isomer form alters the affinity of the β -isomer conjugate and cytochrome P450 isozyme to a greater extent than PEG binding to the α -isomer form. The levels of β -isomer metabolites were lower compared to α -isomer metabolites and non-pegylated naloxone.

Example 10

Activity of various opioid antagonists at the mu-opiate receptor

The biological activity of various conjugates on naloxone, other opioid antagonists and the mu-opiate receptor in vitro was determined in a separate series of experiments. Table six is a summary of the test results.

Watch six

Naloxone and PEG at the mu-opiate receptor_n-6-naloxonol conjugate activity in vitro.

Compound (I)	Molecular weight	EC50(nM)
			Naloxone	327.4	6.8
3-PEG3-O-naloxone	474	2910.0
			6-NH2-naloxone	601	29.2
PEG550-6-NH-naloxone (PEG)13Amides)	951	210.0
			Alpha-6-naloxonol	329	2.0
Beta-6-naloxonol	329	10.8
			α-PEG3-Nal	475.6	7.3
β-PEG3-Nal	475.6	31.7
			α-PEG5-Nal	563	31.5
β-PEG5-6-Nal	563	43.3
			α-PEG7-6-Nal	652	40.6
β-PEG7-6-Nal	652	93.9

In the above table, the biological activity of each PEG conjugate is shown as its relative biological activity compared to the parent drug for each compound. EC (EC)₅₀Is the concentration of agonist that elicits a response in the standard dose response curve halfway between the baseline and the highest response. As shown by the above data, various PEGs_nThe Nal conjugates are biologically active and virtually all 6-naloxone or naloxone conjugates retain at least 5% or greater of the biological activity of the parent drug, which ranges from about 5% to about 35% of the biological activity of the unmodified parent compound. In terms of biological activity, PEG₅₅₀-6-NH-naloxone having its parent compound (6-NH)₂-naloxone) about 13% of the biological activity, alpha-PEG₃Nal has about 30% of the biological activity of its parent compound (α -6-OH-naloxonol), while β -PEG₃Nal has about 35% of the biological activity of its parent compound (. alpha. -6-OH-naloxonol).

Example 11

Process for the preparation of oligo (ethylene glycol) methyl ethers and derivatives thereof of completely monomolecular weight

The monomolecular (monodisperse) PEG of the present invention is prepared according to the method described in detail below. These unimolecular PEGs have particularly significant advantages in providing the modified active agents of the invention and the desirable barrier transport properties of the active agents.

The process illustrated below represents another aspect of the invention, namely a process for the preparation of monodisperse oligo (ethylene oxide) methyl ethers from low molecular weight monodisperse oligo (ethylene glycol) s using halogen-derived (e.g. bromine-derived) oligo (ethylene oxide). In another aspect of the invention, a method of using halogen-derived oligo (ethylene oxide) methyl ethers to combine oligo (ethylene oxide) methyl ethers (from a single molecular weight composition) with an active agent is also provided herein.

The reaction scheme is as follows:

(m＝1，2，3；n＝2，3，4，5，6)

10.A.CH ₃ O-(CH ₂ CH ₂ O) ₅ -H and CH ₃ OCH ₂ CH ₂ Synthesis of Br

Tetra (ethylene glycol) (55 mmol, 10.7 g) was dissolved in 100 ml of tetrahydrofuran ("THF") and KOtBu (55 ml, 1.0M in THF) was added at room temperature. The resulting solution was stirred at room temperature for 30 minutes overnight, then CH was added dropwise₃OCH₂CH₂Br (55 mmol, 5.17 ml in 50 ml THF). The reaction was stirred at room temperature overnight and then treated with H₂O (300 ml) is presentCH₂Cl₂(3X 300 ml) was extracted. The obtained organic extracts are mixed and then added to anhydrous Na₂SO₄And drying. After filtration of the solid desiccant and evaporation of the solution, a crude residue is obtained which is purified by means of a silica gel Column (CH)₂Cl₂∶CH₃OH 60: 1-40: 1) was purified by column chromatography to yield pure pentakis (ethylene glycol) monomethyl ether (yield 35%).¹H NMR(CDCl₃)δ3.75-3.42(m，20H，OCH₂CH₂O)，3.39(s，3H，MeO)。

10.B.CH ₃ O-(CH ₂ CH ₂ O) ₇ -H and MeOH ₂ CH ₂ Synthesis of Br

Sodium hydride (2.55 g, 106 mmol) was slowly added to a solution of hexa (ethylene glycol) (10 g, 35 mmol) and 2-bromoethyl methyl ether (4.9 g, 35 mmol) in THF (100 ml). The solution was stirred at room temperature for two hours. HPLC showed that mPEG was formed in about 54% yield₇-OH. The reaction was stopped by adding dilute hydrochloric acid to eliminate excess sodium hydride. Using a rotary evaporator, all solvent was removed to yield a brown viscous liquid. Using semi-preparative HPLC (20 cm. times.4 cm, C18 column, acetonitrile and water as mobile phase), pure mPEG was obtained₇-OH — colorless liquid (4.9 g, isolated yield 41%).¹H NMR(CDCl₃): 2.57ppm (triplet, 1H, OH); 3.38ppm (singlet, 3H, CH)₃O); 3.62ppm (multiplicities, 30H, OCH)₂CH₂)。

10.C.CH ₃ O-(CH ₂ CH ₂ O) ₅ Synthesis of-Br

Triethylamine (5.7 ml, 40 mmol) was added to CH₃O-(CH₂CH₂O)₅to-OH (5.0 g, 20 mmol), stirring was carried out with addition. Solution in ice bath N₂Cooled under an atmosphere and 2.5 ml of methane were added dropwise over 30 minutesSulfonyl chloride (32 mmol). The solution was stirred at room temperature overnight. Water (40 ml) was then added to the reaction mixture, followed by CH₂Cl₂The solution was extracted (3X 150 ml) and the organic phase obtained was washed with 0.1N HCl (3X 80 ml) and water (2X 80 ml). With Na₂SO₄After drying and removal of the solution, a light brown liquid was obtained. Mixing the product with Bu₄NBr (12.80 g, 39.7 mmol) is dissolved in CH₃CN (50 ml), the resulting solution was at a temperature of 50 ℃ N₂Stirred under atmosphere for 15 hours. After cooling to room temperature, CH was removed by rotary evaporation₃CN, yielded a red liquid, which was dissolved in 150 ml of water and extracted with EtOAc (2 × 200 ml). The resulting organic phases were combined, washed with water and then Na₂SO₄And drying. After removal of the solvent, a pale red liquid was obtained (4.83 g, 77.4%).¹H NMR(300Hz，CDCl₃)：δ3.82(t，2H)，3.67(m，14H)，3.51(m，2H)，3.40(s，3H)。

Example 11

Synthesis of mPEG 3N-Meerquinine

Sodium cyanoborohydride (60 mg, 0.96 mmol) in water (1 ml) was added to mefloquine HCl salt (200 mg, 0.48 mmol) and mPEG₃-butylaldehyde (280 mg, 1.20 mmol) in methanol (5 ml). The resulting solution was heated in an oil bath at 50 ℃ under nitrogen for 16 hours with stirring. HPLC showed the reaction was complete. Using a rotary evaporator, all solvents were removed to yield a crude product. Purification by preparative reverse phase HPLC gave pure mPEG-3-N-melphalan quinine conjugate as a colorless viscous liquid (160 mg, 0.27 mmol, isolated yield 56%),¹H NMR(CDCl₃ppm): 8.15 (multiploid, 3H, aromatic ring); 7.73 (triplet, 1H, aromatic ring); 5.86 (doublet, 1H, CH); 3.67 (multiploid, 14H, PEG backbone); 3.52 (singlet, 3H, PEG-OCH)₃) (ii) a 3.18 (multiplex, 2H, PEG-CH)₂) (ii) a 0.52-2.74 (multiplet, 13H, PEG and cyclohexyl protons).

The reaction scheme is as follows:

。

Claims (8)

1. A monodisperse composition comprising a compound selected from the group consisting of:

6-CH₃-(OCH₂CH₂)₅-O-naloxonol; and

6-CH₃-(OCH₂CH₂)₇-O-naloxonol;

or a pharmaceutically acceptable salt thereof, wherein the compound is the α -6 isomer, the β -6 isomer, or a mixture of the α -6 isomer and the β -6 isomer.

2. The monodisperse composition of claim 1 wherein the compound is selected from the group consisting of:

α，β-6-CH₃-(OCH₂CH₂)₅-O-naloxonol; and

α，β-6-CH₃-(OCH₂CH₂)₇-O-naloxonol;

or a pharmaceutically acceptable salt thereof.

3. The monodisperse composition of claim 2 wherein the compound is α, β -6-CH₃-(OCH₂CH₂)₇-O-naloxonol or a pharmaceutically acceptable salt thereof.

4. The monodisperse composition of claim 1 wherein the compound is selected from the group consisting of:

α-6-CH₃-(OCH₂CH₂)₅-O-naloxonol; and

α-6-CH₃-(OCH₂CH₂)₇-O-naloxonol;

or a pharmaceutically acceptable salt thereof.

5. The monodisperse composition of claim 4 wherein the compound is α -6-CH₃-(OCH₂CH₂)₇-O-naloxonol or a pharmaceutically acceptable salt thereof.

6. The monodisperse composition of claim 1 wherein the compound is selected from the group consisting of:

β-6-CH₃-(OCH₂CH₂)₅-O-naloxonol; and

β-6-CH₃-(OCH₂CH₂)₇-O-naloxonol;

or a pharmaceutically acceptable salt thereof.

7. The monodisperse composition of claim 6 which isWherein the compound is beta-6-CH₃-(OCH₂CH₂)₇-O-naloxonol or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical composition comprising a monodisperse composition according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient.