In other words, a BM can be linked to the functionalized POX polymers either directly or indirectly through the functional groups. In another aspect, in embodiments, a biological material, a bioactive material, a pharmaceutically active agent and the like can be complexed with the polymer of interest without a formal reaction resulting in a covalent bond, instead mere mixing of the polymer with the a biological material, a bioactive material, a pharmaceutically active agent and the like results in a physical relationship between same and a polymer of interest such that the two entities demonstrate a coordinated presence.
Hence, such a composition without a covalent bond of a biological material, a bioactive material, a pharmaceutically active agent and the like and a polymer of interest has the same properties and functions as other compositions of interest. For example, in addition to the BM already attached to a POX at the reactive chain end, an additional BM also can be attached to the same polymer at the initiator end, if the POX utilized is linear.
If the POX is branched, multiple BM molecules can be attached to the same polymer at the multiple initiator ends. In addition, due to the stepwise reaction approach, not only the same but different BM molecules can be attached to the same POX polymer at the initiator and reactive chain ends. Such a differentiated POX can be useful in linking different kinds of BM molecules, and at varying ratios.
Accordingly, various other functional groups can be introduced at the initiator end of the POX polymer in addition to at the terminator end. The functional groups include, but are not limited to, ethyl bromoacetate, methyl bromoacetate, tert-butyl bromoacetate, propyl bromoacetate, benzyl bromoacetate, sulfur-containing compounds, such as, 2- p-toluenesulfonyloxy ethyl disulfide TOEDS , chloromethyl methyl disulfide, bis iodomethyl methyl disulfide and 2-bromoethyl disulfide, silicon-containing compounds, such as, 3-chloropropyl triethoxysilane, 3-bromopropyl trimethoxysilane and 3-iodopropyl trimethoxysilane and protected groups for amines or imines, such as, those comprising a carboxybenzyl group, a p-methoxybenzyl carbonyl group, a tert-butyloxycarbonyl group and a 9-fluorenylmethyloxycarbonyl FMOC group; and so on.
For example, TOEDS, a difunctional initiator, can be utilized to initiate the polymerization of oxazoline monomers at both ends. On termination, for example, with a large excess of ethylene diamine EDA , a POX polymer with amino and imino functional groups at both chain ends can be produced. The chain ends further can be linked with any of a plurality of bioactive materials, such as, small molecule drugs, such as, gemcitabine, camptothecin, paclitaxel and so on, either directly or indirectly through covalent linkages.
When the reagents comprise a disulfide bond, addition of a reducing agent, such as, dithiothreitol DTT , can cleave that bond to generate a sulfhydryl-functionalized POX polymer-bioactive material composition. The sulfhydryl group then can be linked, for example, with a maleimide-functionalized targeting molecule, such as, peptide, protein, such as, antibody, sialic acid, one member of a binding pair and so on to provide a differentiated POX polymer composition with one end linked with any of a plurality of bioactive materials, such as, small molecule drugs, and the other end linked with at least a targeting molecule, such as, peptide, protein, such as, antibody, sialic acid and so on.
The initiator, I, can comprise different functional groups, such as, an alkyl, an aryl, an ester, an amide, a sulfur-containing group, a silica-containing group, a protected amine, a protected imine and so on. MA is methacrylate. EDA is ethylenediamine. The initiator is cleavable. NHS is N-hydroxysuccinimide. DCC is dicyclohexylcarbodiimide. In the second reaction step, EOX is added. In the fourth reaction step, SA is added. DTT is dithiothreitol. MAL is maleimide. PEG is polyethyleneglycol.
The alkyl can comprise one or more saturated bonds or can be unsaturated. Also, the alkyl can comprise one or more pendant groups, functional groups, modifications and the like, which can include other elements, such as, oxygen, nitrogen, sulfur, iodine, bromine or chlorine, or can contain a polar group, such as hydroxyl or amine or a non-polar group, such as, a hydrocarbon, such as, an aliphatic group or an aromatic group, which also can be substituted.
An aryl can comprise multiple aromatic rings, which may be fused or joined, and which can be substituted. An aryl also can comprise a heterocyclic compound, such as, an unsubstituted and a substituted furan or pyridine. Esters are usually derived from an inorganic acid or organic acid in which at least one —OH hydroxyl group is replaced by an —O-alkyl alkoxy group, and most commonly from carboxylic acids and alcohols.
That is, esters are formed by condensing an acid with an alcohol. The compound can be 2- p-toluenesulfonyloxy ethyl disulfide TOEDS , chloromethyl methyl disulfide or bis iodomethyl methyl disulfide, 2-bromoethyl disulfide, for example. The compound can be 3-chloropropyl triethoxysilane, 3-bromopropyl trimethoxysilane or 3-iodopropyl trimethoxysilane, for example. A B can comprise a PAA. A "functional group" is a group that may be used, under normal conditions of organic synthesis, to form a covalent linkage between the structure to which it is attached and another structure, which typically bears a further functional group.
Preferred functional groups for use in the polymers of the invention are described below. The term "reactive" refers to a functional group that reacts readily or at a practical rate under conventional conditions of organic synthesis.
This is in contrast to those groups that either do not react or require strong catalysts or impractical reaction conditions in order to react i. An "activated derivative" of a carboxylic acid refers to a carboxylic acid derivative which reacts readily with nucleophiles, generally much more readily than the underivatized carboxylic acid. Activated carboxylic acids include, for example, acid halides such as acid chlorides , anhydrides, carbonates, and esters.
Such esters include, for example, imidazolyl esters, and benzotriazole esters, and imide esters, such as N-hydroxysuccinimidyl NHS esters. An activated derivative may be formed in situ by reaction of a carboxylic acid with one of various reagents, e.
A "protecting group" is a moiety that prevents or blocks reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions.
The protecting group will vary depending upon the type of chemically reactive group being protected as well as the reaction conditions to be employed and the presence of additional reactive or protecting groups in the molecule.
Functional groups which may be protected include, by way of example, carboxylic acid groups, amino groups, hydroxyl groups, thiol groups, carbonyl groups and the like. Representative protecting groups for carboxylic acids include esters such as a -methoxybenzyl ester , amides and hydrazides; for amino groups, carbamates such as tert-butoxycarbonyl and amides; for hydroxyl groups, ethers and esters; for thiol groups, thioethers and thioesters; for carbonyl groups, acetals and ketals; and the like.
Such protecting groups are well-known to those skilled in the art and are described, for example, in T. Greene and G. A functional group in "protected form" refers to a functional group bearing a protecting group. As used herein, the term "functional group" or any synonym thereof is meant to encompass protected forms thereof. The variable n is 3 to , and the terminal groups and architecture of the overall PEG may vary. When PEG further comprises a spacer as in structure I above to be described in greater detail below , the atoms comprising the spacer X , when covalently attached to a PEG segment, do not result in formation of i an oxygen-oxygen bond -O-O-, a peroxide linkage , or ii a nitrogen-oxygen bond N-O, O-N.
PEGs for use in the invention include PEGs having a variety of molecular weights, structures or geometries to be described in greater detail below. An "end-capping" or "end-capped" group is an inert group present on a terminus of a polymer such as PEG.
An end-capping group is one that does not readily undergo chemical transformation under typical synthetic reaction conditions. An end capping group is generally an alkoxy group, -OR, where R is an organic radical comprised of carbons and is preferably lower alkyl e. Alternatively, the end-capping group can also advantageously comprise a detectable label.
Such labels include, without limitation, fluorescers, chemiluminescers, moieties used in enzyme labeling, colorimetric e. A non-naturally occurring polymer of the invention may however contain one or more subunits or segments of subunits that are naturally occurring, so long as the overall polymer structure is not found in nature.
The polymers of the invention are typically polydisperse, possessing low polydispersity values of less than about 1. The term "linker" is used herein to refer to an atom or a collection of atoms used to link interconnecting moieties, such as an organic radical core and a polymer segment, POLYi. A linker moiety may be hydrolytically stable or may include a physiologically hydrolyzable or enzymatically degradable linkage.
A linker designated herein as Q is hydrolytically stable. The term "spacer" is used herein to refer to a collection of atoms used to link interconnecting moieties, such as POLYi and the active agent, D. A spacer moiety may be hydrolytically stable or may include a physiologically hydrolyzable or enzymatically degradable linkage.
A spacer designated herein as X comprises a hydrolyzable linkage, where the hydrolyzable linkage is attached directly to the active agent, D, such that upon hydrolysis, the active agent is released in its parent form. A "hydrolyzable" bond is a relatively weak bond that reacts with water i. The tendency of a bond to hydrolyze in water will depend not only on the general type of linkage connecting two central atoms but also on the substituents attached to these central atoms.
Illustrative hydrolytically unstable linkages include carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides. An "enzymatically degradable linkage" means a linkage that is subject to degradation by one or more enzymes. Such a linkage requires the action of one or more enzymes to effect degradation. A "hydrolytically stable" linkage or bond refers to a chemical bond, typically a covalent bond, that is substantially stable in water, that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time.
Examples of hydrolytically stable linkages include but are not limited to the following: carbon-carbon bonds e. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks. Thus, a multi-armed polymer may possess 3 polymer arms, 4 polymer arms, 5 polymer arms, 6 polymer arms, 7 polymer arms, 8 polymer arms or more, depending upon its configuration and core structure. One particular type of highly branched polymer is a dendritic polymer or dendrimer, that for the purposes of the invention, is considered to possess a structure distinct from that of a multi-armed polymer.
A multi-arm polymer may have one branch point or multiple branch points. A "dendrimer" is a globular, size monodisperse polymer in which all bonds emerge radially from a central focal point or core with a regular branching pattern and with repeat units that each contribute a branch point. Dendrimers exhibit certain dendritic state properties such as core encapsulation, making them unique from other types of polymers.
Such hydrocarbon chains are preferably but not necessarily saturated and may be branched or straight chain, although typically straight chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1- ethylpropyl, 3-methylpentyl, and the like.
As used herein, "alkyl" includes cycloalkyl when three or more carbon atoms are referenced. The term "substituted" as in, for example, "substituted alkyl," refers to a moiety e. For substitutions on a phenyl ring, the substituents may be in any orientation i. As used herein, "alkenyl" refers to a branched or unbranched hydrocarbon group of 1 to 15 atoms in length, containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, and the like.
The term "alkynyl" as used herein refers to a branched or unbranched hydrocarbon group of 2 to 15 atoms in length, containing at least one triple bond, ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl, octynyl, decynyl, and so forth. Aryl includes multiple aryl rings that may be fused, as in naphthyl or unfused, as in biphenyl. Aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings. As used herein, "aryl" includes heteroaryl.
Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings. Preferred heteroatoms include sulfur, oxygen, and nitrogen. This includes foods, food supplements, nutrients, nutriceuticals, drugs, vaccines, antibodies, vitamins, and other beneficial agents. As used herein, these terms further include any physiologically or pharmacologically active substance that produces a localized or systemic effect in a patient.
The precise amount will depend upon numerous factors, e. Multi-functional polymers of the invention will typically contain from about functional groups, or from functional groups, or from functional groups, or from functional groups, or from 3 to 10 functional groups, i. Typically, in reference to a polymer precursor used to prepare a polymer prodrug of the invention, the polymer possesses 3 or more polymer arms having at the terminus of each arm a functional group suitable for coupling to an active agent moiety via a hydrolyzable linkage.
When the functional groups are the same, the entity is said to be homodifunctional or homobifunctional. When the functional groups are different, the polymer is said to be heterodifunctional or heterobifunctional A basic or acidic reactant described herein includes neutral, charged, and any corresponding salt forms thereof. An exemplary polyolefinic alcohol is polyvinyl alcohol.
As used herein, "non-peptidic" refers to a polymer backbone substantially free of peptide linkages. However, the polymer may include a minor number of peptide linkages spaced along the repeat monomer subunits, such as, for example, no more than about 1 peptide linkage per about 50 monomer units. The term "patient," refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a polymer of the invention, typically but not necessarily in the form of a polymer-active agent conjugate, and includes both humans and animals.
A "small molecule" may be defined broadly as an organic, inorganic, or organometallic compound typically having a molecular weight of less than about Small molecules of the invention encompass oligopeptides and other biomolecules having a molecular weight of less than about An "active agent moiety" in reference to a prodrug conjugate of the invention, refers to the portion or residue of the umodified parent active agent up to the covalent linkage resulting from covalent attachment of the drug or an activated or chemically modified form thereof to a polymer of the invention.
Upon hydrolysis of the hydrolyzable linkage between the active agent moiety and the multi-armed polymer, the active agent per se is released. The conjugates of the invention are typically prodrugs, meaning that the active agent, attached to the polymer via a hydrolytically degradable linkage, is released over time following administration of the conjugate to a subject. Moreover, the conjugates of the invention are well-characterized, isolable, and purifiable compositions, in comparison to, for example, a degradable polymer- matrix having molecules of drug encapsulated therein.
The conjugates of the invention exhibit higher drug loading characteristics when compared to their linear polymer-based counterparts, thus lowering the total dosage weight needed to treat a particular disease state. That is to say, the polymer scaffold of the invention is effective to covalently attach multiple active agent molecules thereto, thereby allowing a greater amount of therapeutic agent i.
The polymers used in the invention are hydrophilic in nature, thereby imparting hydrophilicity to the resulting conjugates, which, particularly in the case of water-insoluble active agents, facilitates their formulation into useful pharmaceutical compositions.
Typically, the total number average molecular weight of the overall multi-arm polymer portion of a polymer conjugate of the invention is about 1, daltons Da to about , Da, more preferably about 10, Da to about 60, Da, most preferably about 15, to about 60, Da.
Multi-armed polymers having a number average molecular weight of about 5, Da, about 8, Da, about 10, Da, about 12, Da, about 15, Da, about 20, Da, about 25, Da, about 30, Da, about 35, Da, about 40, Da, about 45, Da, about 50, Da, and about 60, Da are particularly preferred.
Multi-armed polymers having a molecular weight of 20, Da or greater, i. The actual molecular weight of the multi-armed polymer will depend, of course, on the number of polymer arms and the molecular weight of each polymer arm in the overall multi-armed polymer. The linkage between the multi-armed polymer portion and the active agent is preferably hydrolytically degradable for in vivo release of the parent drug molecule over time.
Representative hydrolytically degradable linkages corresponding to X in structure I include carboxylate ester, carbonate ester, phosphate ester, anhydride, acetal, ketal, acyloxyalkyl ether, imine, orthoester, and oligonucleotides. In some cases depressive symptoms persist for longer. Ecstasy was ranked 18th in dependence, physical harm, and social harm. Motor delays may be temporary during infancy or long-term.
The severity of these developmental delays increases with heavier MDMA use.Multifunctional polymers of the invention will typically contain from about functional groups, or from functional groups, or from functional groups, or from functional groups, or from 3 to 10 functional groups, or will contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups within the polymer backbone. It is therefore likely that no serotonergic neurotoxicity is present in most casual users. In a further embodiment providing a more complex linkage between the polymer and the Diels-Alder adduct, the polymer has the formula POLY-L3-Y, wherein POLY is a water soluble and non-peptidic polymer, L3 is a hydrolytically stable linkage, and Y is a functional group reactive with the Diels-Alder adduct reagent, such as an amino group. Alternately or in addition, the method includes the step of purifying the oligomer once it is formed.
An end-capping group is one that does not readily undergo chemical transformation under typical synthetic reaction conditions. In addition to providing alternative rates of hydrolysis, the oligomeric reagents provided herein have additional advantages over prior art oligomeric reagents. All atoms are understood to have their normal number of valences for bond formation i. The existence of the opioid delta receptor is a recent discovery which followed the isolation and characterization of endogenous enkephalin peptides which are ligands for the delta receptor. In some instances, opioid diarylpiperazines can be obtained from commercial sources. Alternatively, the end-capping group can also advantageously comprise a detectable label.
Examples include Ecallantide. As stated above, for the PEG oligomers, the variable n ranges from about 1 to 30, and the terminal groups and architecture of the overall PEG can vary.
It has been hypothesized that these conjugates are inactive due to the relatively long polyethylene glycol chain, which may "wrap" itself around the entire active agent, thereby blocking access to potential ligands required for activity. Exemplary phospholipids include, without limitation, phosphatidylcholines, phospatidylserine, phospatidylinositol, phospatidylglycerol, and phospatidylethanolamine.
In comparison, conjugation of an active agent to a polymer such as polyethylene glycol offers a more well-defined alternative, since the conjugate itself is often although not necessarily well-characterized, particularly in the case of site-specific attachment of the polymer to the active agent. In some instances, it is preferred that X is not an amide, i. Appropriate hydrolytically unstable or weak linkages include but are not limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides, oligonucleotides, thioesters, and carbonates. Suitable detectors include photometers, films, spectrometers, and the like. For instance, a bimodal oligomer may have any one of the following exemplary combinations of monomer subunits: , , , , , , , , , and so forth; , , , , , , , , and so forth; , , , , , 3- 9, , and so forth; , , , , , , and so forth; , , , , , and so forth; , , , , and so forth; , , , and so forth; and , , and so forth.
POX polymers comprising, for example, poly unsubstituted oxazoline or poly substituted oxazoline , can be produced by cationic ring opening polymerization. The termination of a reactive POX polymer with a polyfunctional polymer to generate star, comb, Starburst or Combburst polymers is described, for example, in Tomalia, U.
The substituted alkyl groups may be substituted once, twice or three times with the same or with different substituents.
Maleimide groups can be used to facilitate covalent attachment of proteins and other molecules to polymers. Finally, the synthesis of a modified or drug-delivery enhanced active agent must result in reasonable yields, to make any such approach economically attractive.
The combined organic layers were washed with water and saturated sodium chloride, and dried over sodium sulfate. Preferably, R contains from about 3 to about 50 carbon atoms, and even more preferably, R contains from about 3 to about 10 carbon atoms. The conjugates of the invention exhibit higher drug loading characteristics when compared to their linear polymer-based counterparts, thus lowering the total dosage weight needed to treat a particular disease state. Oligomer compositions possessing a well-defined mixture of oligomers i. Focusing on organic cores derived from polyols or polya ines, although the number of intervening atoms between each hydroxyl or amino group will vary, preferred cores are those having a length of from about 1 to about 20 intervening core atoms, such as carbon atoms, between each hydroxyl or amino group, preferably from about 1 to about 5. Molecular weight in the context of a water soluble polymer of the invention, such as PEG, can be expressed as either a number average molecular weight or a weight average molecular weight.
Exemplary phospholipids include, without limitation, phosphatidylcholines, phospatidylserine, phospatidylinositol, phospatidylglycerol, and phospatidylethanolamine. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks. Alternately or in addition, the method includes the step of purifying the oligomer once it is formed. In one embodiment involving formation of a maleimide group from a Diels-Alder adduct prior to reaction with a polymer, the method comprises: i providing a linker molecule comprising a hydrocarbon chain, the hydrocarbon chain comprising a bivalent saturated cycloalkyl group, an alkylene group, or a combination thereof, and having a total number of carbons ranging from 1 to about 30; ii reacting the linker molecule with a Diels-Alder adduct comprising a polycyclic imide ring or a polycyclic anhydride ring to form a Diels-Alder adduct linker; iii treating the Diels-Alder linker under conditions sufficient to initiate a retro Diels-Alder reaction, optionally in the presence of a reactive dienophile, wherein a diene is released from the Diels-Alder adduct to form a maleimide functionalized linker; and iv reacting a water soluble and non-peptidic polymer with the maleimide functionalized linker to form a maleimide functionalized polymer. C,
Preferred oligomers used in connection with the present invention are homo-oligomers. Preferred oligomers used in connection with present the invention are homo-oligomers. Again, chromatography is not a viable option because of the sensitivity of the maleimide group to the functional groups of the ion exchange column. Thus, using techniques well known in the art, the oligomer can be functionalized to include a reactive group e. Such a linkage requires the action of one or more enzymes to effect degradation. A non-naturally occurring polymer of the invention may however contain one or more subunits or segments of subunits that are naturally occurring, so long as the overall polymer structure is not found in nature.
The laminated structure can contain a single reservoir, or it can contain multiple reservoirs. However, preliminary experiments have suggested that BWU86 may produce an increase in hyperactivity, including convulsions Comer, S. In addition, the term "active agent" is intended to encompass the active agent prior to conjugation as well as the active agent "residue" following conjugation.